WO2020007227A1 - 检测慢性鼻窦炎伴鼻息肉亚型的方法和试剂盒及alox15基因作为生物标志物的应用 - Google Patents

检测慢性鼻窦炎伴鼻息肉亚型的方法和试剂盒及alox15基因作为生物标志物的应用 Download PDF

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WO2020007227A1
WO2020007227A1 PCT/CN2019/093281 CN2019093281W WO2020007227A1 WO 2020007227 A1 WO2020007227 A1 WO 2020007227A1 CN 2019093281 W CN2019093281 W CN 2019093281W WO 2020007227 A1 WO2020007227 A1 WO 2020007227A1
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gene
alox15
nasal
rna
detecting
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French (fr)
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张罗
王成硕
闫冰
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Beijing Institute Of Otolaryngology
Beijing Tongren Hospital
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Beijing Institute Of Otolaryngology
Beijing Tongren Hospital
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Priority claimed from CN201810717413.XA external-priority patent/CN108949954A/zh
Priority claimed from CN201810720285.4A external-priority patent/CN108913764A/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material

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  • the present disclosure belongs to the technical field of biomedicine, and particularly relates to a kit for detecting chronic sinusitis with nasal polyp subtype and the application of ALOX15 gene as a biomarker, and a method for detecting the expression level of ALOX15 gene in nasal exfoliated cells. .
  • CRSwNP Chronic rhinosinusitis with nasal polyps
  • CRSwNP is often accompanied by asthma and allergic rhinitis. It has been reported that 7% of asthma patients have CRSwNP and 26-48% of CRSwNP have asthma. The pathogenesis of CRSwNP is still uncertain.
  • CRSwNP can be divided into Eosinophilic (CRSwNP, ECRSwNP) and Nonosinophilic (CRSwNP, nonECRSwNP) according to the degree of eosinophil infiltration.
  • the clinical manifestations, medications and prognosis of the two are different.
  • the clinical symptoms of eosinophils are severe, mainly nasal congestion and decreased olfactory symptoms. Most patients have asthma, and the recurrence rate is higher.
  • the degree of eosinophil infiltration in nasal polyp tissue is most closely related to recurrence. When the percentage of cells in the tissue exceeds 27%, the risk of recurrence exceeds 90%.
  • the sensitivity of eosinophilic polyps to glucocorticoids is significantly higher than that of non-eosinophilic polyps.
  • the clinical symptoms of non-eosinophils are generally mild, and there is less chance of asthma, airway inflammation is lighter, and the postoperative recurrence rate is lower than that of eosinophils, and the response to macrolide therapy is good.
  • the western countries are mainly eosinophils, which mainly show TH2 inflammatory response, while the proportion of eosinophils and non-eosinophils in China is about half, and the non-eosinophils mainly show TH1 / TH17 is predominantly inflammatory.
  • the eosinophilic and non-eosinophilic types are significantly different in immunopathological types, clinical symptoms, drug response, and prognosis.
  • Different chronic sinusitis with nasal polyps have different inflammatory / pathological typing treatment strategies. Therefore, the identification of the pathological classification of chronic sinusitis with nasal polyps is particularly important.
  • the judgment of the two subtypes is mainly based on the staining of histopathological specimens after nasal mucosal biopsy, and the lack of non-invasive biological markers for differential diagnosis.
  • the patients were routinely treated with pathological specimens such as paraffin fixation, and then stained with hematoxylin and eosin, and then the tissue-infiltrated inflammatory cells were observed with a high-power microscope (mainly inflammatory cells include eosinophil , Neutrophils, lymphocytes, plasma cells) infiltration number for cell typing.
  • pathological specimens such as paraffin fixation
  • hematoxylin and eosin mainly inflammatory cells include eosinophil , Neutrophils, lymphocytes, plasma cells
  • the disadvantages of nasal mucosal biopsy are as follows: 1.
  • kits for detecting chronic sinusitis with nasal polyp subtype wherein the kit includes a specific primer for the ALOX15 gene.
  • the present disclosure also provides an application of ALOX15 gene as a biomarker in the preparation of a product for detecting chronic sinusitis with nasal polyp subtypes.
  • the present disclosure also provides the use of the ALOX15 gene as a biomarker for detecting chronic sinusitis with nasal polyp subtypes.
  • the present disclosure also provides the use of the kit according to the present disclosure for detecting chronic sinusitis with nasal polyp subtypes.
  • the present disclosure also provides the use of a reagent for detecting ALOX15 for detecting chronic sinusitis with nasal polyp subtypes.
  • the present disclosure also provides a method for detecting the expression level of ALOX15 gene in nasal cavity exfoliated cells, including the following steps: extracting RNA from nasal cavity exfoliated cells, reverse transcription of total RNA into cDNA, and using quantitative polymerase chain reaction to convert ALOX15 in cDNA Genes and internal reference genes were amplified by real-time fluorescent quantitative PCR using specific primers of the ALOX15 gene and specific primers of the internal reference gene, and the expression of ALOX15 gene was calculated based on the detection results of the amplified products.
  • the present disclosure also provides an application of the above method for detecting the expression level of ALOX15 gene in nasal exfoliated cells in preparing a kit for detecting a chronic sinusitis subtype with nasal polyps.
  • the present disclosure also provides a method for detecting a chronic sinusitis subtype with nasal polyps, which comprises detecting the ALOX15 gene expression amount in nasal exfoliated cells by the method described in the present disclosure.
  • the present disclosure also provides a method for detecting a chronic sinusitis subtype with nasal polyps, comprising detecting and detecting the expression level of ALOX15 gene in nasal exfoliated cells, preferably using the kit of the present disclosure to detect Expression of ALOX15 gene.
  • FIG. 1 is a real-time fluorescent quantitative PCR amplification curve diagram of ALOX15 gene as part of an experimental example of the present disclosure
  • FIG. 2 is a real-time quantitative PCR amplification curve diagram of ALOX15 gene in another part of the experimental example of the present disclosure
  • FIG. 3 is a real-time quantitative PCR amplification curve diagram of ALOX15 gene in the experimental example of the present disclosure
  • FIG. 4 is a real-time fluorescent quantitative PCR amplification curve diagram of ALOX15 gene in another part of the experimental example of the present disclosure
  • FIG. 5 is a melting curve diagram of ALOX15 gene real-time quantitative PCR
  • FIG. 6 is a melting curve diagram of ALOX15 gene real-time quantitative PCR in another part of the experimental example of the present disclosure
  • FIG. 7 is a melting curve diagram of the ALOX15 gene real-time quantitative PCR in the experimental example of the present disclosure.
  • FIG. 8 is a melting curve diagram of the ALOX15 gene real-time quantitative PCR, which is part of the experimental example of the present disclosure.
  • FIG. 9 is an optional ROC curve for detecting chronic sinusitis with nasal polyp typing in Experimental Example 1 of the present disclosure.
  • FIG. 10 is a real-time fluorescent quantitative PCR amplification curve of ALOX15 gene in the second part of the experimental example of the present disclosure.
  • FIG. 11 is a real-time quantitative PCR amplification curve diagram of ALOX15 gene in another part of Experimental Example 2 of the present disclosure.
  • FIG. 12 is a real-time fluorescent quantitative PCR amplification curve diagram of ALOX15 gene in a second part of Experimental Example 2 of the present disclosure
  • FIG. 15 is a melting curve diagram of the ALOX15 gene real-time quantitative PCR in a second part of the experimental example of the present disclosure.
  • 16 is a real-time fluorescent quantitative PCR amplification curve of ALOX15 gene in Experimental Example 3 of the present disclosure
  • FIG. 17 is a melting curve diagram of real-time fluorescent quantitative PCR of ALOX15 gene in Experimental Example 3 of the present disclosure.
  • FIG. 19 is a melting curve diagram of ALOX15 gene real-time quantitative PCR in Experimental Example 4 of the present disclosure.
  • primer refers to an oligonucleotide that is capable of specifically annealing to an RNA or DNA region site adjacent to a target sequence, and as appropriate under conditions Primer for DNA synthesis. It is usually single-stranded, which can be naturally occurring or synthetic.
  • a “primer” or “oligonucleotide primer” typically comprises a sequence between about 5 to about 50 nucleotides, more preferably about 10 to about 30 nucleotides, or more preferably about 15 to 25 cores Glycylic acid.
  • the term "upstream primer” as used herein generally binds to a region closer to the 5 ' end of the nucleic acid molecule.
  • the term “downstream primer” as used herein generally binds to a region closer to the 3 'end of the nucleic acid molecule relative to a region on the nucleic acid to be amplified.
  • the term “internal reference gene” generally refers to a gene that is expressed relatively constant in various tissues and cells, such as GAPDH gene in nasal polyp tissue or cells exfoliated from nasal mucosa, and is mainly used as a reference when detecting gene expression levels.
  • polymerase chain reaction is the amplification of a nucleic acid consisting of: the initial denaturation step of separating the strands of a double-stranded nucleic acid sample, followed by repeating (i) allowing the amplification primers to be specific to the flanking position of the target sequence Annealing step of thermal annealing; (ii) an extending step of extending primers in the 5 'to 3' direction to form an amplicon polynucleotide complementary to the target sequence, and (iii) denaturation causing separation of the amplicon from the target sequence step.
  • the above steps can be performed at different temperatures, preferably using an automatic thermal cycler.
  • Real-time quantitative PCR also known as quantitative PCR, or QPCR for short, refers to adding a fluorescent group to a PCR reaction system, and using fluorescence signal accumulation to monitor the entire PCR process in real time. Method for quantitative analysis of unknown template by standard curve.
  • Ct cycle threshold
  • Ct value generally refers to the number of cycles that a fluorescent signal undergoes when a fluorescent signal reaches a set threshold.
  • the upstream primer of the ALOX15 gene is shown in SEQ ID NO: 2
  • the downstream primer of the ALOX15 gene is shown in SEQ ID NO: 3.
  • the kit further includes specific primers for internal reference genes.
  • the internal reference gene is GAPDH
  • the upstream primer of the internal reference gene is shown in SEQ ID NO: 4
  • the downstream primer of the ALOX15 gene is shown in SEQ ID NO: 5.
  • the kit further includes: a reagent for extracting RNA from nasal polyp tissue or detached cells from nasal mucosa; a reagent for reverse transcription of total RNA into cDNA; using quantitative polymerase chain reaction Reagent for performing real-time quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA.
  • the reagent for reverse transcription of total RNA into cDNA includes: a reverse transcription mixture and deRNase and deDNase water;
  • the reagent for reverse transcription of the total RNA into cDNA includes: 1 ⁇ L to 40 ⁇ L of a reverse transcription mixed solution, and 0 ⁇ L to 160 ⁇ L of deRNase and deDNase water. Further preferably, the reagent for reverse transcription of total RNA into cDNA includes: 2 ⁇ L of a reverse transcription mixed solution, and 0 ⁇ L to 8 ⁇ L of deRNase and DNAse dehydrating water.
  • a reagent for performing a real-time fluorescent quantitative PCR reaction of the ALOX15 gene and an internal reference gene in a cDNA using a quantitative polymerase chain reaction includes a PCR premix solution, double distilled water, machine fluorescence compensation, and a corrector. , The upstream primer of the ALOX15 gene, the downstream primer of the ALOX15 gene, the upstream primer of the internal reference gene, and the downstream primer of the internal reference gene.
  • a reagent for performing a real-time fluorescent quantitative PCR reaction of the ALOX15 gene and an internal reference gene in the cDNA by quantitative polymerase chain reaction includes: 1 ⁇ L to 25 ⁇ L of a PCR master mix, and 0 ⁇ L to 50 ⁇ L of a double-distillation Water, 0 ⁇ L to 2 ⁇ L of machine fluorescence compensation and correction agent, 0.01 to 100 ⁇ M of the ALOX15 gene upstream primer, 0.01 to 100 ⁇ M of the ALOX15 gene downstream primer, 0.01 to 100 ⁇ M of the internal reference gene upstream primer, 0.01 to 100 ⁇ M of the internal reference gene Downstream primers; Further preferably, the reagent for performing real-time quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction includes: 5 ⁇ L of PCR premix, 0 ⁇ L to 10 ⁇ L of double distilled water, according to the total volume Make up to 10 ⁇ L
  • the reagent for extracting RNA from nasal polyp tissue can be selected from the following two reagents.
  • the first type includes: RNA extraction solution, chloroform, isopropanol, 65% to 90% ethanol, RNase and DNAse-free water; among these, preferably, 0.1 mL to 20 mL of RNA extraction solution Trizol or RNAiso Blood or RNAiso Plus or other substances containing phenol, guanidine isothiocyanate, 8-hydroxyquinoline, guanidine isothiocyanate or ⁇ -mercaptoethanol, the Trizol or the RNAiso Blood or the RNAiso Plus Or 0.1 to 0.5 times the volume of the other substance containing phenol, guanidine isothiocyanate, 8-quinolinol, guanidine isothiocyanate, or ⁇ -mercaptoethanol, and 0.5 to 3 times the volume of the chloroform Isopropyl alcohol, 0.5 to 5 times the volume of isopropyl alcohol, 65% to 90% ethanol, and 0.01mL to 5mL of RNase and DNase water; further preferably
  • the reagent for extracting RNA from nasal polyp tissue includes: a cell lysate, a first buffer solution for removing impurities from the purification column to which RNA is adsorbed, and an impurity for removing impurities from the purification column to which RNA is adsorbed And saline second buffer solution and deRNase and DNase water;
  • the tool for extracting RNA from nasal polyp tissue includes an RNA purification column; wherein the reagent for extracting RNA from nasal polyp tissue also includes DNase
  • the reaction solution or the tool for extracting RNA from nasal polyp tissue also includes a genomic DNA adsorption column;
  • the DNase reaction solution includes a DNase buffer solution, a recombinant DNase, and a double-distilled water of deRNase.
  • the reagent for extracting RNA from nasal polyp tissue includes: 0.1 mL to 2 mL of a cell lysate for lysing cells and inhibiting RNA degradation, and 0.1 mL to 0.7 mL of a washing agent
  • a washing agent One buffer, 0.1 mL to 0.7 mL of second buffer for washing, 0.01 mL to 1 mL of RNase and DNase-free water, 0 to 10 ⁇ L of genomic DNA-recombinant DNase, and 0 to 10 ⁇ L of genomics DNA DNase buffer, 20 to 100 ⁇ L of RNase-free double-distilled water
  • the tool for extracting RNA from nasal polyp tissue includes an RNA purification column; or includes 0.1 mL to 2 mL for lysing cells and inhibiting RNA degradation Cell lysate, 0.1 mL to 0.7 mL of first buffer for washing, 0.1 mL to 0.7 mL of second buffer for washing, 0.01 mL to 1 m
  • the reagent for extracting RNA from nasal polyp tissue includes: 300 ⁇ L of a cell lysate for lysing cells and inhibiting RNA degradation, 500 ⁇ L of a first buffer for washing, and 600 ⁇ L of washing Use a second buffer, 0.02 mL of RNase and DNase-free water, 4 ⁇ L of genomic DNA-recombinant DNase, 5 ⁇ L of 10 ⁇ genomic DNA-removed DNase buffer, 41 ⁇ L of RNase-free double-distilled water
  • the tool for extracting RNA from nasal polyp tissue includes an RNA purification column; or includes 300 ⁇ L of a cell lysate for lysing cells and inhibiting RNA degradation, 500 ⁇ L of a first buffer for washing, and 600 ⁇ L of a second buffer for washing Solution, 0.02 mL of RNase and DNase water
  • the tools for extracting RNA from nasal polyp tissue include genomic DNA adsorption column and RNA purification column.
  • the nasal polyp tissue is nasal polyp tissue obtained from nasal pathological biopsy, or the nasal mucosa exfoliated cells are nasal polyp cells obtained by brushing or sticking to the surface of the nasal polyp.
  • the ⁇ Ct (Ct (ALOX15) -Ct (GAPDH)) analysis method is used to analyze the data result of the amplification product, and the cutoff value compared with the ⁇ Ct is 1.675.
  • the upstream primer of the ALOX15 gene is shown in SEQ ID NO: 2
  • the downstream primer of the ALOX15 gene is shown in SEQ ID NO: 3.
  • the internal reference gene is GAPDH
  • the upstream primer of the internal reference gene is shown in SEQ ID NO: 4
  • the downstream primer is shown in SEQ ID NO: 5.
  • the nasal cavity exfoliated cells are obtained by using a hair brush on the surface of the nasal polyp, and the hair brush after the nasal exfoliated cells are obtained is placed in a cell lysate and stored below 4 ° C.
  • the method for extracting RNA from exfoliated cells in the nasal cavity includes two methods, wherein the first method includes the following steps:
  • Step 1 Dissolve the nasal cavity exfoliated cells in 100-2000 ⁇ L of cell lysate, add an equal volume of ethanol, mix well and add it to the RNA purification column. After centrifugation, remove the filtrate from the collection tube. The RNA purification column is placed in a collection tube;
  • Step 2 Add 300 ⁇ L to 700 ⁇ L of the first buffer to the RNA purification column obtained in step 1, and centrifuge to remove the first filtrate; continue to add 400 ⁇ L to 800 ⁇ L of the second buffer to the RNA purification column, After centrifugation, the second filtrate was removed, and an RNA purification column was eluted to obtain RNA.
  • the method for extracting RNA from exfoliated cells in the nasal cavity further includes the following steps: adding 10 to 100 ⁇ L of a DNase reaction solution to the RNA purification column after removing the second filtrate, and After the standing treatment, 300 ⁇ L to 700 ⁇ L of the second buffer solution was added, and after centrifugation, the third filtrate was removed. After the RNA purification column was eluted, the RNA purity was measured using a spectrophotometer to obtain RNA;
  • the method for preparing the DNase reaction solution includes the following steps: a DNase buffer solution, a recombinant DNase, and double-distilled water from which the RNase is removed are mixed to obtain a DNase reaction solution.
  • the method for preparing the DNase reaction solution includes the following steps: 5 ⁇ L of 10 ⁇ DNase buffer solution, 4 ⁇ L of recombinant DNase, and 41 ⁇ L of RNase-free double-distilled water are mixed to obtain a DNase reaction solution.
  • the method for extracting RNA from nasal cavity exfoliated cells further includes the following steps: in step 1 After the nasal cavity exfoliated cells are dissolved in the cell lysate, they are first added to a genomic DNA adsorption column to take a filtrate, and then an equal volume of ethanol is added to the filtrate.
  • the method for extracting RNA from exfoliated cells in the nasal cavity further includes the following steps: adding an RNA purification column to be eluted and adding distilled water to remove RNA hydrolase Or diethyl pyrocarbonate treated water, left at room temperature, centrifuged, and eluted the RNA purification column, and measured the purity of the RNA using a spectrophotometer to obtain RNA.
  • Step 1 uses a cell lysate that can rapidly break down nasal cells and inhibit nucleases released by nasal cells; genomic DNA adsorption columns are used to remove genomic DNA; RNA purification columns in step 2 are used to enrich RNA; The collection tube is used for collecting the solution after removing the genomic DNA, the first buffer solution for removing impurities in the purification column to which the RNA is adsorbed, and the second buffer solution for removing impurities and salts in the RNA solution.
  • the first method for extracting RNA from exfoliated nasal cells includes the following steps:
  • Step 1 Dissolve the nasal cavity exfoliated cells in 300 ⁇ L of cell lysate, add an equal volume of 70% ethanol, and mix the solution evenly with a pipette; immediately add the mixed solution to the RNA purification column at 12000 rpm , Centrifuge for 1min, remove the filtrate, and place the RNA purification column in a 2mL collection tube;
  • Step 2 Add 500 ⁇ L of the first buffer to the RNA purification column obtained in step 1, centrifuge at 12,000 rpm for 30 s to remove the first filtrate; continue to add 600 ⁇ L of the second buffer to the RNA purification column, Centrifuge at 12000 rpm for 30 seconds to remove the second filtrate;
  • Step 3 Take 5 ⁇ L of 10 ⁇ DNase buffer, 4 ⁇ L of recombinant DNase, and 41 ⁇ L of RNase-free double-distilled water to obtain a DNase reaction solution. Add 50 ⁇ L of DNase to the RNA purification column after removing the second filtrate. The reaction solution was left at room temperature for 15 minutes, 350 ⁇ L of the second buffer solution was added, 12000 rpm, and centrifuged for 30 seconds to remove the third filtrate;
  • Step 4 Place the RNA purification column with the third filtrate removed in step 3 in a 1.5 mL RNA hydrolase-free collection tube. Add 50 ⁇ L of RNA hydrolase-free distilled water or 0.1% diethyl pyrocarbonate-treated water to the RNA purification column. After standing at room temperature for 5 minutes, 12000 rpm, centrifugation for 2 minutes, the RNA purification column was eluted, and the OD260 / OD280 ratio of the RNA solution was measured using a spectrophotometer to obtain an RNA of 1.7 to 2.1.
  • the method for extracting RNA from exfoliated cells in the nasal cavity includes the following steps:
  • Step 1 Take the genomic DNA adsorption column in a 2mL collection tube, dissolve the nasal cavity exfoliated cells in 100-2000 ⁇ L of cell lysate, and add it to the genomic DNA adsorption column. Take the filtrate and add an equal volume of 70% ethanol, mixed well and added to the RNA purification column, 12000 rpm, centrifugation for 1min, the filtrate was removed, and the RNA purification column was placed in a 2mL collection tube;
  • Step 2 Add 500 ⁇ L of the first buffer to the RNA purification column obtained in step 1, centrifuge at 12,000 rpm for 30 s to remove the first filtrate; continue to add 600 ⁇ L of the second buffer to the RNA purification column, Centrifuge at 12000 rpm for 30 seconds to remove the second filtrate;
  • Step 3 Place the RNA purification column with the second filtrate removed in step 2 into a 1.5 mL RNA hydrolase-free collection tube. Add 50 ⁇ L of RNA hydrolase-free distilled water or 0.1% diethyl pyrocarbonate-treated water to the RNA purification column. After standing at room temperature for 5 minutes, 12000 rpm, centrifugation for 2 minutes, the RNA purification column was eluted, and the OD260 / OD280 ratio of the RNA solution was measured using a spectrophotometer to obtain an RNA of 1.7 to 2.1.
  • the second method for extracting RNA from nasal cavity exfoliated cells includes the following steps: adding 0.1 mL to 20 mL of RNA extraction solution to a centrifuge tube containing nasal cavity exfoliated cells for lysis 1. Add chloroform 0.1-0.5 times the volume of the RNA extraction solution after shaking, mix by shaking, leave it at room temperature, centrifuge the centrifuge tube, take the supernatant, and add 0.5-3 times the volume of chloroform.
  • Isopropyl alcohol stand still, centrifuge after mixing, discard the supernatant, retain the first precipitate, and add 0.5 to 5 times the volume of 65% to 90% ethanol of the isopropyl alcohol to the first precipitate, After washing, mix and centrifuge, discard the supernatant, and retain the second pellet; cover the centrifuge tube, centrifuge again, remove the supernatant, and continue to add 0.01 to 5 mL of RNase and DNAase to the centrifuge tube The second precipitate was dissolved in water, and the purity of the RNA was measured by a spectrophotometer to obtain RNA.
  • the RNA extraction solution is Trizol, RNAiso Blood, RNAiso Plus or other phenol, guanidinium isothiocyanate, 8-hydroxyquinoline, guanidine isothiocyanate, and ⁇ -mercapto group. Any one or several reagents in ethanol.
  • the second method for extracting RNA from nasal cavity exfoliated cells includes the following steps: adding 0.1 to 20 mL of RNA extraction solution to a centrifuge tube containing nasal cavity exfoliated cells for dissolution and shaking After that, let stand at room temperature for 3 ⁇ 7min; add 40 ⁇ L ⁇ 5mL of chloroform, shake and mix well, let stand at room temperature for 3 ⁇ 7min, centrifuge at 3 °C ⁇ 5 °C, 10000 ⁇ 14000r / min for 10 ⁇ 20min; take the supernatant 40 ⁇ L ⁇ 8mL Add equal volume of isopropanol, mix and let stand for 8 ⁇ 12min, centrifuge at 3 °C ⁇ 5 °C, 10000 ⁇ 14000r / min for 10 ⁇ 20min, discard the supernatant and keep the first precipitate; Add 65% to 90% ethanol with the same volume as isopropanol, and centrifuge at 7000 to 14000 r / min for 10
  • the second method for extracting RNA from nasal cavity exfoliated cells includes the following steps: After adding 1 mL of RNA extraction solution to a centrifuge tube containing nasal cavity exfoliated cells for dissolution and shaking, Let stand at room temperature for 5min; add 200 ⁇ L of chloroform, shake and mix, stand at room temperature for 5min, and centrifuge at 12,000r / min for 15min at 4 °C; take 200 ⁇ L of supernatant, add 200 ⁇ L of isopropanol, and let stand for 10min after mixing, and stand at 4 °C The supernatant was discarded by centrifugation at 12000 r / min for 15 min, and the first pellet was retained; 75% ethanol with an equal volume of isopropyl alcohol was added to the first pellet, and centrifuged at 7500 r / min for 15 min at 4 ° C, and the supernatant was discarded.
  • the method for reverse transcription of total RNA into cDNA includes the following steps: taking 1 to 3 ⁇ L of the reverse transcription mixture, 0 to 10 ⁇ L of de-hydrolase distilled water and the extracted RNA at a temperature of 37 ° C. A reverse transcription reaction occurred under the conditions for 15 min, and then a reverse transcriptase inactivation reaction occurred at a temperature of 84 ° C to obtain a reverse transcription product cDNA.
  • the method for reverse transcription of total RNA into cDNA includes the following steps: taking 2 ⁇ L of the reverse transcription mixture, 8 ⁇ L of the deRNA hydrolase distilled water, and a total amount not exceeding 500 ng or The volume does not exceed 8 ⁇ L of total RNA, and the de-RNA hydrolase distilled water is used to make up to 10 ⁇ L.
  • the reverse transcription reaction is performed under the following conditions: at 37 ° C, a reverse transcription reaction is performed for 15 minutes; at 85 Under the condition of °C, the reverse transcriptase inactivation reaction was performed for 5 seconds; the product was left at 4 °C.
  • the reaction system can be scaled up according to requirements.
  • a 10 ⁇ L reaction system can use a maximum of 500 ng of total RNA. Those skilled in the art can choose according to actual needs.
  • the real-time quantitative PCR amplification includes the following steps:
  • Step 1 Prepare a real-time quantitative PCR reaction solution: include 1 ⁇ L to 25 ⁇ L of PCR premix, 0 ⁇ L to 10 ⁇ L of double distilled water to make up the total volume of water to 10 ⁇ L, 0 ⁇ L to 2 ⁇ L of machine fluorescence compensation and correction agent, 0.01 to 100 ⁇ M Upstream primer of ALOX15 gene, downstream primer of ALOX15 gene of 0.01 to 100 ⁇ M, upstream primer of internal reference gene of 0.01 to 100 ⁇ M, downstream primer of internal reference gene of 0.01 to 100 ⁇ M, 0.01 ⁇ L to 5 ⁇ L of cDNA;
  • Step 2 Real-time quantitative PCR detection using standard two-step PCR amplification standard program or three-step PCR amplification standard program;
  • Step 3 Calculate the expression of ALOX15 gene.
  • a real-time quantitative PCR reaction solution is prepared: including 5 ⁇ L of a PCR premix, 2.8 ⁇ L of double distilled water to make up a total volume of water to 10 ⁇ L, 0.2 ⁇ L of a machine fluorescence compensation and correction agent, 0.5 ⁇ L of the ALOX15 upstream primer, 0.5 ⁇ L of the ALOX15 downstream primer, 0.5 ⁇ L of the internal reference gene upstream primer, 0.5 ⁇ L of the internal reference gene downstream primer, 1 ng / ⁇ L of the cDNA or 0.01 ⁇ L to 5 ⁇ L of all primers Mentioned RNA.
  • the reaction conditions of the standard two-step PCR amplification procedure include the following steps: Stage 1: Pre-denaturation for 30 seconds at 95 ° C; Stage 2 PCR reaction: at 95 The reaction was carried out at a temperature of 15 ° C for 15 seconds, and at a temperature of 60 ° C for 60 seconds, followed by annealing and extension, and 40 cycles were performed.
  • the reaction conditions of the standard three-step PCR amplification procedure include the following steps: Stage 1: Pre-denaturation at 95 ° C for 2 minutes; Stage 2 PCR reaction: at 95 The reaction was performed at a temperature of 1 ° C for 1 minute, at 55 ° C for 1 minute, and at 72 ° C for 1 minute, and 40 cycles were thus performed. Finally, 72 ° C, annealed and extended for 7 minutes.
  • the reagent for detecting ALOX15 includes a specific primer for the ALOX15 gene, and preferably the upstream primer of the specific primer for the ALOX15 gene is as shown in SEQ ID NO: 2 and the ALOX15 gene The downstream primers are shown in SEQ ID NO: 3.
  • the reagent for detecting ALOX15 is selected from the group consisting of a primer, an antibody, an aptamer, a probe, or a combination thereof for ALOX15.
  • detection of ALOX15 gene level by fluorescent PCR method is used to detect chronic sinusitis with nasal polyp subtypes.
  • the amount of ALOX15 gene expression in nasal exfoliated cells is detected by fluorescent PCR.
  • chronic sinusitis with nasal polyp subtype is determined according to ⁇ Ct (Ct (ALOX15) -Ct (GAPDH)), Ct (ALOX15) is the Ct value of ALOX15 gene, and Ct (GAPDH) is Ct value of internal reference gene GAPDH.
  • the ⁇ Ct greater than or equal to 1.675 represents non-eosinophilic chronic sinusitis with nasal polyps, and the ⁇ Ct less than 1.675 represents eosinophilic chronic sinusitis with nasal polyps.
  • the chronic sinusitis with nasal polyp subtype is non-eosinophilic chronic sinusitis with nasal polyps or eosinophilic chronic sinusitis with nasal polyps.
  • the present disclosure provides a kit for detecting chronic sinusitis with nasal polyp subtypes.
  • the proteomics and transcriptomics methods are used to select the ALOX15 gene as a biomarker and apply it to the kit to achieve A kit was used to detect chronic sinusitis with nasal polyp subtypes, so that the final kit obtained included specific primers for the ALOX15 gene.
  • the kit of the present disclosure can quickly identify nasal polyp subtypes, and is more accurate than traditional pathological detection methods.
  • the kit can simultaneously perform large-scale, rapid Testing, saving labor costs and medical treatment costs. And the systematic kit has high identification accuracy, which can fully reflect the histopathological characteristics.
  • the kit provided by the present disclosure can detect nasal polyp cells from the surface of the nasal polyp by brushing or sticking, so as to determine the chronic sinusitis of the patient with nasal polyp subtypes, avoiding causing the patient
  • the wound surface improves the safety of patient examination, and the operation is more convenient, saving labor costs and medical treatment costs.
  • the method for detecting the expression level of ALOX15 gene in nasal exfoliated cells provided by the present disclosure, using the effectively screened ALOX15 gene as a biomarker, and providing a method for detecting its gene expression level, thereby realizing the expression of ALOX15 gene in nasal exfoliated cells.
  • the calculation can effectively obtain the expression level of ALOX15 gene, and the provided method is simple, fast, sensitive, and reproducible, which is suitable for wide application.
  • ALOX15 gene in nasal exfoliated cells
  • the ALOX15 gene can encode a lipoxygenase protein
  • the encoded enzyme acts on various polyunsaturated fatty acid substrates to produce various biologically active lipids Medium, such as eicosanoid, heparin, lipoxin, etc.
  • the arachidonic acid was converted into 12-eicosatetraenoic acid / 12-HPETE and 15-eicosatetraenoic acid / 15-HPETE. It also converts linoleic acid into 13-hydroperoxyoctadecadienoic acid.
  • ALOX15 can also act on (12S) -hydrogen peroxytetraenoic acid / (12S) -HPETE to produce heparin A3.
  • the encoded enzyme and its reaction products can regulate inflammation and immune response.
  • Diseases related to ALOX15 include softening of white matter around the ventricle. It helps clear apoptotic cells during inflammation by oxidizing membrane-bound phosphatidylethanolamine in macrophages, and inhibits autoimmune responses associated with inflammatory monocytes clearing apoptotic cells.
  • the ALOX15 gene has not been disclosed in the prior art, and the use of the gene has not been found.
  • the method for the expression of ALOX15 gene in exfoliated nasal cells can be used to detect the expression of ALOX15 in exfoliated nasal cells, and it can be used to further provide a basis for the gene screening technology for detecting chronic sinusitis subtypes with nasal polyps, It provides a reliable basis for clinical guidance and drug treatment. The feasibility of the kit for detecting chronic sinusitis subtype with nasal polyps in clinical application is guaranteed.
  • nasal shedding method of detecting expression of genes in cells of the present disclosure provides ALOX15, using ⁇ Ct or 2 - ⁇ relative quantification method Ct method according to actual needs, the expression of selected relatively constant reference gene, carried out with the number of reference gene Standardization. Calculate the expression of the target gene by measuring the difference between the Ct value of the target gene and the internal reference gene. The method is simple and fast, the detection accuracy is high, the detection cost can be reduced, and the detection time can be saved. The result is easy to interpret and so on. Greatly improved the experimental efficiency.
  • chronic sinusitis with nasal polyp subtype is detected by detecting the level of ALOX15 protein in nasal lesion tissue of sinusitis with nasal polyps.
  • a nasal cavity tissue sample of chronic sinusitis with nasal polyps is obtained by, for example, nasal endoscopy, and the protein level of ALOX15 in the sample is determined by, for example, immunohistochemistry.
  • the protein level of ALOX15 in the sample is determined using an antibody against ALOX15.
  • an antibody against ALOX15 for detecting chronic sinusitis with nasal polyp subtypes is provided.
  • the embodiment of the present disclosure provides a kit for detecting chronic sinusitis with nasal polyp subtype, the kit includes a specific primer of ALOX15 gene.
  • the present disclosure obtains a kit for detecting chronic sinusitis with nasal polyp subtypes by using the ALOX15 gene as a biomarker through proteomics and transcriptomics methods through a large number of creative experiments and screenings, which are not currently provided in the existing technology. Any reports accordingly.
  • the ALOX15 gene is a known gene, the gene ID is 246, the DNA sequence is shown in SEQ ID NO: 1, and the gene NM number is 001140.4.
  • the upstream primer of the ALOX15 gene is shown in SEQ ID NO: 2
  • the downstream primer of the ALOX15 gene is shown in SEQ ID NO: 3.
  • the kit of the present disclosure has the highest accuracy and more effective in identifying nasal polyp subtypes, which makes the kit suitable for large-scale and rapid detection.
  • the kit further includes a reference gene. More preferably, the internal reference gene is GAPDH, the upstream primer of the internal reference gene is shown in SEQ ID NO: 4, and the downstream primer of the ALOX15 gene is shown in SEQ ID NO: 5.
  • the upstream and downstream primers of the internal reference gene determined by the kit of the present disclosure can effectively obtain the appropriate ⁇ CT value by displaying the expression of the ALOX15 gene compared to GAPDH when identifying the nasal polyp subtypes of the kit of the present disclosure. Identification of nasal polyp subtypes. And has a higher accuracy.
  • the kit further comprises: a reagent for extracting RNA from nasal polyp tissue or detached cells from nasal mucosa; a reagent for reverse transcription of total RNA into cDNA; and a quantitative polymerase chain reaction for converting cDNA Reagents for the ALOX15 gene and internal reference genes for real-time quantitative PCR reactions.
  • the reagent for reverse transcription of total RNA into cDNA includes: a reverse transcription mixed solution and de-RNase and de-DNase water; using quantitative polymerase chain reaction to perform real-time ALOX15 gene and internal reference gene in cDNA
  • the reagents for the quantitative PCR reaction include: PCR premix, double-distilled water, machine fluorescence compensation and correction agent, upstream primer of ALOX15 gene, downstream primer of ALOX15 gene, upstream primer of internal reference gene, and downstream primer of internal reference gene.
  • RNA extraction solution chloroform, isopropanol, 65% to 90% ethanol, RNase and DNA. Enzyme water
  • the reagent for extracting RNA from nasal polyp tissue includes: a cell lysate, a first buffer solution for removing impurities from the purification column to which RNA is adsorbed, and an impurity for removing impurities from the purification column to which RNA is adsorbed And saline second buffer solution and deRNase and DNase water;
  • the tool for extracting RNA from nasal polyp tissue includes an RNA purification column; wherein the reagent for extracting RNA from nasal polyp tissue also includes DNase
  • the reaction solution or the tool for extracting RNA from nasal polyp tissue also includes a genomic DNA adsorption column;
  • the DNase reaction solution includes a DNase buffer solution, a recombinant DNase, and a double-distilled water of deRNase.
  • the reagent for reverse transcription of total RNA into cDNA includes: 1 ⁇ L to 40 ⁇ L of a reverse transcription mixed solution, and 0 ⁇ L to 160 ⁇ L of deRNase and DNAse dehydrating water. Further preferably, the reagent for reverse transcription of the total RNA into cDNA includes: 2 ⁇ L of a reverse transcription mixed solution, and 0 ⁇ L to 8 ⁇ L of deRNase and DNAase water (completed to 8 ⁇ L with water according to the amount of RNA).
  • the reverse transcription step can be realized, and for those skilled in the art, it can be selected according to actual needs.
  • the reagents for performing real-time fluorescent quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction include: 1 ⁇ L to 25 ⁇ L PCR premix, 0 ⁇ L to 50 ⁇ L double-distilled water, and 0 ⁇ L to 2 ⁇ L machine Fluorescence compensation and correction agent, 0.01 to 100 ⁇ M upstream primer of ALOX15 gene, 0.01 to 100 ⁇ M downstream primer of ALOX15 gene, 0.01 to 100 ⁇ M internal reference gene upstream primer, 0.01 to 100 ⁇ M internal reference gene downstream primer; more preferably,
  • the reagents for real-time fluorescent quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction include: 5 ⁇ L of PCR premix, 0 ⁇ L to 10 ⁇ L of double distilled water (make up to 10 ⁇ L with water based on the total volume), 0.2 ⁇ L of machine fluorescence compensation and correction agent, 1 ⁇ M of the A
  • the following two reagents can be selected.
  • the first includes: 0.1 mL to 20 mL of RNA extraction solution Trizol or RNAiso, Blood or RNAiso Plus or other phenol, isothiocyanate Guanidine acid, 8-hydroxyquinoline, guanidine isothiocyanate, or ⁇ -mercaptoethanol, the Trizol or the RNAisoBlood or the RNAisoPlus or the other substances containing phenol, guanidine isothiocyanate, 8- Hydroxyquinoline, guanidine isothiocyanate, or ⁇ -mercaptoethanol are 0.1 to 0.5 times the volume of chloroform, 0.5 to 3 times the volume of isopropyl alcohol, and 0.5 to 5 times the volume of isopropyl alcohol. 65% to 90% ethanol, and 0.01mL to 5mL of RNase and DNase water;
  • Another type includes: 0.1 mL to 2 mL of a cell lysate for lysing cells and inhibiting RNA degradation, 0.1 mL to 0.7 mL of a first buffer for washing, 0.1 mL to 0.7 mL of a second buffer for washing, 0.01mL to 1mL of RNase and DNase-free water, 0 to 10 ⁇ L of genomic DNA-recombinant DNase, 0 to 10 ⁇ L of genomic DNA-removed DNase buffer, 20 to 100 ⁇ L of RNase-free double distillation Water
  • the tool for extracting RNA from nasal polyp tissue includes an RNA purification column; or includes 0.1 mL to 2 mL of a cell lysate for lysing cells and inhibiting RNA degradation, and 0.1 mL to 0.7 mL of a first buffer for washing 0.1 to 0.7 mL of a second buffer solution for washing, 0.01 to 1 mL of RNase and DNase water, and the tools for extract
  • the following two reagents can be selected, the first includes: 1 mL of RNA extraction solution Trizol or RNAiso Blood or RNAiso Plus or other phenol, guanidine isothiocyanate , 8-hydroxyquinoline, guanidine isothiocyanate or ⁇ -mercaptoethanol, 200 ⁇ L of chloroform, 200 ⁇ L of isopropanol, 200 ⁇ L of 65% to 90% ethanol by volume, and 0.02 mL of deRNase and DNAse-free water; the names of the RNA extraction solutions are Trizol, RNAiso Blood, and RNAiso Plus are all trade names.
  • Another type includes: 300 ⁇ L of cell lysate for lysing cells and inhibiting RNA degradation, 500 ⁇ L of first buffer for washing, 600 ⁇ L of second buffer for washing, 0.02 mL of RNase and DNase Water, 4 ⁇ L of genomic DNA-removed recombinant DNase, 5 ⁇ L of 10 ⁇ genomic DNA-removed DNase buffer, 41 ⁇ L of de-RNase double-distilled water, the tool for extracting RNA from nasal polyp tissue includes an RNA purification column; Alternatively, it includes 300 ⁇ L of a cell lysate for lysing cells and inhibits RNA degradation, 500 ⁇ L of a first buffer for washing, 600 ⁇ L of a second buffer for washing, 0.02 mL of RNase and DNase water, Tools for extracting RNA from nasal polyp tissue include genomic DNA adsorption columns and RNA purification columns. For the above-mentioned limited numerical ranges, the steps of extracting RNA from nasal polyp tissue can be realized
  • the cell lysate described above is used to rapidly disrupt cells and inhibit the release of nucleases from cells.
  • the first buffer is used to remove impurities from the purification column to which RNA is adsorbed, and the second buffer is used to remove the RNA that is adsorbed.
  • the impurities and salts of the purification column, de-RNase and DNase-free water are used to dissolve the RNA.
  • reagents for extracting RNA from nasal polyp tissue reagents for reverse transcription of total RNA into cDNA
  • reagents for quantitative real-time PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction reagents for quantitative real-time PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction .
  • the nasal polyp tissue is nasal polyp tissue obtained from nasal pathological biopsy, or the nasal mucosa exfoliated cells are nasal polyp cells obtained by brushing or sticking to the surface of the nasal polyp.
  • the brushing or sticking method is used to avoid wounds to the patient, improve the safety of patient examination, and make the operation more convenient, saving labor costs and medical treatment costs.
  • the ⁇ Ct (Ct (ALOX15) -Ct (GAPDH)) analysis method is used to analyze the data result of the amplification product, and the limit value for comparison with the ⁇ Ct is 1.675.
  • the defined value can enable the kit provided by the present disclosure to achieve an accuracy rate of more than 75% when detecting chronic sinusitis with nasal polyp subtypes.
  • An embodiment of the present disclosure also provides an application of the ALOX15 gene as a biomarker in preparing a product for detecting chronic sinusitis with nasal polyp subtypes.
  • the product may be a detection reagent, a chip or a kit.
  • the above embodiments only describe the specific technical content of the kit, for those skilled in the art, based on the disclosure of the technical solution of the present application, combined with common general knowledge, the specific technical content of the detection reagents and chip products can be directly obtained. .
  • An embodiment of the present disclosure provides a method for detecting the expression level of ALOX15 gene in nasal exfoliated cells, including the following steps: extracting RNA from nasal exfoliated cells, reverse-transcribe total RNA into cDNA, and use quantitative polymerase chain reaction to convert the The ALOX15 gene and internal reference gene were amplified by real-time fluorescent quantitative PCR using specific primers of the ALOX15 gene and specific primers of the internal reference gene, respectively. The expression of ALOX15 gene was calculated based on the detection results of the amplified products.
  • the present disclosure uses proteomics and transcriptomics methods to screen a large number of creative experiments to obtain a subtype of chronic sinusitis with nasal polyps by calculating the expression of ALOX15 gene, and provides a simple and reliable method for calculating the expression of ALOX15 gene. , High accuracy. No corresponding reports have been provided in the existing technology.
  • the ALOX15 gene is a known gene, the gene ID is 246, the DNA sequence is shown in SEQ ID NO: 1, and the gene NM number is 001140.4.
  • the upstream primer of the ALOX15 gene is shown in SEQ ID NO: 2
  • the downstream primer of the ALOX15 gene is shown in SEQ ID NO: 3.
  • the design of the upstream and downstream primers of the ALOX15 gene has higher sensitivity. When detecting the expression of the ALOX15 gene, the results are more accurate and repeatable.
  • the internal reference gene is GAPDH
  • the upstream primer of the internal reference gene is shown in SEQ ID NO: 4
  • the downstream primer is shown in SEQ ID NO: 5.
  • the nasal cavity exfoliated cells are obtained by using a hair brush on the surface of the nasal polyps, and the hair brush after obtaining the nasal cavity exfoliated cells is placed in a cell lysate and stored below 4 ° C. Based on this method, the method of the present disclosure avoids wounds to patients, improves the safety of patient examination, and is more convenient to operate, saving labor costs and medical treatment costs.
  • the method for extracting RNA from exfoliated nasal cells includes two methods, wherein the first method includes the following steps:
  • Step 1 Dissolve the nasal cavity exfoliated cells in 100-2000 ⁇ L of cell lysate, add an equal volume of ethanol, mix well and add it to the RNA purification column. After centrifugation, remove the filtrate from the collection tube. The RNA purification column is placed in a collection tube;
  • Step 2 Add 300 ⁇ L to 700 ⁇ L of the first buffer to the RNA purification column obtained in step 1, and centrifuge to remove the first filtrate; continue to add 400 ⁇ L to 800 ⁇ L of the second buffer to the RNA purification column, After centrifugation, the second filtrate was removed, and an RNA purification column was eluted to obtain RNA.
  • the method for extracting RNA from exfoliated cells in the nasal cavity further comprises the following steps: adding 10 to 100 ⁇ L of a DNase reaction solution to the RNA purification column after removing the second filtrate, and after standing treatment, adding 300 ⁇ L ⁇ 700 ⁇ L of the second buffer solution, after centrifugation, remove the third filtrate, take the RNA purification column and elute the RNA purity using a spectrophotometer to obtain RNA;
  • the method for preparing the DNase reaction solution includes the following steps: a DNase buffer solution, a recombinant DNase, and double-distilled water from which the RNase is removed are mixed to obtain a DNase reaction solution.
  • the method for preparing the DNase reaction solution includes the following steps: 5 ⁇ L of 10 ⁇ DNase buffer solution, 4 ⁇ L of recombinant DNase, and 41 ⁇ L of RNase-free double-distilled water are mixed to obtain a DNase reaction solution.
  • the method for extracting RNA from nasal cavity exfoliated cells when the genome content is low or the starting amount of material is low when performing RNA extraction further includes the following steps: in step 1, the nasal exfoliated cells are lysed After the cell lysate is added to a genomic DNA adsorption column to take a filtrate, an equal volume of ethanol is added to the filtrate.
  • the method for extracting RNA from nasal cavity exfoliated cells further includes the following steps: adding an RNA purification column to be eluted and adding distilled water to remove RNA hydrolase or diethyl pyrocarbonate treatment After the water was left at room temperature, the RNA purification column was eluted by centrifugation, and the RNA purity was measured by a spectrophotometer to obtain RNA.
  • Step 1 uses a cell lysate that can rapidly break down nasal cells and inhibit nucleases released by nasal cells; genomic DNA adsorption columns are used to remove genomic DNA; RNA purification columns in step 2 are used to enrich RNA; The collection tube is used for collecting the solution after removing the genomic DNA, the first buffer solution for removing impurities in the purification column to which the RNA is adsorbed, and the second buffer solution for removing impurities and salts in the RNA solution.
  • the first method for extracting RNA from exfoliated nasal cells includes the following steps:
  • Step 1 Dissolve the nasal cavity exfoliated cells in 300 ⁇ L of cell lysate, add an equal volume of 70% ethanol, and mix the solution evenly with a pipette; immediately add the mixed solution to the RNA purification column at 12000 rpm , Centrifuge for 1min, remove the filtrate, and place the RNA purification column in a 2mL collection tube;
  • Step 2 Add 500 ⁇ L of the first buffer to the RNA purification column obtained in step 1, centrifuge at 12,000 rpm for 30 s to remove the first filtrate; continue to add 600 ⁇ L of the second buffer to the RNA purification column, Centrifuge at 12000 rpm for 30 seconds to remove the second filtrate;
  • Step 3 Take 5 ⁇ L of 10 ⁇ DNase buffer, 4 ⁇ L of recombinant DNase, and 41 ⁇ L of RNase-free double-distilled water to obtain a DNase reaction solution. Add 50 ⁇ L of DNase to the RNA purification column after removing the second filtrate. The reaction solution was left at room temperature for 15 minutes, 350 ⁇ L of the second buffer solution was added, 12000 rpm, and centrifuged for 30 seconds to remove the third filtrate;
  • Step 4 Place the RNA purification column with the third filtrate removed in step 3 in a 1.5 mL RNA hydrolase-free collection tube. Add 50 ⁇ L of RNA hydrolase-free distilled water or 0.1% diethyl pyrocarbonate-treated water to the RNA purification column. After standing at room temperature for 5 minutes, 12000 rpm, centrifugation for 2 minutes, the RNA purification column was eluted, and the OD260 / OD280 ratio of the RNA solution was measured using a spectrophotometer to obtain an RNA of 1.7 to 2.1.
  • the method for extracting RNA from exfoliated nasal cells includes the following steps:
  • Step 1 Take the genomic DNA adsorption column in a 2mL collection tube, dissolve the nasal cavity exfoliated cells in 100-2000 ⁇ L of cell lysate, and add it to the genomic DNA adsorption column. Take the filtrate and add an equal volume of 70% ethanol, mixed well and added to the RNA purification column, 12000 rpm, centrifugation for 1min, the filtrate was removed, and the RNA purification column was placed in a 2mL collection tube;
  • Step 2 Add 500 ⁇ L of the first buffer to the RNA purification column obtained in step 1, centrifuge at 12,000 rpm for 30 s to remove the first filtrate; continue to add 600 ⁇ L of the second buffer to the RNA purification column, Centrifuge at 12000 rpm for 30 seconds to remove the second filtrate;
  • Step 3 Place the RNA purification column with the second filtrate removed in step 2 into a 1.5 mL RNA hydrolase-free collection tube. Add 50 ⁇ L of RNA hydrolase-free distilled water or 0.1% diethyl pyrocarbonate-treated water to the RNA purification column. After standing at room temperature for 5 minutes, 12000 rpm, centrifugation for 2 minutes, the RNA purification column was eluted, and the OD260 / OD280 ratio of the RNA solution was measured using a spectrophotometer to obtain an RNA of 1.7 to 2.1.
  • step 1 uses a cell lysate to rapidly break down nasal cells and inhibit nuclease released by nasal cells;
  • step 1 uses a genomic DNA adsorption column to remove genomic DNA; and an RNA purification column in step 2 is used for enrichment RNA;
  • the collection tube is used to collect the solution after removing the genomic DNA, the first buffer is used to remove impurities from the purification column to which the RNA is adsorbed, and the second buffer is used to remove impurities and salts from the RNA solution.
  • the second method for extracting RNA from nasal cavity exfoliated cells includes the following steps: adding 0.1 mL to 20 mL of RNA extraction solution to a centrifuge tube containing nasal cavity exfoliated cells, lysing, shaking and adding 0.1 to 0.5 times the volume of the chloroform of the RNA extraction solution, shake and mix, and stand at room temperature, centrifuge the centrifuge tube, take the supernatant, and add 0.5 to 3 times the volume of chloroform in isopropyl alcohol.
  • RNA purity is measured by a spectrophotometer to obtain RNA.
  • the RNA extraction solution is any one or more of Trizol, RNAisoBlood, RNAisoPlus or other phenol, guanidine isothiocyanate, 8-hydroxyquinoline, guanidine isothiocyanate and ⁇ -mercaptoethanol.
  • Trizol Trizol
  • RNAisoBlood RNAisoPlus or other phenol
  • guanidine isothiocyanate 8-hydroxyquinoline
  • guanidine isothiocyanate and ⁇ -mercaptoethanol.
  • reagents are examples of Trizol, RNAisoBlood, RNAisoPlus or other phenol, guanidine isothiocyanate, 8-hydroxyquinoline, guanidine isothiocyanate and ⁇ -mercaptoethanol.
  • the second method for extracting RNA from nasal cavity exfoliated cells includes the following steps: adding 0.1 to 20 mL of RNA extraction solution to a centrifuge tube containing nasal exfoliated cells for dissolution, shaking, and room temperature Let stand for 3 ⁇ 7min; add 40 ⁇ L ⁇ 5mL of chloroform, shake and mix well, let stand at room temperature for 3 ⁇ 7min, centrifuge at 3 °C ⁇ 5 °C, 10000 ⁇ 14000r / min for 10 ⁇ 20min; take the supernatant 40 ⁇ L ⁇ 8mL, add etc.
  • the volume of isopropanol was mixed and left to stand for 8-12 minutes, centrifuged at 3 ° C to 5 ° C, 10,000 to 14000 r / min for 10 to 20 minutes, the supernatant was discarded, and the first precipitate was retained;
  • Propanol equal volume of 65% to 90% ethanol was centrifuged at 3 ° C to 5 ° C, 7000 to 14000r / min for 10 to 20min, the supernatant was discarded, and the second pellet was retained;
  • the centrifuge tube was tightly closed at 3 ° C ⁇ 5 ° C, centrifuge at 7000 ⁇ 14000r / min for 1 ⁇ 3min, remove the supernatant, and let stand for 10 ⁇ 20min, then continue to add 0.01 ⁇ 5mL of deRNase and deDNase water to the centrifuge tube to dissolve the second Precipitate and measure RNA purity using a spectrophotometer to obtain RNA.
  • the second method for extracting RNA from nasal cavity exfoliated cells includes the following steps: adding 1 mL of RNA extraction solution to a centrifuge tube containing nasal cavity exfoliated cells for dissolution, shaking, and standing at room temperature for 5 min; adding 200 ⁇ L of chloroform, Mix by shaking, let stand at room temperature for 5 minutes, and centrifuge at 12,000 r / min for 15 min at 4 ° C. Take the supernatant 200 ⁇ L, add 200 ⁇ L isopropanol, mix and let stand for 10 min, and centrifuge at 4 ° C for 15 min at 12000 r / min to discard the supernatant.
  • the method for reverse transcription of total RNA into cDNA includes the following steps: 1 to 3 ⁇ L of the reverse transcription mixed solution, 0 to 10 ⁇ L of de-hydrolase-distilled water and the extracted RNA undergo a reverse transcription reaction at a temperature of 37 ° C. After 15 min, a reverse transcriptase inactivation reaction occurred at a temperature of 84 ° C to obtain a reverse transcription product cDNA.
  • the method for reverse transcription of total RNA into cDNA includes the following steps: taking 2 ⁇ L of the reverse transcription mixture, 8 ⁇ L of the deRNA hydrolase distilled water, and a total amount not exceeding 500 ng or a volume not exceeding
  • the de-hydrolase enzyme distilled water is used to make up to 10 ⁇ L
  • the reverse transcription reaction is performed after gentle mixing, the conditions are as follows: at 37 ° C, the reverse transcription reaction is performed for 15 minutes; at 85 ° C, Under the conditions, an inactivation reaction of reverse transcriptase was performed for 5 seconds; the product was left at 4 ° C.
  • the real-time quantitative PCR amplification includes the following steps:
  • Step 1 Prepare a real-time quantitative PCR reaction solution: include 1 ⁇ L to 25 ⁇ L of PCR premix, 0 ⁇ L to 10 ⁇ L of double distilled water to make up the total volume of water to 10 ⁇ L, 0 ⁇ L to 2 ⁇ L of machine fluorescence compensation and correction agent, 0.01 to 100 ⁇ M Upstream primer of ALOX15 gene, downstream primer of ALOX15 gene of 0.01 to 100 ⁇ M, upstream primer of internal reference gene of 0.01 to 100 ⁇ M, downstream primer of internal reference gene of 0.01 to 100 ⁇ M, 0.01 ⁇ L to 5 ⁇ L of cDNA;
  • Step 2 Real-time quantitative PCR detection using standard two-step PCR amplification standard program or three-step PCR amplification standard program;
  • Step 3 Calculate the expression of ALOX15 gene.
  • a real-time quantitative PCR reaction solution was prepared: including 5 ⁇ L of a PCR premix, 2.8 ⁇ L of double distilled water to make up a total volume of water to 10 ⁇ L, 0.2 ⁇ L of a machine fluorescence compensation and correction agent, and 0.5 ⁇ L of the upstream ALOX15 gene.
  • the reaction conditions of the standard two-step PCR amplification procedure include the following steps: first stage: pre-denaturing at 95 ° C for 30 seconds; second stage PCR reaction: at 95 ° C Under the conditions, the reaction is performed for 15 seconds, and the reaction is performed at 60 ° C for 60 seconds, and the annealing is extended, so that 40 cycles are performed;
  • the reaction conditions of the standard three-step PCR amplification procedure include the following steps: first stage: pre-denaturation at 95 ° C for 2 minutes; second stage PCR reaction: at 95 ° C, reaction for 1 minute, at The reaction is performed at 55 ° C for 1 minute, and at 72 ° C for 1 minute, so that 40 cycles are performed; finally, 72 ° C, 7 minutes of annealing and extension;
  • Relative quantification method using ⁇ CT method or 2- ⁇ Ct method selecting internal reference genes with relatively constant expression, normalizing with the number of internal reference genes, and calculating the target gene expression by measuring the difference between the Ct value of the target gene and the internal reference gene. It is simple and fast, and the detection accuracy is high, which can reduce the detection cost and save the detection time. The result is easy to interpret and so on. Greatly improved the experimental efficiency.
  • the expression of ALOX15 and the reference gene are different.
  • the reference gene is a gene that is more stable in vivo and usually does not change with diseases. Therefore, the comparison with the reference gene can reflect the relative abundance of the target gene and the reference gene. , You can use ⁇ CT method. For different subjects, the expression of ALOX15 and the reference gene can be different using the 2- ⁇ Ct method.
  • kits for detecting chronic sinusitis with nasal polyp subtypes provided by the embodiments of the present disclosure and the application of ALOX15 gene as a biomarker, and a method for detecting nasal cavity shedding provided by the embodiments of the present invention
  • ALOX15 gene expression in cells will be described below with reference to specific examples.
  • a kit for detecting chronic sinusitis with nasal polyp subtypes comprising the following reagents:
  • RNA extraction solution Trizol or RNAiso Blood or RNAisoPlus or other products containing phenol, guanidine isothiocyanate, 8-hydroxyquinoline, guanidine isothiocyanate, ⁇ -mercaptoethanol, etc.
  • Substances that can rapidly disrupt cells and inhibit nucleases released by the cells 2mL of chloroform; 20mL of isopropanol; 40mL of 65-90% ethanol; 5mL of RNase and DNAse-free water;
  • Reagent for reverse transcription of extracted RNA into cDNA 40 ⁇ L of reverse transcription mixture (containing reverse transcription enzymes, RNase inhibitors, random 6-nucleotide primers, polythymine, T repeat oligonucleotides , Deoxyribonucleotide triphosphate mixture, buffer solution, etc.), 160 ⁇ L of RNase and DNAse-free water; RNase and DNAse-free water are used to complete the system, solubilize and dilute RNA;
  • Reagent for real-time fluorescent quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction 25 ⁇ L of premixed solution (containing enzymes and buffers required for PCR), 0 to 50 ⁇ L of double distilled water (based on total Make up volume to 50 ⁇ L with water), 0 to 2 ⁇ L of dye (for fluorescence compensation and correction of the machine), 100 ⁇ M upstream primer of ALOX15 gene, 100 ⁇ M downstream primer of ALOX15 gene, 100 ⁇ M upstream primer of internal reference gene, 100 ⁇ M
  • the downstream primer of the internal reference gene 10 ⁇ g positive control, 10 ⁇ g negative control, the positive control is a plasmid containing ALOX15, and the negative control is an empty plasmid (plasmid vector).
  • a kit for detecting chronic sinusitis with nasal polyp subtypes comprising the following reagents:
  • RNA extraction solution Trizol or RNAiso Blood or RNAiso Plus or other substances containing phenol, guanidine isothiocyanate, 8-hydroxyquinoline, guanidine isothiocyanate, ⁇ -mercaptoethanol, etc.
  • Substances that can rapidly disrupt cells and inhibit the release of nucleases from cells 0.2mL of chloroform; 0.2mL of isopropanol; 0.2mL of 65-90% ethanol; 0.05mL of RNase and DNAse-free water;
  • Reagent for reverse transcription of extracted RNA into cDNA 2 ⁇ L of reverse transcription mixture (containing the enzyme required for reverse transcription, RNase inhibitor, random 6 nucleotide primers, polythymine, T repeat oligonucleotide , Deoxyribonucleotide triphosphate mixture, buffer solution, etc.), 7 ⁇ L of RNase and DNAse-free water; of which RNase and DNAse-free water are used to complete the system, solubilize and dilute RNA;
  • Reagent for real-time fluorescent quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction 5 ⁇ L of premix (containing enzymes and buffers required for PCR), 0-10 ⁇ L of double-distilled water (based on total Make up volume to 10 ⁇ L with water), 0 to 2 ⁇ L of dye (for fluorescence compensation and correction of the machine), 50 ⁇ M upstream primer of ALOX15 gene, 50 ⁇ M downstream primer of ALOX15 gene, 50 ⁇ M upstream primer of internal reference gene, 50 ⁇ M
  • the downstream primer of the internal reference gene 5 ⁇ g positive control, 5 ⁇ g negative control, the positive control is a plasmid containing ALOX15, and the negative control is an empty plasmid (plasmid vector).
  • a kit for detecting chronic sinusitis with nasal polyp subtypes comprising the following reagents:
  • RNA extraction solution Trizol or RNAiso Blood or RNAiso Plus or other containing phenol, guanidine isothiocyanate, 8-hydroxyquinoline, guanidine isothiocyanate, ⁇ -mercaptoethanol And other substances that can rapidly break down cells and inhibit the nuclease released by the cells; 0.05mL of chloroform; 0.015mL of isopropanol; 0.0075mL of 65-90% ethanol; 0.01mL of RNase and DNase-free water ;
  • Reagent for reverse transcription of total RNA into cDNA 1 ⁇ L of reverse transcription mixture (containing enzymes required for reverse transcription, RNase inhibitors, random 6 nucleotide primers, polythymine, T repeat oligonucleotides, Deoxyribonucleotide triphosphate mixtures, buffers, etc.), 0-10 ⁇ L of RNase and DNAse-free water; RNase and DNAse-free water are used to complete the system, solubilize and dilute RNA;
  • Quantitative polymerase chain reaction reagent for real-time fluorescent quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA 1 ⁇ L of premixed solution (containing enzymes and buffers required for PCR), 0 to 10 ⁇ L of double-distilled water (based on total Make up volume to 10 ⁇ L with water), 0 to 2 ⁇ L of dye (for fluorescence compensation and correction of the machine), 1 ⁇ M upstream primer of ALOX15 gene, 1 ⁇ M downstream primer of ALOX15 gene, 1 ⁇ M upstream primer of internal reference gene, 1 ⁇ M
  • a kit for detecting chronic sinusitis with nasal polyp subtypes comprising the following reagents and tools:
  • Reagents for RNA extraction from nasal polyp tissue 100 ⁇ L of cell lysate (RL buffer with 50 x dithiothreitol (DTT)), genomic DNA adsorption column for removing genomic DNA, and for collecting and removing genomic DNA
  • RL buffer with 50 x dithiothreitol (DTT) genomic DNA adsorption column for removing genomic DNA
  • genomic DNA adsorption column for removing genomic DNA
  • genomic DNA adsorption column for removing genomic DNA
  • genomic DNA adsorption column for removing genomic DNA
  • genomic DNA for collecting and removing genomic DNA
  • a collection tube for the solution an RNA purification column for enriching RNA, 0.1 mL of a first buffer for removing impurities from the purification column to which the RNA is adsorbed, and 0.1 mL of a second buffer for removing impurities and salts from the RNA solution Solution, centrifuge tube for collecting RNA, 0.01 mL of RNase and DNase water for dissolving
  • Reagent for reverse transcription of total RNA into cDNA 1 ⁇ L of reverse transcription mixture (containing enzymes required for reverse transcription, RNase inhibitors, random 6 nucleotide primers, polythymine, T repeat oligonucleotides, Deoxyribonucleotide triphosphate mixtures, buffers, etc.), 0-10 ⁇ L of RNase and DNAse-free water; RNase and DNAse-free water are used to complete the system, solubilize and dilute RNA;
  • Reagent for real-time fluorescent quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction 25 ⁇ L premix (containing enzymes and buffers required for PCR), 0-10 ⁇ L double-distilled water (based on total Make up volume to 10 ⁇ L with water), 0 to 2 ⁇ L of dye (for fluorescence compensation and correction of the machine), 0.01 ⁇ M upstream primer of ALOX15 gene, 0.01 ⁇ M downstream primer of ALOX15 gene, 0.01 ⁇ M upstream of reference gene Primer, 0.01 ⁇ M of the internal reference gene downstream primer, 1 ⁇ g positive control, 1 ⁇ g negative control, the positive control is a plasmid containing ALOX15, and the negative control is an empty plasmid (plasmid vector).
  • a kit for detecting chronic sinusitis with nasal polyp subtypes comprising the following reagents and tools:
  • Reagent for RNA extraction from nasal polyp tissue 0.3 mL of cell lysate (RL buffer with 50 x dithiothreitol (DTT)), RNA purification column for enriching RNA, 0.5 mL for removing adsorbed The first buffer of impurities in the RNA purification column, 0.6 mL of the second buffer for removing impurities and salts from the RNA solution, 4 ⁇ L of genomic DNA-removing recombinant DNase, 5 ⁇ L of genomic DNA-removing DNase buffer , 41 ⁇ L double-distilled water for RNase, centrifuge tube for collecting RNA, 0.05 mL of RNase and DNase water for dissolving RNA;
  • Reagent for reverse transcription of total RNA into cDNA 2 ⁇ L of reverse transcription mixture (containing the enzyme required for reverse transcription, RNase inhibitor, random 6 nucleotide primers, polythymine, T repeat oligonucleotide, Deoxyribonucleotide triphosphate mixtures, buffers, etc.), 0-10 ⁇ L of RNase and DNAse-free water; RNase and DNAse-free water are used to complete the system, solubilize and dilute RNA;
  • Reagent for real-time fluorescent quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction 5 ⁇ L of premix (containing enzymes and buffers required for PCR), 0-10 ⁇ L of double-distilled water (based on total Make up volume to 10 ⁇ L with water), 0 to 2 ⁇ L of dye (for fluorescence compensation and correction of the machine), 10 ⁇ mol / L upstream primer of ALOX15 gene, 10 ⁇ mol / L downstream primer of ALOX15 gene, 10 ⁇ mol / L internal reference
  • a kit for detecting chronic sinusitis with nasal polyp subtypes comprising the following reagents and tools:
  • Reagents for RNA extraction from nasal polyps 2 mL of cell lysate (RL buffer with 50 x dithiothreitol (DTT)), a genomic DNA adsorption column for removing genomic DNA, and after collecting and removing genomic DNA
  • RL buffer with 50 x dithiothreitol (DTT) 2 mL of cell lysate
  • DTT dithiothreitol
  • a collection tube for the solution an RNA purification column for enriching RNA, 0.7 mL of a first buffer for removing impurities from the purification column to which the RNA is adsorbed, and 0.7 mL of a second buffer for removing impurities and salts from the RNA solution Liquid, centrifuge tube for collecting RNA, 1 mL of RNase and DNase water for dissolving RNA;
  • Reagent for reverse transcription of total RNA into cDNA 2 ⁇ L of reverse transcription mixture (containing the enzyme required for reverse transcription, RNase inhibitor, random 6 nucleotide primers, polythymine, T repeat oligonucleotide, Deoxyribonucleotide triphosphate mixtures, buffers, etc.), 0 ⁇ 8 ⁇ L of RNase and DNase water (make up to 8 ⁇ L with water according to the amount of RNA); RNase and DNase-free water is used for replenishment. Uniform system, dissolve and dilute RNA;
  • Reagent for real-time fluorescent quantitative PCR reaction of ALOX15 gene and internal reference gene in cDNA by quantitative polymerase chain reaction 5 ⁇ L of premix (containing enzymes and buffers required for PCR), 0-10 ⁇ L of double distilled water (based on total Make up volume to 10 ⁇ L with water), 0 to 2 ⁇ L of dye (for fluorescence compensation and correction of the machine), 1 ⁇ M upstream primer of ALOX15 gene, 1 ⁇ M downstream primer of ALOX15 gene, 1 ⁇ M upstream primer of internal reference gene, 1 ⁇ M Downstream primers for the internal reference gene, 1 ⁇ g positive control, 1 ⁇ g negative control.
  • the manufacturer of the first buffer RWA buffer used was Takara Company, No. 9767; the manufacturer of the second buffer RWB buffer was Takara Company, No. 9767.
  • kits provided in Examples 1 to 6 of the present disclosure can all realize the detection of chronic rhinosinusitis with nasal polyp subtypes.
  • the following are specific test experiments of the kits for detecting chronic rhinosinusitis with nasal polyp subtypes:
  • nasal polyps were obtained under nasal endoscope. Nasal polyps were cut into tissues with a diameter of about 0.5 cm, immersed in RNA stabilization and storage solution (RNAlater), stored at 4 ° C for a short period of time, and then transferred to a storage temperature below -20 ° C for a long time.
  • RNA stabilization and storage solution RNAlater
  • Step 1 Weigh the tissue immersed in RNA stabilization and storage solution (RNAlater). Weigh about 0.01g of tissue into a magnetic beaded centrifuge tube, place it in liquid nitrogen, and grind it on a homogenizer. (3000r, 5min) (or manual grinding). Add 1mL Trizol to the test tube containing the tissue cells to dissolve it, collect it in a centrifuge tube, shake it thoroughly, and leave it at room temperature for 5 minutes; then add 200 ⁇ L of chloroform (trichloromethane) to the RNA extraction reagent group, and shake vigorously to mix. Let stand at room temperature for 5 minutes.
  • Step 2 12,000 rpm, 4 ° C, and centrifuge for 15 minutes.
  • Step 3 Take the supernatant to obtain a volume of about 200 ⁇ L, add it to a centrifuge tube, and add an equal amount (about 200 ⁇ L) of isopropanol to the above RNA extraction reagent group. After mixing, let stand for 10 minutes, 12000 rpm, and centrifuge at 4 ° C for 15 minutes. Discard the supernatant and retain the pellet.
  • Step 4 Add about 200 ⁇ L of 75% ethanol (equivalent to about 150 ⁇ L of anhydrous ethanol and 50 ⁇ L of DNAse and RNase water) of the above-mentioned RNA extraction reagent group (equivalent to isopropanol) to wash the pellet and mix. Centrifuge at 7500 rpm, 4 ° C for 15 minutes. Discard the supernatant and retain the pellet.
  • Step 5 Cap the centrifuge tube tightly and centrifuge at 7500 rpm and 4 ° C for 2 minutes.
  • Step 6 Open the lid, discard the supernatant, and leave it in a fume hood for 15 minutes.
  • Step 7 Add 0.02 mL of RNA hydrolase-free and DNA hydrolase-free (RNase-free and DNase-free) water-soluble precipitates to the above-mentioned RNA extraction reagent group.
  • Step 8 Measure the RNA concentration with a spectrophotometer, and the OD260 / OD280 ratio is between 1.7-2.1.
  • the reverse transcription reaction conditions are as follows:
  • the product was left at 4 ° C.
  • Stage 1 Pre-denaturation: 95 ° C, 30 seconds.
  • Phase 2 PCR reaction: 95 ° C, 15 seconds; 60 ° C, 1 minute annealing extension, for a total of 40 cycles; Phase 2: Melting curve: 60 ° C gradually rises to 95 ° C, the rate is 0.1 ° C / second, Collecting fluorescence;
  • Step 1 Judgment of the quality control of the experiment: The positive control Ct value ⁇ 20 and the negative control Ct value> 38 are regarded as valid experiments, otherwise the experiments are invalid.
  • Step 2 Judgment of typing: The Ct value of the target gene minus the Ct value of the reference gene.
  • the optimal cutoff value of the Ct value of ALOX15 is 1.675. If the Ct value of ALOX15 is greater than 1.675, it is a non-eosinophil. Cellular chronic sinusitis with nasal polyps; if the Ct value of ALOX15 is less than 1.675, it is a typical eosinophilic chronic sinusitis with nasal polyps.
  • Eosinophil type Eosinophil type 44 0.296 Eosinophil type Eosinophil type 45 -1.137 Eosinophil type Eosinophil type 46 3.202 Non-eosinophil type Eosinophil type 47 0.442 Eosinophil type Eosinophil type 48 0.765 Eosinophil type Eosinophil type 49 2.253 Non-eosinophil type Eosinophil type 50 0.769 Eosinophil type Eosinophil type 51 2.422 Non-eosinophil type Eosinophil type 52 0.749 Eosinophil type Eosinophil type 53 -3.834 Eosinophil type Eosinophil type 54 1.930 Non-eosinophil type Eosinophil type 55 0.767 Eosinophil type Eosinophil type 56 0.778 Eosinophil type Eosinophil type 57 0.061 Eosinophil type Eosinophil type 58 4.640
  • Step 1 Add 1mL Trizol to the test tube containing the detached cells, dissolve it, shake it thoroughly, and let it stand at room temperature for 5 minutes, then add 200 ⁇ L of chloroform (trichloromethane), mix with vigorous shaking, and let it stand at room temperature for 5 minutes;
  • chloroform trichloromethane
  • Step 2 12,000 rpm, 4 ° C, and centrifuge for 15 minutes;
  • Step 3 Take the supernatant to obtain a volume of about 200 ⁇ L, add to the centrifuge tube, and add the same amount of isopropyl alcohol (about 200 ⁇ L) as chloroform. After mixing, let stand for 10 minutes, 12000 rpm, and centrifuge at 4 ° C for 15 minutes. Discard the supernatant and keep the pellet;
  • Step 4 Add approximately 200 ⁇ L of 75% ethanol (a mixture of approximately 150 ⁇ L of absolute ethanol and 50 ⁇ L of DNAse and RNAse water) to the RNA extraction reagent group (equivalent to isopropyl alcohol), and mix well. 7500 rpm, 4 ° C, centrifugation for 15 minutes, discard the supernatant and retain the pellet;
  • Step 5 Cap the centrifuge tube tightly at 7500 rpm, 4 ° C, and centrifuge for 2 minutes;
  • Step 6 Open the lid, discard the supernatant, and leave it in a fume hood for 15 minutes;
  • Step 7 Add 0.02 mL of RNA-hydrolase-free and DNA-hydrolase-free (RNase-free and DNase-free) water-soluble precipitates to the above-mentioned RNA extraction reagent group;
  • Step 8 Measure the RNA concentration with a spectrophotometer.
  • the OD260 / OD280 ratio is preferably 1.7 to 2.1.
  • Step 1 Place the genomic DNA adsorption column (genomic DNA Spinner Column) on a 2mL collection tube (Collection Tube);
  • Step 2 Transfer the lysate (cell lysate) containing the detached cells into a genomic DNA adsorption column;
  • Step 3 12,000 rpm, centrifuge for 1 minute
  • Step 4 Discard the genomic DNA adsorption column and retain the filtrate in the 2mL collection tube;
  • Step 5 Add 300 ⁇ L of 70% ethanol to the above step 4 (precipitation may occur at this time), and use a pipette to mix the solution uniformly;
  • Step 6 Immediately transfer all the mixed solution (including the precipitate) into an RNA purification column (containing a 2 mL collection tube);
  • Step 7 12,000 rpm, centrifuge for 1 minute, and discard the filtrate. Place the RNA purification column back into the 2mL collection tube;
  • Step 8 Add 500 ⁇ L of the first buffer (Buffer RWA) to the RNA purification column, 12,000 rpm, centrifuge for 30 seconds, and discard the filtrate;
  • Buffer RWA the first buffer
  • Step 9 Add 600 ⁇ L of the second buffer (Buffer RWB) to the RNA purification column, 12,000 rpm, centrifuge for 30 seconds, and discard the filtrate.
  • Buffer RWB buffer RWB
  • Step 10 Place the RNA purification column on a 1.5 mL RNAase-free collection tube (RNase Free Colletion Tube), and add 50 ⁇ L of RNA hydrolysis-free distilled water (RNase Free dH2O) or 0.1% to the center of the RNA purification column membrane. Diethyl pyrocarbonate (DEPC) treated water, and allowed to stand at room temperature for 5 minutes;
  • RNase Free Colletion Tube 1.5 mL RNAase-free collection tube
  • RNase Free dH2O RNA hydrolysis-free distilled water
  • DEPC Diethyl pyrocarbonate
  • Step 11 Centrifuge at 12,000 rpm and elute RNA for 2 minutes with de-RNase and de-DNase water;
  • Step 12 Measure the RNA concentration with a spectrophotometer, and the OD260 / OD280 ratio is 2.0.
  • Step 1 Configure 45 ⁇ L of SYBR Green 1 pre-mixed solution; and ROX: 1.8 ⁇ L, mix and divide into 3 parts, respectively A.11.7 ⁇ L; B.11.7 ⁇ L; C.23.4 ⁇ L, add 1ng / Solution A was obtained from ⁇ L positive control, solution B was added to B with 1ng / ⁇ L negative control, and solution C was added from C with 2ng to obtain solution C (SYBR Green Green 1 premix and ROX are products of Takara Company, article number RR820A);
  • Step 2 Configure 8 groups of parallel holes
  • the first and second parallel wells solution A, specific primers for ALOX15 gene, 3.8 ⁇ L sterilized double distilled water;
  • solution B specific primers for ALOX15 gene, 3.8 ⁇ L sterilized double distilled water
  • Step 3 Seal the plate with clear plastic film, centrifuge, and perform the PCR operation.
  • Step 4 Two-step PCR standard procedure:
  • Stage 1 Pre-denaturation: 95 ° C, 30 seconds.
  • Phase 2 Phase 2: PCR reaction: 95 ° C, 15 seconds; 60 ° C, 1 minute annealing extension, 40 cycles were performed. ;
  • kits provided in Examples 1 to 6 of the present disclosure can all realize the detection of chronic rhinosinusitis with nasal polyp subtypes.
  • Example 5 the effect of the kit for detecting chronic rhinosinusitis with nasal polyp subtypes is performed as follows. Detection experiment:
  • RNA stabilization and storage solution RNAlater
  • Step 1 Weigh the tissue immersed in RNA stabilization and storage solution (RNAlater). Weigh about 0.01g of tissue into a magnetic beaded centrifuge tube, place it in liquid nitrogen, and grind it on a homogenizer. (3000r, 5min) (or manual grinding); add 0.3mL cell lysate, 12,000 rpm, centrifuge for 15 minutes;
  • Step 2 Aspirate the supernatant, add 70% ethanol (70% absolute ethanol and 30% DEPC or RNase and DNase water) equal to the volume of the supernatant, and use a pipette to mix the solution uniformly;
  • Step 3 Transfer all the mixed solution (including the precipitate) to the RNA purification column (including the 2mL collection tube) immediately;
  • Step 4 12,000 rpm, centrifuge for 1 minute, and discard the filtrate. Put the RNA purification back into the 2mL collection tube;
  • Step 5 Add 500 ⁇ L of the first buffer solution (Buffer RWA) to the RNA purification column, 12,000 rpm, centrifuge for 30 seconds, and discard the filtrate;
  • Buffer RWA the first buffer solution
  • Step 6 Add 600 ⁇ L of a second buffer (Buffer RWB) to the RNA purification column, 12,000 rpm, centrifuge for 30 seconds, and discard the filtrate;
  • Buffer RWB a second buffer
  • Step 7 Preparation of DNase I reaction solution: Take 5 ⁇ L of 10 ⁇ DNase I buffer, 4 ⁇ L of recombinant DNase I ((RNase-free, 5U / ⁇ L), 41 ⁇ L of RNase-free Double distilled water into a new 1.5mL tube (no RNase) and mix well;
  • Step 8 Add 50 ⁇ L DNase I reaction solution to the center of the RNA purification column membrane, and leave it at room temperature for 15 minutes;
  • Step 9 Add 350 ⁇ L of a second buffer to the center of the RNA purification column membrane, 12,000 rpm, centrifuge for 30 seconds, and discard the filtrate;
  • Step 10 Repeat step 6;
  • Step 11 Reposition the RNA purification column on a 2mL collection tube, 12,000 rpm, and centrifuge for 2 minutes;
  • Step 12 Place the RNA purification column on a 1.5 mL RNAase-free collection tube (RNase Free Colletion Tube), and add 50 ⁇ L of RNAase-free distilled water (RNase Free HdO) or 0.1% to the center of the RNA purification column membrane. Diethyl pyrocarbonate (DEPC) treated water, and allowed to stand at room temperature for 5 minutes;
  • RNase Free Colletion Tube 1.5 RNAase-free collection tube
  • RNase Free HdO RNAase-free distilled water
  • DEPC Diethyl pyrocarbonate
  • Step 13 Centrifuge at 12,000 rpm, and elute RNA for 2 minutes with RNase and DNase-free water;
  • Step 14 Measure the RNA concentration with a spectrophotometer, and the OD260 / OD280 ratio is 2.0;
  • reaction conditions used a standard three-step PCR amplification procedure:
  • Stage 1 Pre-denaturation: 95 ° C, 2 minutes;
  • Phase 2 Phase 2: PCR reaction: 95 ° C, 1 minute; 55 ° C, 1 minute, 72 ° C for 1 minute, a total of 40 cycles were performed, and finally 72 ° C, 7 minutes annealing extension.
  • the present disclosure provides a kit for detecting chronic sinusitis with nasal polyp subtypes.
  • the screening uses the ALOX15 gene as a biomarker and applies it to the kit to realize the use of the kit to detect chronic
  • the method of sinusitis with nasal polyp subtypes can quickly, accurately and comprehensively identify patients with nasal polyp subtypes through the kit, in order to carry out targeted treatment based on the inflammatory subtypes of nasal polyps as soon as possible, and effectively guide chronic sinusitis.
  • the determination of drug treatment methods and surgical methods for patients with nasal polyps accurately predict the response to drug treatment, and judge the prognostic effect.
  • a method for detecting the expression of ALOX15 gene in exfoliated nasal cells comprising the following steps:
  • Step 1 Extract RNA from exfoliated nasal cells:
  • Step 1 Take the genomic DNA adsorption column into a 2mL collection tube, dissolve the nasal cavity exfoliated cells in 300 ⁇ L of cell lysate, and add it to the genomic DNA adsorption column. Take the filtrate and add an equal volume of 70% to the filter. Ethanol, after mixing, added to the RNA purification column, 12000 rpm, centrifugation for 1min, the filtrate was removed, and the RNA purification column was placed in a 2mL collection tube;
  • Step 2 Add 500 ⁇ L of the first buffer to the RNA purification column obtained in step 1, centrifuge at 12,000 rpm for 30 s to remove the first filtrate; continue to add 600 ⁇ L of the second buffer to the RNA purification column, Centrifuge at 12000 rpm for 30 seconds to remove the second filtrate;
  • Step 3 Place the RNA purification column with the second filtrate removed in step 2 into a 1.5 mL RNA hydrolase-free collection tube. Add 50 ⁇ L of RNA hydrolase-free distilled water or 0.1% diethyl pyrocarbonate-treated water to the RNA purification column. Leave at room temperature for 5 minutes, 12000 rpm, centrifuge for 2 minutes, elute the RNA purification column, and use a spectrophotometer to measure the OD260 / OD280 ratio of the RNA solution to 2.0 to obtain RNA;
  • Step 2 Prepare cDNA by reverse transcription, including the following steps: take 2 ⁇ L of the reverse transcription mixture, 0-8 ⁇ L of the deRNA hydrolase distilled water (make up to 8 ⁇ L with water according to the amount of RNA), and the total amount does not exceed 500ng Or the total RNA volume is not more than 8 ⁇ L, and the de-RNA hydrolase distilled water is used to make up to 10 ⁇ L.
  • reverse transcription reaction is performed under the following conditions: at 37 ° C, a reverse transcription reaction is performed for 15 minutes; Under the condition of 85 ° C, the reverse transcriptase inactivation reaction was performed for 5 seconds; the product was left at 4 ° C.
  • Step 3 Real-time quantitative PCR amplification detection, including the following steps:
  • Step 1 Prepare a real-time PCR reaction solution: include 1 ⁇ L of PCR premix, 0-10 ⁇ L of double distilled water (to make up to 10 ⁇ L with water based on the total volume), 0.2 ⁇ L of machine fluorescence compensation and correction agent, 1 ⁇ M of ALOX15 Gene upstream primer, 1 ⁇ M ALOX15 gene downstream primer, 1 ⁇ M internal reference gene upstream primer, 1 ⁇ M internal reference gene downstream primer, 0.01 ⁇ L of the cDNA, 1 ⁇ g positive control, 1 ⁇ g negative control, positive control contains ALOX15 plasmid, the negative control is an empty plasmid (plasmid vector);
  • Step 2 Standard procedure for two-step PCR amplification: The reaction conditions for the standard procedure for two-step PCR amplification include the following steps: Stage 1: Pre-denaturation for 30 seconds at 95 ° C; Stage 2 PCR reaction : Reaction was performed at 95 ° C for 15 seconds, and at 60 ° C, reaction was performed for 60 seconds, followed by annealing extension, and 40 cycles were performed.
  • Step 4 Calculate the expression of the ALOX15 gene:
  • ⁇ CT method was used to compare the expression of ALOX15 and the reference gene: the average CT value of ALOX15 was 20.9, the average CT value of GAPDH was 18.9, and the ⁇ CT value was 2.0.
  • a method for detecting the expression of ALOX15 gene in exfoliated nasal cells comprising the following steps:
  • Step 1 Extract RNA from exfoliated nasal cells:
  • Step 1 Take the genomic DNA adsorption column into a 2mL collection tube, dissolve the nasal cavity exfoliated cells in 100 ⁇ L of cell lysate, add it to the genomic DNA adsorption column, centrifuge at 12000 rpm for 60s, and take the filtrate. Add an equal volume of 70% ethanol to the filter, mix it and add it to the RNA purification column, 12000 rpm, centrifuge for 1min, remove the filtrate, and place the RNA purification column in a 2mL collection tube;
  • Step 2 Add 300 ⁇ L of the first buffer to the RNA purification column obtained in step 1, centrifuge at 12,000 rpm for 30 seconds to remove the first filtrate; continue to add 400 ⁇ L of the second buffer to the RNA purification column Centrifuge at 12000 rpm for 30 seconds to remove the second filtrate;
  • Step 3 Place the RNA purification column with the second filtrate removed in step 2 into a 1.5 mL RNA hydrolase-free collection tube. Add 50 ⁇ L of RNA hydrolase-free distilled water or 0.1% diethyl pyrocarbonate-treated water to the RNA purification column. Leave at room temperature for 5 minutes, 12000 rpm, centrifuge for 2 minutes, elute the RNA purification column, and use a spectrophotometer to measure the OD260 / OD280 ratio of the RNA solution to 2.0 to obtain RNA;
  • Step 2 Reverse transcription to prepare cDNA, including the following steps: take 1 ⁇ L of the reverse transcription mixture, 0 to 10 ⁇ L of the deRNA hydrolase distilled water, and total RNA not exceeding 500 ng or 8 ⁇ L in volume. Make up the RNA hydrolase distilled water to make up to 10 ⁇ L; gently mix the reverse transcription reaction under the following conditions: at 37 ° C for 15 minutes, and at 85 ° C for 5 seconds Reverse transcriptase inactivation reaction; the product was left at 4 ° C.
  • Step 3 Real-time quantitative PCR amplification detection, including the following steps:
  • Step 1 Prepare a real-time quantitative PCR reaction solution: include 25 ⁇ L of PCR premix, 0 to 10 ⁇ L of double distilled water (to make up to 10 ⁇ L with water based on the total volume), and 0 to 2 ⁇ L of dye (for fluorescence compensation of the machine) And correction), 0.01 ⁇ M ALOX15 gene upstream primer, 0.01 ⁇ M ALOX15 gene downstream primer, 0.01 ⁇ M internal reference gene upstream primer, 0.01 ⁇ M internal reference gene downstream primer, 5 ⁇ L cDNA, 1 ⁇ g positive control, 1 ⁇ g negative Control, the positive control is a plasmid containing ALOX15, and the negative control is an empty plasmid (plasmid vector);
  • Step 2 Standard procedure for two-step PCR amplification: The reaction conditions for the standard procedure for two-step PCR amplification include the following steps: Stage 1: Pre-denaturation for 30 seconds at 95 ° C; Stage 2 PCR reaction : Reaction was performed at 95 ° C for 15 seconds, and at 60 ° C, reaction was performed for 60 seconds, followed by annealing extension, and 40 cycles were performed.
  • Step 4 Calculate the expression of the ALOX15 gene:
  • ⁇ CT method was used to compare the expression difference of ALOX15 and internal reference genes: the average CT value of ALOX15 was 19.5, the average CT value of GAPDH was 18.0, and the ⁇ CT value was 1.5.
  • a method for detecting the expression of ALOX15 gene in exfoliated nasal cells comprising the following steps:
  • Step 1 Extract RNA from exfoliated nasal cells:
  • Step 1 Take the genomic DNA adsorption column into a 2mL collection tube, dissolve the nasal cavity exfoliated cells in 2000 ⁇ L of cell lysate, add it to the genomic DNA adsorption column, centrifuge at 12000 rpm for 60 seconds, and take the filtrate. Add an equal volume of 70% ethanol to the filter, mix it and add it to the RNA purification column, 12000 rpm, centrifuge for 1min, remove the filtrate, and place the RNA purification column in a 2mL collection tube;
  • Step 2 Add 700 ⁇ L of the first buffer to the RNA purification column obtained in step 1, centrifuge at 12,000 rpm for 30 s to remove the first filtrate; continue to add 800 ⁇ L of the second buffer to the RNA purification column, Centrifuge at 12000 rpm for 30 seconds to remove the second filtrate;
  • Step 3 Place the RNA purification column with the second filtrate removed in step 2 into a 1.5 mL RNA hydrolase-free collection tube. Add 50 ⁇ L of RNA hydrolase-free distilled water or 0.1% diethyl pyrocarbonate-treated water to the RNA purification column. Leave at room temperature for 5 minutes, 12000 rpm, centrifuge for 2 minutes, elute the RNA purification column, and use a spectrophotometer to measure the OD260 / OD280 ratio of the RNA solution to 2.0 to obtain RNA;
  • Step 2 Reverse transcription to prepare cDNA, including the following steps: take 3 ⁇ L of the reverse transcription mixture, 0-8 ⁇ L of RNase and DNAse-free water (make up to 8 ⁇ L with water according to the amount of RNA), and the total amount does not exceed 500ng or total volume of no more than 8 ⁇ L, the deRNA hydrolase distilled water was made up to 10 ⁇ L; the reverse transcription reaction was performed after gentle mixing, the conditions were as follows: at 37 ° C, a reverse transcription reaction was performed for 15 minutes; Under the condition of 85 ° C, the reverse transcriptase inactivation reaction was performed for 5 seconds; the product was left at 4 ° C.
  • Step 3 Real-time quantitative PCR amplification detection, including the following steps:
  • Step 1 Prepare a real-time PCR reaction solution: include 5 ⁇ L of premixed solution (containing enzymes and buffers required for PCR), 0-10 ⁇ L of double-distilled water (make up to 10 ⁇ L with water based on the total volume), and 0 to 2 ⁇ L of Dye (for fluorescence compensation and correction of the machine), 1 ⁇ M upstream primer of ALOX15 gene, 1 ⁇ M downstream primer of ALOX15 gene, 1 ⁇ M upstream reference gene of primer, 1 ⁇ M downstream reference gene of primer, 2 ⁇ L of cDNA, 1 ⁇ g Positive control, 1 ⁇ g negative control;
  • Step 2 Standard procedure for two-step PCR amplification:
  • the reaction conditions for the standard procedure for two-step PCR amplification include the following steps: Stage 1: Pre-denaturation for 30 seconds at 95 ° C; Stage 2 PCR : Reaction was performed at 95 ° C for 15 seconds, and at 60 ° C, reaction was performed for 60 seconds, followed by annealing extension, and 40 cycles were performed.
  • Step 4 Calculate the expression of the ALOX15 gene:
  • ⁇ CT method was used to compare the expression of ALOX15 and internal reference genes: the average CT value of ALOX15 was 20.1, the average CT value of GAPDH was 17.9, and the ⁇ CT value was 2.2.
  • the preferred manufacturer of the first buffer RWA buffer used is Takara Company, article number 9767; the manufacturer of the second buffer RWB buffer is Takara Company, article number 9767.
  • the protection scope of the present application is not limited to the first buffer solution and the second buffer solution described above. Those skilled in the art can choose according to the actual application needs.
  • nasal endoscope was used to press the surface of the nasal polyps with a brush (Copan) for 30 seconds, rotated 3-4 times to brush the surface of the polyp, and the brush was placed in a subsequent place.
  • the lysate is stored at 4 ° C for short-term storage (no more than 24 hours), or transferred to long-term storage below -20 ° C.
  • a method for detecting the expression of ALOX15 gene in exfoliated nasal cells comprising the following steps:
  • Step 1 Extract RNA from exfoliated nasal cells: add 1mL RNA extraction solution to the centrifuge tube containing detached nasal cells, dissolve and shake, and let stand at room temperature for 5min; add 200 ⁇ L of chloroform, mix by shaking, and let stand at room temperature for 5min. Centrifuge at 12000 r / min for 15 min at 4 ° C. Take 200 ⁇ L of the supernatant, add 200 ⁇ L isopropanol, mix and let stand for 10 min. Centrifuge at 4 ° C, 12000 r / min for 15 min. Discard the supernatant and retain the first pellet.
  • Step 2 The steps for preparing cDNA by reverse transcription are the same as in Example 7.
  • Step 3 The detection steps of real-time quantitative PCR amplification are the same as those in Example 7.
  • Step 4 Calculate the expression of the ALOX15 gene:
  • Step 1 Calculate the average ⁇ CT of the healthy control group:
  • subjects 1 to 10 are healthy control groups.
  • Step 2 Calculate the relative expression of the subject:
  • the patient was instructed to flush the nasal cavity with normal saline, and under a nasal endoscope, press the surface of the nasal polyp with a brush (Copan) for 30 seconds, rotate 3-4 times, brush the surface of the polyp, and place the brush in the lysate , Short-term storage at 4 ° C (not more than 24 hours), or transfer to long-term storage below -20 ° C.
  • a brush Copan
  • a method for detecting the expression of ALOX15 gene in exfoliated nasal cells comprising the following steps:
  • Step 1 Extract RNA from the nasal cavity exfoliated cells: Add 20mL RNA extraction solution to the centrifuge tube containing the nasal cavity exfoliated cells, dissolve and shake, and let stand at room temperature for 7min; add 10mL of chloroform, shake and mix, and leave at room temperature for 7min Centrifuge at 5 ° C, 14000r / min for 20min; take 20mL of supernatant, add 20mL of isopropanol, mix well and let stand for 12min, centrifuge at 5 ° C, 14000r / min for 20min, discard the supernatant, and retain the first precipitate; 40 mL of 90% ethanol was added to the first pellet, centrifuged at 14000 r / min for 3 min at 5 ° C, the supernatant was discarded, and the second pellet was retained; the centrifuge tube was capped, and centrifuged at 5 ° C, 14000 r / min for 3 min After removing the supernatant, let it stand for 20 minutes,
  • Step 2 The steps for preparing cDNA by reverse transcription are the same as in Example 7.
  • Step 3 The preparation of the real-time quantitative PCR reaction solution in the real-time quantitative PCR amplification detection step is the same as in Example 7.
  • a standard three-step method for PCR amplification is adopted.
  • the reaction conditions of the standard three-step PCR standard procedure include The following steps: Phase 1: Pre-denaturation at 95 ° C for 2 minutes; Phase 2 PCR reaction: Reaction at 95 ° C for 1 minute, at 55 ° C for 1 minute, at 72 ° C Under the conditions, the reaction was performed for 1 minute, and thus 40 cycles were performed; finally, 72 ° C, 7 minutes of annealing extension.
  • Step 4 Calculate the expression of the ALOX15 gene:
  • the patient was instructed to flush the nasal cavity with normal saline, and under a nasal endoscope, press the surface of the nasal polyp with a brush (Copan) for 30 seconds, rotate 3-4 times, brush the surface of the polyp, and place the brush in the lysis solution , Short-term storage at 4 ° C (not more than 24 hours), or transfer to long-term storage below -20 ° C.
  • a brush Copan
  • a method for detecting the expression of ALOX15 gene in exfoliated nasal cells comprising the following steps:
  • Step 1 Extract RNA from nasal cavity exfoliated cells. Add 0.1mL RNA extraction solution to the centrifuge tube containing nasal exfoliated cells, dissolve and shake, and let stand at room temperature for 7min. Add 0.03mL of chloroform, shake and mix, and leave at room temperature.
  • Step 2 The steps for preparing cDNA by reverse transcription are the same as in Example 7.
  • Step 3 The preparation of the real-time quantitative PCR reaction solution in the real-time quantitative PCR amplification detection step is the same as in Example 7.
  • a standard three-step method for PCR amplification is adopted.
  • the reaction conditions of the standard three-step PCR standard procedure include The following steps: Phase 1: Pre-denaturation at 95 ° C for 2 minutes; Phase 2 PCR reaction: Reaction at 95 ° C for 1 minute, at 55 ° C for 1 minute, at 72 ° C Under the conditions, the reaction was performed for 1 minute, and thus 40 cycles were performed; finally, 72 ° C, 7 minutes of annealing extension.
  • Step 4 Calculate the expression of the ALOX15 gene:
  • the expression of ALOX15 in this patient is 0.51 times (1/2 1.4 ) of GAPDH.
  • the present disclosure provides a kit for detecting chronic sinusitis with nasal polyp subtypes.
  • the proteomics and transcriptomics methods are used to select the ALOX15 gene as a biomarker and apply it to the kit to realize the use of reagents.
  • the kit is used to detect chronic sinusitis with nasal polyp subtypes, so that the final obtained kit includes a specific primer for the ALOX15 gene. Based on the specific primers, the kit of the present disclosure can quickly identify nasal polyp subtypes, and is more accurate than traditional pathological detection methods.
  • the kit can simultaneously perform large-scale, rapid Testing, saving labor costs and medical treatment costs. And the systematic kit has high identification accuracy, which can fully reflect the histopathological characteristics.
  • the kit provided by the present disclosure can detect nasal polyp cells from the surface of the nasal polyp by brushing or sticking, so as to determine the chronic sinusitis of the patient with nasal polyp subtypes, thereby avoiding wounds to the patient.
  • the safety of patient examination is improved, the operation is more convenient, and the labor cost and medical treatment cost are saved.
  • the method for detecting the expression level of ALOX15 gene in nasal exfoliated cells uses the effectively selected ALOX15 gene as a biomarker to provide a method for detecting the expression level of the gene and realize calculation of the expression level of ALOX15 gene in nasal exfoliated cells. It can effectively obtain the expression level of ALOX15 gene.
  • the method provided is simple and fast, high sensitivity, good reproducibility, and is suitable for wide application.
  • the present disclosure provides a method for detecting the expression level of ALOX15 gene in nasal exfoliated cells, wherein the ALOX15 gene can encode a lipoxygenase protein, and the encoded enzyme acts on various polyunsaturated fatty acid substrates to produce various biologically active lipid mediators, For example, eicosanoid, heparin, lipoxin and so on.
  • the arachidonic acid was converted into 12-eicosatetraenoic acid / 12-HPETE and 15-eicosatetraenoic acid / 15-HPETE. It also converts linoleic acid into 13-hydroperoxyoctadecadienoic acid.
  • ALOX15 can also act on (12S) -hydrogen peroxytetraenoic acid / (12S) -HPETE to produce heparin A3.
  • the encoded enzyme and its reaction products can regulate inflammation and immune response.
  • Diseases related to ALOX15 include softening of white matter around the ventricle. It helps clear apoptotic cells during inflammation by oxidizing membrane-bound phosphatidylethanolamine in macrophages, and inhibits autoimmune responses associated with inflammatory monocytes clearing apoptotic cells.
  • the ALOX15 gene has not been disclosed in the prior art, and the use of the gene has not been found.
  • the method for the expression of ALOX15 gene in exfoliated nasal cells can be used to detect the expression of ALOX15 in exfoliated nasal cells, and it can be used to further provide a basis for the gene screening technology for detecting chronic sinusitis subtypes with nasal polyps, It provides a reliable basis for clinical guidance and drug treatment. The feasibility of the kit for detecting chronic sinusitis subtype with nasal polyps in clinical application is guaranteed.
  • Nasal shedding was detected ALOX15 gene expression in cells of the present disclosure provides, according to actual needs or relative quantitation method using ⁇ Ct 2 - ⁇ Ct method, the expression of selected relatively constant reference gene, normalized by the number of reference genes by The difference between the Ct value of the target gene of the sample and the internal reference gene is calculated to calculate the expression amount of the target gene.
  • the method is simple and fast, the detection accuracy is high, the detection cost can be reduced, and the detection time can be saved. The result is easy to interpret and so on. Greatly improved the experimental efficiency.

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Abstract

本公开提出一种用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒及ALOX15基因作为生物标志物的应用，以及检测鼻腔脱落细胞中ALOX15基因表达量的方法及应用。其中所述试剂盒包括ALOX15基因的特异性引物。所述ALOX15基因作为生物标志物能够制备用于检测慢性鼻窦炎伴鼻息肉亚型的产品。本公开的检测方法包括以下步骤：从鼻腔脱落细胞中提取RNA，将总RNA逆转录为cDNA，采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因分别采用ALOX15基因的特异性引物和内参基因的特异性引物进行实时荧光定量PCR扩增，基于扩增产物的检测结果计算ALOX15基因表达量。

Description

检测慢性鼻窦炎伴鼻息肉亚型的方法和试剂盒及ALOX15基因作为生物标志物的应用
相关申请的交叉引用
本公开要求于2018年7月3日提交中国国家知识产权局的申请号为201810717413.X、名称为“用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒及ALOX15基因作为生物标志物的应用”的中国专利申请，和于2018年7月3日提交中国国家知识产权局的申请号为201810720285.4、名称为“检测鼻腔脱落细胞中ALOX15基因表达量的方法及应用”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开属于生物医药技术领域，尤其涉及一种用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒及ALOX15基因作为生物标志物的应用，以及一种检测鼻腔脱落细胞中ALOX15基因表达量的方法。
背景技术
慢性鼻窦炎伴鼻息肉(Chronic rhinosinusitis with nasal polyps，CRSwNP)是鼻窦黏膜的慢性炎症，查体可见鼻腔或中鼻道息肉形成。CRSwNP常见症状为鼻堵、流涕或鼻涕倒流、嗅觉减退、面部闷胀感或压力感，持续时间超过12周。患病率约为0.5-4％，CRSwNP常伴有哮喘和过敏性鼻炎，有报道7％的哮喘患者患有CRSwNP，而26-48％的CRSwNP患有哮喘。CRSwNP发病机制目前还不确定，黏膜上皮细胞破坏、宿主免疫系统失衡和病原微生物入侵可能是其发病的主要原因。CRSwNP主要的治疗方式是手术和药物治疗。但研究表明，即使通过规范的药物或手术治疗，慢性鼻窦炎伴鼻息肉的复发率仍高达56％，严重影响患者生活质量，同时带来高额医疗支出，但临床却缺乏根治性治疗方法，因而成为鼻科学研究领域的重点。
依据嗜酸性粒细胞浸润的程度可以将CRSwNP分为嗜酸性粒细胞型(Eosinophilic CRSwNP，ECRSwNP)与非嗜酸性粒细胞型(Noneosinophilic CRSwNP，nonECRSwNP)，二者的临床表现、用药和预后均不同，嗜酸性粒细胞型的临床症状较重，以鼻堵和嗅觉减退为主，多合并有哮喘，术后复发率较高。鼻息肉组织中嗜酸性粒细胞浸润程度与复发关系最为密切，当组织中该细胞百分比超过27％时，复发风险超过90％。嗜酸性粒细胞型息肉对糖皮质激素类药物的敏感度显著高于非嗜酸性粒细胞型。非嗜酸性粒细胞型临床症状一般较轻，且合并哮喘的几率较小，气道炎症较轻，且术后复发率较嗜酸性粒细胞型低，对大环内酯类药物治疗反应好。西方国家以嗜酸性粒细胞型为主，主要表现为TH2炎症反应，而我国的嗜酸性粒细胞型和非嗜酸性粒细胞型比例均约占一半，非嗜酸性粒细胞型主要表现为TH1/TH17炎症反应为主。综上所述，嗜酸性粒细胞型和非嗜酸性粒细胞型在免疫病理类型、临床症状、药物治疗反应和预后存在明显不同。不同慢性鼻窦炎伴鼻息肉的炎症/病理分型治疗策略不同。所以对慢性鼻窦炎伴鼻息肉的病理分型的鉴定显得尤为重要。
目前对两种亚型的判断主要依据鼻腔黏膜活检后组织病理标本染色，缺乏无创性生物学标志物用于鉴别诊断。患者在鼻内镜下获得息肉标本后，进行石蜡固定等病理标本的常规处理，然后进行苏木素伊红的染色，接下来通过高倍显微镜观察组织浸润的炎细胞(主要的炎细胞包括嗜酸性粒细胞，中性粒细胞，淋巴细胞，浆细胞)浸润个数，进行细胞分型。鼻腔黏膜病理活检的缺点如下：1.为有创检查：增加了患者的感染风险，不适用于免疫力较低人群如儿童、老年人等；取材时鼻腔出血，往往引起患者恐惧和担忧。2.难以获得疾病的实时动态变化信息：不能在术后对愈合期黏膜进行病理活检。但临床数据表明慢性鼻窦炎伴鼻息肉的病理分类会随药物治疗、手术治疗等转归，用治疗前的病理活检结果无法代表全部疾病时期的特征。3.费时且增加就医成本，从获取组织样本到获取病理结果一般3-4个工作日。由于无法当日或次日获得结果，外地患者就医等产生额外的交通费、住宿费、挂号费，增加了就医成本。4.存在一定的人为误差，不同的病理医生计数的炎细胞数量有可能不同，影响息肉分型的判断。5.组织病理切片较局限，只能反映切片位置的标本炎症状态，不能反映组织的全貌，可能会造成误诊。6.每张片子都需要病理科医生人工计数，难以批量操作。
由此可见，提供一种能够不依赖于鼻腔黏膜病理活检且能快速准确大批量检测慢性鼻窦炎伴鼻息肉亚型的方法成为本领域技术人员亟待解决的技术难题。
近年兴起的实时荧光定量PCR弥补了上述技术的不足。该方法是在PCR反应体系中加入荧光基团，利用荧光信号的积累实时监测整个PCR过程，最后根据荧光信号对所测DNA样品进行定量分析，该方法操作简便，敏感性高，重复性好且结果准确性高。然而现有技术中尚未公开有效提供检测鼻腔脱落细胞中ALOX15基因表达量的方法，以更好的获取到鼻腔脱落细胞内ALOX15基因含量情况。
发明内容
本公开提供一种用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒，其中，所述试剂盒包括ALOX15基因的特异性引物。
本公开还提供了一种ALOX15基因作为生物标志物在制备用于检测慢性鼻窦炎伴鼻息肉亚型的产品中的应用。
本公开还提供一种ALOX15基因作为生物标志物用于检测慢性鼻窦炎伴鼻息肉亚型中的用途。
本公开还提供了本公开所述的试剂盒用于检测慢性鼻窦炎伴鼻息肉亚型中的用途。
本公开还提供了检测ALOX15的试剂用于检测慢性鼻窦炎伴鼻息肉亚型中的用途。
本公开还提供了一种检测鼻腔脱落细胞中ALOX15基因表达量的方法，包括以下步骤：从鼻腔脱落细胞中提取RNA，将总RNA逆转录为cDNA，采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因分别采用ALOX15基因的特异性引物和内参基因的特异性引物进行实时荧光定量PCR扩增，基于扩增产物的检测结果计算ALOX15基因表达量。
本公开还提供了一种上述检测鼻腔脱落细胞中ALOX15基因表达量的方法在制备用于检测伴有鼻息肉的慢性鼻窦炎亚型的试剂盒中的应用。
本公开还提供了一种检测伴有鼻息肉的慢性鼻窦炎亚型方法，包括通过本公开所述的方法检测鼻腔脱落细胞中ALOX15基因表达量。
本公开还提供了一种检测伴有鼻息肉的慢性鼻窦炎亚型方法，包括检测检测鼻腔脱落细胞中的ALOX15基因表达量，优选地使用本公开所述的试剂盒检测所述腔脱落细胞中的ALOX15基因表达量。
附图说明
图1为本公开实验例一部分ALOX15基因实时荧光定量PCR扩增曲线图；
图2为本公开实验例一另一部分ALOX15基因实时荧光定量PCR扩增曲线图；
图3为本公开实验例一再一部分ALOX15基因实时荧光定量PCR扩增曲线图；
图4为本公开实验例一再一部分ALOX15基因实时荧光定量PCR扩增曲线图；
图5为本公开实验例一部分ALOX15基因实时荧光定量PCR熔解曲线图；
图6为本公开实验例一另一部分ALOX15基因实时荧光定量PCR熔解曲线图；
图7为本公开实验例一再一部分ALOX15基因实时荧光定量PCR熔解曲线图；
图8为本公开实验例一再一部分ALOX15基因实时荧光定量PCR熔解曲线图；
图9为本公开实验例一的一种可选的进行慢性鼻窦炎伴鼻息肉分型检测的ROC曲线。
图10为本公开实验例二部分ALOX15基因实时荧光定量PCR扩增曲线图；
图11为本公开实验例二另一部分ALOX15基因实时荧光定量PCR扩增曲线图；
图12为本公开实验例二再一部分ALOX15基因实时荧光定量PCR扩增曲线图；
图13为本公开实验例二部分ALOX15基因实时荧光定量PCR熔解曲线图；
图14为本公开实验例二另一部分ALOX15基因实时荧光定量PCR熔解曲线图；
图15为本公开实验例二再一部分ALOX15基因实时荧光定量PCR熔解曲线图；
图16为本公开实验例三ALOX15基因实时荧光定量PCR扩增曲线图；
图17为本公开实验例三ALOX15基因实时荧光定量PCR熔解曲线图；
图18为本公开实验例四ALOX15基因实时荧光定量PCR扩增曲线图；
图19为本公开实验例四ALOX15基因实时荧光定量PCR熔解曲线图。
具体实施方式
下面将对本公开实施方式中的技术方案进行清楚、完整地描述，显然，所描述的实施方式仅仅是本公开一部分实施方式，而不是全部的实施方式。基于本公开中的实施方式，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施方式，都属于本公开保护的范围。
如本文所用术语“引物”、“扩增引物”或“寡核苷酸引物”是指寡核苷酸，其能够与邻近靶序列的RNA或DNA区位点特异性地退火，并作为合适条件下DNA合成的起始引物。其通常为单链，其可以是天然发生的或合成的。“引物”或“寡核苷酸引物”通常包含约5至约50个核苷酸之间的序列，更优选为约10至约30个核苷酸，或更优选为约15至25个核苷酸。
相对于待扩增的核酸的区域，如本文所用术语“上游引物”一般与距离核酸分子5’末端较近的区域结合。另一方面，相对于待扩增的核酸上的区域，如本文所用术语“下游引物”一般与距离核酸分子3’末端较近的区域结合。如本文所用术语“内参基因”通常是指在各个组织和细胞中表达相对恒定的基因，例如鼻息肉组织或从鼻黏膜脱落细胞中的GAPDH基因，主要在检测基因表达水平时作为参照物使用。
如本文所用，“聚合酶链反应”或PCR是由以下项组成的核酸的扩增：分离双链核酸样本的链的初始变性步骤，随后重复(i)允许扩增引物对靶序列侧翼位置特异性退火的退火步骤；(ii)在5'至3'方向上延伸引物从而形成与靶序列互补的扩增子多核苷酸的延伸步骤，以及(iii)引起扩增子与靶序列分离的变性步骤。上述各步骤可以在不同的温度下进行，优选地使用自动热循环仪。
如本文所用，“荧光定量PCR(Real-time quantitative PCR)”，也称为定量PCR，简称为QPCR，是指在PCR反应体系中加入荧光基团，利用荧光信号累积实时监测整个PCR进程，最后通过标准曲线对未知模板进行定量分析的方法。
如本文所用术语“Ct“(循环阈值)或“Ct值”通常是指荧光定量PCR中荧光信号到达设定的阈值时所经历的循环数。
如本文所用，术语“包含”、“包括”、“涵盖”、“内含”、“含有”、“其特征在于”、“具有”或它们的任何其它变型旨在涵盖非排它性的包含之意。
在一种或多种实施方式中，所述ALOX15基因的上游引物如SEQ ID NO:2所示，所述ALOX15基因的下游引物如SEQ ID NO:3所示。
在一种或多种实施方式中，所述试剂盒进一步还包括内参基因的特异性引物。
在一种或多种实施方式中，所述内参基因为GAPDH，所述内参基因的上游引物如SEQ ID NO:4所示，所述ALOX15基因的下游引物如SEQ ID NO:5所示。
在一种或多种实施方式中，所述试剂盒进一步还包括：从鼻息肉组织或从鼻黏膜脱落细胞中提取RNA的试剂；将总RNA逆转录为cDNA的试剂；采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因进行实时荧光定量PCR反应的试剂。
在一种或多种实施方式中，所述将总RNA逆转录为cDNA的试剂包括：逆转录混合液和去RNA酶及去DNA酶的水；
在一种或多种实施方式中，将总RNA进行逆转录为cDNA的试剂包括：1μL～40μL的逆转录混合液，以及0μL～160μL的去RNA酶及去DNA酶的水。进一步优选的，所述将总RNA逆转录为cDNA的试剂包括：2μL的逆转录混合液，以及0μL～8μL的去RNA酶及去DNA酶的水。
在一种或多种实施方式中，采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因进行实 时荧光定量PCR反应的试剂包括：PCR预混合液、双蒸水、机器荧光补偿及矫正剂、ALOX15基因的上游引物、ALOX15基因的下游引物、内参基因的上游引物和内参基因的下游引物。
在一种或多种实施方式中，采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因进行实时荧光定量PCR反应的试剂包括：1μL～25μL的PCR预混合液，0μL～50μL的双蒸水，0μL～2μL的机器荧光补偿及矫正剂，0.01～100μM的ALOX15基因的上游引物，0.01～100μM的ALOX15基因的下游引物，0.01～100μM的内参基因的上游引物，0.01～100μM的内参基因的下游引物；进一步优选的，采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因进行实时荧光定量PCR反应的试剂包括：5μL的PCR预混合液，0μL～10μL的双蒸水，根据总体积用水补齐至10μL，0.2μL的机器荧光补偿及矫正剂，1μM的ALOX15基因的上游引物，1μM的ALOX15基因的下游引物，1μM的内参基因的上游引物，1μM的内参基因的下游引物。
在一种或多种实施方式中，所述从鼻息肉组织中提取RNA的试剂可以选择以下两种试剂。
第一种包括：RNA抽提液、氯仿、异丙醇、浓度为65％～90％的乙醇、去RNA酶及去DNA酶的水；其中优选的，包括0.1mL～20mL的RNA抽提液Trizol或RNAiso Blood或RNAiso Plus或其它含有苯酚、异硫氰酸胍、8-羟基喹啉、异硫氰酸胍或β-巯基乙醇的物质，所述Trizol或所述RNAiso Blood或所述RNAiso Plus或所述其它含有苯酚、异硫氰酸胍、8－羟基喹啉、异硫氰酸胍或β-巯基乙醇的物质的体积的0.1～0.5倍的氯仿，所述氯仿体积的0.5～3倍的异丙醇，所述异丙醇体积的0.5～5倍的65％至90％的乙醇，以及0.01mL至5mL的去RNA酶及去DNA酶的水；进一步优选的，针对从鼻息肉组织中提取RNA的试剂可选择以下两种试剂，第一种包括：1mL的RNA抽提液Trizol或RNAiso Blood或RNAiso Plus或其它含有苯酚、异硫氰酸胍、8-羟基喹啉、异硫氰酸胍或β-巯基乙醇的物质，200μL的氯仿，200μL的异丙醇，200μL体积浓度为65％至90％的乙醇，以及0.02mL的去RNA酶及去DNA酶的水；
另外一种包括：所述从鼻息肉组织中提取RNA的试剂包括：细胞裂解液、用于去除吸附有RNA的纯化柱的杂质的第一缓冲液、用于去除吸附有RNA的纯化柱的杂质和盐分的第二缓冲液和去RNA酶及去DNA酶的水；所述从鼻息肉组织中提取RNA的工具包括RNA纯化柱；其中所述从鼻息肉组织中提取RNA的试剂还包括DNA酶反应液或所述从鼻息肉组织中提取RNA的工具还包括基因组DNA吸附柱；所述DNA酶反应液包括DNA酶缓冲液、重组DNA酶和去RNA酶的双蒸水。
在一种或多种实施方式中，所述从鼻息肉组织中提取RNA的试剂包括：0.1mL～2mL的用于裂解细胞和抑制RNA降解的细胞裂解液、0.1mL～0.7mL的洗涤用第一缓冲液、0.1mL～0.7mL的洗涤用第二缓冲液、0.01mL～1mL的去RNA酶及去DNA酶的水、0～10μL的去除基因组DNA的重组DNA酶、0～10μL的去除基因组DNA的DNA酶缓冲液、20～100μL的去RNA酶的双蒸水，所述从鼻息肉组织中提取RNA的工具包括RNA纯化柱；或者包括0.1mL～2mL的用于裂解细胞和抑制RNA降解的细胞裂解液、0.1mL～0.7mL的洗涤用第一缓冲液、0.1mL～0.7mL的洗涤用第二缓冲液、0.01mL～1mL的去RNA酶及去DNA酶的水，所述从鼻息肉组织中提取RNA的工具包括基因组DNA吸附柱和RNA纯化柱。
在一种或多种实施方式中，所述从鼻息肉组织中提取RNA的试剂包括：300μL的用于裂解细胞和抑制RNA降解的细胞裂解液、500μL的洗涤用第一缓冲液、600μL的洗涤用第二缓冲液、0.02mL的去RNA酶及去DNA酶的水、4μL的去除基因组DNA的重组DNA酶、5μL 10×去除基因组DNA的DNA酶缓冲液、41μL的去RNA酶的双蒸水，所述从鼻息肉组织中提取RNA的工具包括RNA纯化柱；或者包括300μL的用于裂解细胞和抑制RNA降解的细胞裂解液、500μL的洗涤用第一缓冲液、600μL的洗涤用第二缓冲液、0.02mL的去RNA酶及去DNA酶的水，所述从鼻息肉组织中提取RNA的工具包括基因组DNA吸附柱和RNA纯化柱。
在一种或多种实施方式中，所述鼻息肉组织为鼻腔病理活检获得的鼻息肉组织，或者所述鼻黏膜脱落细胞为刷取或粘取鼻息肉表面获取的鼻息肉细胞。
在一种或多种实施方式中，选用△Ct(Ct(ALOX15)-Ct(GAPDH))分析法分析扩增产物的数据结果，且与所述△Ct进行比较的界定值为1.675。
在一种或多种实施方式中，ALOX15基因的上游引物如SEQ ID NO:2所示，ALOX15基因的下游引物如SEQ ID NO:3所示。
在一种或多种实施方式中，所述内参基因为GAPDH，所述内参基因的上游引物如SEQ ID NO:4所示，所述下游引物如SEQ ID NO:5所示。
在一种或多种实施方式中，所述鼻腔脱落细胞采用毛刷于鼻息肉表面获取，且将获取鼻腔脱落细胞后的毛刷置于细胞裂解液中于4℃以下保存。
在一种或多种实施方式中，从鼻腔脱落细胞中提取RNA的方法，包括两种方法，其中第一种方法包括以下步骤：
步骤1：将所述鼻腔脱落细胞溶解于100～2000μL细胞裂解液中，并加入等体积的乙醇，混合均匀后加入至RNA纯化柱中，离心处理后，除去收集管中的滤液，将所述RNA纯化柱置于收集管中；
步骤2：向步骤1中获取的所述RNA纯化柱中加入300μL～700μL的第一缓冲液，离心处理后，除去第一滤液；继续向所述RNA纯化柱加入400μL～800μL第二缓冲液，离心处理后，除去第二滤液，取RNA纯化柱经洗脱得到RNA。
在一种或多种实施方式中，所述从鼻腔脱落细胞中提取RNA的方法，还包括以下步骤：向除去所述第二滤液后的RNA纯化柱中添加10～100μL DNA酶反应液，经静置处理后，加入300μL～700μL所述第二缓冲液，离心处理后，除去第三滤液，取RNA纯化柱经洗脱后采用分光光度计测量RNA纯度，得到RNA；
所述DNA酶反应液的制备方法包括以下步骤：取DNA酶缓冲液、重组DNA酶，去RNA酶的双蒸水经混合得到DNA酶反应液。优选所述DNA酶反应液的制备方法包括以下步骤：取5μL 10×DNA酶缓冲液、4μL重组DNA酶，41μL去RNA酶的双蒸水经混合得到DNA酶反应液。
在一种或多种实施方式中，当进行RNA提取时基因组含量较低或材料起始量较少时，所述从鼻腔脱落细胞中提取RNA的方法，还包括以下步骤：所述步骤1中，所述鼻腔脱落细胞溶解于细胞裂解液中后先加入至基因组DNA吸附柱中取滤液，再向所述滤液中加入等体积的乙醇。
在一种或多种实施方式中，为了获取更高浓度的RNA，所述从鼻腔脱落细胞中提取RNA的方法，还包括以下步骤：取待洗脱的RNA纯化柱加入去RNA水解酶的蒸馏水或焦碳酸二乙酯处理水，室温静置后，经离心处理、洗脱所述RNA纯化柱，采用分光光度计测量RNA纯度，得到RNA。
其中步骤1采用细胞裂解液能够迅速破碎鼻腔脱落细胞并抑制鼻腔脱落细胞释放出的核酸酶的物质；采用基因组DNA吸附柱用于去除基因组DNA；步骤2中的RNA纯化柱用于富集RNA；其中收集管用于收集去除基因组DNA后的溶液，用于去除吸附有RNA的纯化柱的杂质的第一缓冲液、用于去除RNA溶液中的杂质和盐分的第二缓冲液。
在一种或多种实施方式中，所述从鼻腔脱落细胞中提取RNA的第一种方法，包括以下步骤：
步骤1：将所述鼻腔脱落细胞溶解于300μL细胞裂解液中，并加入等体积的70％乙醇，使用移液枪将溶液混合均匀；立即将混合液加入至RNA纯化柱中，12000转/分钟，离心1min，除去滤液，将所述RNA纯化柱置于2mL收集管中；
步骤2：向步骤1中获取的所述RNA纯化柱中加入500μL的第一缓冲液，12000转/分钟，离心30s，除去第一滤液；继续向所述RNA纯化柱加入600μL第二缓冲液，12000转/分钟，离心30s，除去第二滤液；
步骤3：取5μL 10×DNA酶缓冲液、4μL重组DNA酶，41μL去RNA酶的双蒸水经混合得到DNA酶反应液，向除去所述第二滤液后的RNA纯化柱中添加50μL DNA酶反应液，室温静置15分钟，加入350μL所述第二缓冲液，12000转/分钟，离心30s，除去第三滤液；
步骤4：将步骤3中除去第三滤液的RNA纯化柱安置于1.5mL无RNA水解酶收集管，向RNA纯化柱加入50μL的去RNA水解酶的蒸馏水或0.1％焦碳酸二乙酯处理水，室温静置5分钟，12000转/分钟，离心2min，洗脱RNA纯化柱，采用分光光度计测量RNA溶液的OD260/OD280比值为1.7～2.1时，得到RNA。
在一种或多种实施方式中，所述从鼻腔脱落细胞中提取RNA的方法，包括以下步骤：
步骤1：取基因组DNA吸附柱置于2mL收集管内，将所述鼻腔脱落细胞溶解于100～2000μL细胞裂解液中后加入至基因组DNA吸附柱中，取滤液，向所述滤中加入等体积的70％乙醇，混合均匀后加入至RNA纯化柱中，12000转/分钟，离心1min，除去滤液，将所述RNA纯化柱置于2mL收集管中；
步骤2：向步骤1中获取的所述RNA纯化柱中加入500μL的第一缓冲液，12000转/分钟，离心30s，除去第一滤液；继续向所述RNA纯化柱加入600μL第二缓冲液，12000转/分钟，离心30s，除去第二滤液；
步骤3：将步骤2中除去第二滤液的RNA纯化柱安置于1.5mL无RNA水解酶收集管，向RNA纯化柱加入50μL的去RNA水解酶的蒸馏水或0.1％焦碳酸二乙酯处理水，室温静置5分钟，12000转/分钟，离心2min，洗脱RNA纯化柱，采用分光光度计测量RNA溶液的OD260/OD280比值为1.7～2.1时，得到RNA。
在一种或多种实施方式中，所述从鼻腔脱落细胞中提取RNA的第二种方法，包括以下步骤：向装有鼻腔脱落细胞的离心管中加入0.1mL～20mL RNA抽提液进行溶解、振荡后加入所述RNA抽提液体积的0.1～0.5倍的氯仿，震荡混匀，室温静置，将所述离心管离心，取上清液，加入所述氯仿体积的0.5～3倍的异丙醇，混匀后静置、离心，弃上清，保留第一沉淀，向所述第一沉淀中加入所述异丙醇体积的0.5～5倍的浓度65％～90％的乙醇，经洗涤后混匀、离心，弃上清，保留第二沉淀；盖紧所述离心管，再次离心，除去上清液，继续向所述离心管中加入0.01～5mL去RNA酶及去DNA酶的水溶解所述第二沉淀，采用分光光度计测量RNA纯度，得到RNA。
在一种或多种实施方式中，所述RNA抽提液为Trizol、RNAiso Blood、RNAiso Plus或其它含有苯酚、异硫氰酸胍、8－羟基喹啉、异硫氰酸胍和β-巯基乙醇中的任一种或几种的试剂。
在一种或多种实施方式中，所述从鼻腔脱落细胞中提取RNA的第二种方法，包括以下步骤：向装有鼻腔脱落细胞的离心管中加入0.1～20mLRNA抽提液进行溶解、振荡后，室温静置3～7min；加入40μL～5mL氯仿，震荡混匀，室温静置3～7min，于3℃～5℃，10000～14000r/min离心10～20min；取上清液40μL～8mL，加入等体积的异丙醇，混匀后静置8～12min，于3℃～5℃，10000～14000r/min离心10～20min弃上清，保留第一沉淀；向所述第一沉淀中加入与异丙醇等体积的浓度65％～90％的乙醇，于3℃～5℃，7000～14000r/min离心10～20min，弃上清，保留第二沉淀；盖紧所述离心管，于3℃～5℃，7000～14000r/min离心1～3min，除去上清液，静置10～20min后继续向所述离心管中加入0.01～5mL去RNA酶及去DNA酶的水溶解所述第二沉淀，采用分光光度计测量RNA纯度，得到RNA。
在一种或多种实施方式中，所述从鼻腔脱落细胞中提取RNA的第二种方法，包括以下步骤：向装有鼻腔脱落细胞的离心管中加入1mLRNA抽提液进行溶解、振荡后，室温静置5min；加入200μL氯仿，震荡混匀，室温静置5min，于4℃，12000r/min离心15min；取上清液200μL，加入200μL异丙醇，混匀后静置10min，于4℃，12000r/min离心15min弃上清，保留第一沉淀；向所述第一沉淀中加入与异丙醇等体积的浓度75％的乙醇，于4℃，7500r/min离心15min，弃上清，保留第二沉淀；盖紧所述离心管，于4℃，7500r/min离心2min，除去上清液，静置15min后继续向所述离心管中加入50 μL去RNA酶及去DNA酶的水溶解所述第二沉淀，采用分光光度计测量RNA溶液OD260/OD280比值为1.7～2.1，得到RNA。
在一种或多种实施方式中，将总RNA逆转录为cDNA的方法包括以下步骤：取1～3μL的逆转录混合液、0～10μL的去RNA水解酶蒸馏水和提取的RNA于37℃温度条件下发生反转录反应15min，后于84℃温度条件下发生反转录酶的失活反应，得到逆转录产物cDNA。
在一种或多种实施方式中，将总RNA逆转录为cDNA的方法包括以下步骤：取2μL的所述逆转录混合液，8μL的所述去RNA水解酶蒸馏水，以及总量不超过500ng或体积不超过8μL的总RNA，所述去RNA水解酶蒸馏水补齐至10μL；轻柔混匀后进行逆转录反应,条件如下:在37℃的条件下，进行15分钟的反转录反应；在85℃的条件下，进行5秒的反转录酶的失活反应；产物4℃放置。其中反应体系可按需求相应放大，10μL反应体系可最大使用500ng的总RNA，本领域技术人员可根据实际需要进行选择。
在一种或多种实施方式中，所述实时荧光定量PCR扩增包括以下步骤：
步骤1：制备实时荧光定量PCR反应液：包括1μL～25μL的PCR预混合液，0μL～10μL的双蒸水补齐总体积用水至10μL，0μL～2μL的机器荧光补偿及矫正剂，0.01～100μM的ALOX15基因的上游引物，0.01～100μM的ALOX15基因的下游引物，0.01～100μM的内参基因的上游引物，0.01～100μM的内参基因的下游引物，0.01μL～5μL的cDNA；
步骤2：采用两步法PCR扩增标准程序或三步法PCR扩增标准程序进行实时荧光定量PCR检测；
步骤3：计算ALOX15基因表达量。
在一种或多种实施方式中，制备实时荧光定量PCR反应液：包括5μL的PCR预混合液，2.8μL的双蒸水补齐总体积用水至10μL，0.2μL的机器荧光补偿及矫正剂，0.5μL的ALOX15基因的上游引物，0.5μL的ALOX15基因的下游引物，0.5μL的内参基因的上游引物，0.5μL的内参基因的下游引物，1ng/μL的所述cDNA或0.01μL至5μL的所述RNA。
在一种或多种实施方式中，所述两步法PCR扩增标准程序的反应条件包括以下步骤：第1阶段：在95℃的条件下预变性30秒；第2阶段PCR反应：在95℃的条件下，反应15秒，在60℃的条件下，反应60秒，退火延伸，如此进行40个循环。
在一种或多种实施方式中，所述三步法PCR扩增标准程序的反应条件包括以下步骤：第1阶段：在95℃的条件下预变性2分钟；第2阶段PCR反应：在95℃的条件下，反应1分钟，在55℃的条件下，反应1分钟，在72℃的条件下，反应1分钟，如此进行40个循环；最后72℃，7分钟退火延伸。
在一种或多种实施方式中，计算所述ALOX15基因表达量的方法为：计算ΔCT或2 -ΔΔCT来计算目的基因的表达量；其中ΔCT＝(CT(ALOX15)-CT(GAPDH))，-ΔΔCT＝-(ΔCT(处理标本CT(ALOX15)-CT(GAPDH))-健康对照组平均ΔCT)；健康对照组平均ΔCT＝∑每个对照组ΔCT(CT(ALOX15)-CT(GAPDH))/对照组样本数。
在一种或多种实施方式中，所述检测ALOX15的试剂包括ALOX15基因的特异性引物，优选地所述ALOX15基因的特异性引物的上游引物如SEQ ID NO:2所示，所述ALOX15基因的下游引物如SEQ ID NO:3所示。
在一种或多种实施方式中，检测ALOX15的试剂选自由针对ALOX15的引物、抗体、适配体、探针或其组合组成的组。
在一种或多种实施方式中，通过荧光PCR法测定ALOX15基因水平来实现检测慢性鼻窦炎伴鼻息肉亚型。
在一种或多种实施方式中，其中，通过荧光PCR检测鼻腔脱落细胞中ALOX15基因表达量。
在一种或多种实施方式中，根据△Ct(Ct(ALOX15)-Ct(GAPDH))确定慢性鼻窦炎伴鼻息肉亚 型，Ct(ALOX15)为ALOX15基因的Ct值，Ct(GAPDH)为内参基因GAPDH的Ct值。
在一种或多种实施方式中，所述△Ct大于或等于1.675代表非嗜酸性粒细胞型慢性鼻窦炎伴鼻息肉，而△Ct小于1.675代表嗜酸性粒细胞型慢性鼻窦炎伴鼻息肉。
在一种或多种实施方式中，所述慢性鼻窦炎伴鼻息肉亚型为非嗜酸性粒细胞型慢性鼻窦炎伴鼻息肉或嗜酸性粒细胞型慢性鼻窦炎伴鼻息肉。
本公开的优点和积极效果包括但不限于以下中至少一种。
1、本公开提供一种用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒，通过蛋白质组学和转录组学方法筛选采用ALOX15基因作为生物标志物，将其应用到试剂盒中，以实现采用试剂盒进行检测慢性鼻窦炎伴鼻息肉亚型的方法，使最终获取的试剂盒包括ALOX15基因的特异性引物。在具有该特异性引物的基础上，本公开的试剂盒能够快速对鼻息肉亚型进行鉴别，且相比较传统的病理检测方法准确性更高，此试剂盒可以同时对样品进行大批量、快速检测，节约了人力成本和就医成本。且系统化试剂盒鉴别准确率较高，可以全面的反应组织病理学特点。解决现有技术中人为误差的影响，避免了组织切片反应了组织局部特征造成误诊的弊端。通过试剂盒进行快速、准确、全面的鼻息肉亚型鉴别对临床诊疗至关重要，以尽早根据鼻息肉的炎症亚型进行针对化治疗，有效指导针对慢性鼻窦炎伴鼻息肉患者的药物治疗方式及手术方式的确定，准确预估药物治疗的反应、判断预后效果。
2、本公开所提供的试剂盒能够通过采用刷取或粘取的方式从鼻息肉的表面获取鼻息肉细胞来进行检测，从而确定患者的慢性鼻窦炎伴鼻息肉亚型，避免了对患者造成创面，提高了患者检查的安全性，且操作更便捷，节约了人力成本和就医成本。
3、本公开提供的检测鼻腔脱落细胞中ALOX15基因表达量的方法，以有效筛选的ALOX15基因作为生物标志物，提供对其基因表达量的检测方法，实现对鼻腔脱落细胞中ALOX15基因表达量的计算，能够有效获取ALOX15基因表达量，提供的方法简单快捷，敏感性高，重复性好，适宜广泛推广应用。
4、本公开提供的检测鼻腔脱落细胞中ALOX15基因表达量的方法，其中ALOX15基因能编码脂氧合酶蛋白质，编码的酶作用于各种多不饱和脂肪酸底物以产生各种生物活性脂质介质，例如类花生酸，肝素，脂氧素等。将花生四烯酸转化为12-氢过氧化二十碳四烯酸/12-HPETE和15-氢过氧化二十碳四烯酸/15-HPETE。还将亚油酸转化为13-氢过氧十八碳二烯酸。也可以作用于(12S)-过氧化氢二十一碳四烯酸/(12S)-HPETE以产生肝霉素A3。编码的酶及其反应产物能够调节炎症和免疫反应，与ALOX15相关的疾病包括脑室周围白质软化等。其通过氧化巨噬细胞中膜结合的磷脂酰乙醇胺帮助清除炎症期间的凋亡细胞，并且抑制与炎性单核细胞清除凋亡细胞相关的自身免疫应答。目前现有技术中尚未公开有ALOX15基因，更未发现该基因的用途。其中公开提供的针对鼻腔脱落细胞中ALOX15基因表达量的方法可用于检测鼻腔脱落细胞中ALOX15表达情况，可用于进一步为鼻腔伴有鼻息肉的慢性鼻窦炎亚型的检测基因筛选技术提供了基础，为临床指导和药物治疗提供了可靠的基础。保证了用于检测伴有鼻息肉的慢性鼻窦炎亚型的试剂盒在临床应用的可行性。
5、本公开提供的检测鼻腔脱落细胞中ALOX15基因表达量的方法，根据实际需求采用ΔCt或2 -ΔΔ Ct法的相对定量法，选择了表达量相对恒定的内参基因，用内参基因的数量进行标准化，通过测定样品目的基因与内参基因的Ct值差异计算目的基因表达量，方法简便快速，检测精度高，可降低检测成本，节约检测时间。结果便于判读等优点。大大提升了实验效率。在一种或多种实施方式中，通过检测鼻窦炎伴鼻息肉的鼻腔病变组织中的ALOX15蛋白质水平来检测检测慢性鼻窦炎伴鼻息肉亚型。
在一种或多种实施方式中，通过例如鼻内镜切除获得慢性鼻窦炎伴鼻息肉的鼻腔病变组织样本，通过例如免疫组织化学法测定样本中ALOX15的蛋白质水平。优选地，使用抗ALOX15的抗体测定样本中ALOX15的蛋白质水平。
在一种或多种实施方式中，提供抗ALOX15的抗体用于检测慢性鼻窦炎伴鼻息肉亚型的用途。
本公开实施例提供了一种用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒，所述试剂盒包括ALOX15基因的特异性引物。
本公开通过蛋白质组学和转录组学方法经大量的创造性实验筛选得到采用ALOX15基因作为生物标志物来制备用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒，目前现有的技术中尚未提供相应的任何报道。其中针对ALOX15基因为已知基因，基因ID为246，其DNA序列如SEQ ID NO:1所示，基因NM号为001140.4。
在一可选实施方式中，所述ALOX15基因的上游引物如SEQ ID NO:2所示，所述ALOX15基因的下游引物如SEQ ID NO:3所示。针对本公开试剂盒中所确定的上游引物和下游引物，针对本公开的试剂盒在进行鼻息肉亚型鉴别时，准确性最高，且更加有效，使试剂盒适宜进行大批量、快速检测。
在一可选实施方式中，所述试剂盒进一步还包括内参基因。更为优选的，将所述内参基因为GAPDH，所述内参基因的上游引物如SEQ ID NO:4所示，所述ALOX15基因的下游引物如SEQ ID NO:5所示。本公开试剂盒所确定的内参基因上游引物和下游引物，针对本公开的试剂盒在进行鼻息肉亚型鉴别时，能够有效通过显示ALOX15基因与GAPDH相比的表达，获取合适的ΔCT值，以进行鼻息肉亚型鉴别。且具有较高的准确率。
在一可选实施方式中，所述试剂盒进一步还包括：从鼻息肉组织或从鼻黏膜脱落细胞中提取RNA的试剂；将总RNA逆转录为cDNA的试剂；采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因进行实时荧光定量PCR反应的试剂。更为优选的，所述将总RNA逆转录为cDNA的试剂包括：逆转录混合液和去RNA酶及去DNA酶的水；采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因进行实时荧光定量PCR反应的试剂包括：PCR预混合液、双蒸水、机器荧光补偿及矫正剂、ALOX15基因的上游引物、ALOX15基因的下游引物、内参基因的上游引物和内参基因的下游引物。
另外针对从鼻息肉组织中提取RNA的试剂可选择以下两种试剂，第一种包括：RNA抽提液、氯仿、异丙醇、浓度为65％～90％的乙醇、去RNA酶及去DNA酶的水；
另外一种包括：所述从鼻息肉组织中提取RNA的试剂包括：细胞裂解液、用于去除吸附有RNA的纯化柱的杂质的第一缓冲液、用于去除吸附有RNA的纯化柱的杂质和盐分的第二缓冲液和去RNA酶及去DNA酶的水；所述从鼻息肉组织中提取RNA的工具包括RNA纯化柱；其中所述从鼻息肉组织中提取RNA的试剂还包括DNA酶反应液或所述从鼻息肉组织中提取RNA的工具还包括基因组DNA吸附柱；所述DNA酶反应液包括DNA酶缓冲液、重组DNA酶和去RNA酶的双蒸水。本领域技术人员可根据实际的制备需求进行选择。
具体的，所述将总RNA进行逆转录为cDNA的试剂包括：1μL～40μL的逆转录混合液，以及0μL～160μL的去RNA酶及去DNA酶的水。进一步优选的，所述将总RNA逆转录为cDNA的试剂包括：2μL的逆转录混合液，以及0μL～8μL的去RNA酶及去DNA酶的水(根据RNA量用水补齐至8μL)。针对上述限定的数值范围，均能够实现逆转录步骤，且对于本领域技术人员来说能够根据实际需要进行选择。
具体的，采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因进行实时荧光定量PCR反应的试剂包括：1μL～25μL的PCR预混合液，0μL～50μL的双蒸水，0μL～2μL的机器荧光补偿及矫正剂，0.01～100μM的ALOX15基因的上游引物，0.01～100μM的ALOX15基因的下游引物，0.01～100μM的内参基因的上游引物，0.01～100μM的内参基因的下游引物；进一步优选的，采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因进行实时荧光定量PCR反应的试剂包括：5μL的PCR预混合液，0μL～10μL的双蒸水(根据总体积用水补齐至10μL)，0.2μL的机器荧光补偿及矫正剂，1μM的ALOX15基因的上游引物，1μM的ALOX15基因的下游引物，1μM的内参基因的上游引物，1μM的内参基因的下游引物。针对上述限定的数值范围，均能够实现定量聚合酶链反应将cDNA中的 ALOX15基因和内参基因进行实时荧光定量PCR扩增的步骤，且对于本领域技术人员来说能够根据实际需要进行选择。
具体的，针对从鼻息肉组织中提取RNA的试剂可选择以下两种试剂，第一种包括：包括0.1mL～20mL的RNA抽提液Trizol或RNAiso Blood或RNAiso Plus或其它含有苯酚、异硫氰酸胍、8-羟基喹啉、异硫氰酸胍或β-巯基乙醇的物质，所述Trizol或所述RNAiso Blood或所述RNAiso Plus或所述其它含有苯酚、异硫氰酸胍、8－羟基喹啉、异硫氰酸胍或β-巯基乙醇的物质的体积的0.1～0.5倍的氯仿，所述氯仿体积的0.5～3倍的异丙醇，所述异丙醇体积的0.5～5倍的65％至90％的乙醇，以及0.01mL至5mL的去RNA酶及去DNA酶的水；
另外一种包括：包括0.1mL～2mL的用于裂解细胞和抑制RNA降解的细胞裂解液、0.1mL～0.7mL的洗涤用第一缓冲液、0.1mL～0.7mL的洗涤用第二缓冲液、0.01mL～1mL的去RNA酶及去DNA酶的水、0～10μL的去除基因组DNA的重组DNA酶、0～10μL的去除基因组DNA的DNA酶缓冲液、20～100μL的去RNA酶的双蒸水，所述从鼻息肉组织中提取RNA的工具包括RNA纯化柱；或者包括0.1mL～2mL的用于裂解细胞和抑制RNA降解的细胞裂解液、0.1mL～0.7mL的洗涤用第一缓冲液、0.1mL～0.7mL的洗涤用第二缓冲液、0.01mL～1mL的去RNA酶及去DNA酶的水，所述从鼻息肉组织中提取RNA的工具包括基因组DNA吸附柱和RNA纯化柱。
进一步优选的，针对从鼻息肉组织中提取RNA的试剂可选择以下两种试剂，第一种包括：1mL的RNA抽提液Trizol或RNAiso Blood或RNAiso Plu s或其它含有苯酚、异硫氰酸胍、8-羟基喹啉、异硫氰酸胍或β-巯基乙醇的物质，200μL的氯仿，200μL的异丙醇，200μL体积浓度为65％至90％的乙醇，以及0.02mL的去RNA酶及去DNA酶的水；其中RNA抽提液为Trizol、RNAiso Blood和RNAiso Plus的名称均为商品名。
另外一种包括：包括300μL的用于裂解细胞和抑制RNA降解的细胞裂解液、500μL的洗涤用第一缓冲液、600μL的洗涤用第二缓冲液、0.02mL的去RNA酶及去DNA酶的水、4μL的去除基因组DNA的重组DNA酶、5μL 10×去除基因组DNA的DNA酶缓冲液、41μL的去RNA酶的双蒸水，所述从鼻息肉组织中提取RNA的工具包括RNA纯化柱；或者包括300μL的用于裂解细胞和抑制RNA降解的细胞裂解液、500μL的洗涤用第一缓冲液、600μL的洗涤用第二缓冲液、0.02mL的去RNA酶及去DNA酶的水，所述从鼻息肉组织中提取RNA的工具包括基因组DNA吸附柱和RNA纯化柱。针对上述限定的数值范围，均能够实现从鼻息肉组织中提取RNA的步骤，且对于本领域技术人员来说能够根据实际需要进行选择。
上述中的所述细胞裂解液用于迅速破碎细胞并抑制细胞释放出的核酸酶的物质，第一缓冲液用于去除吸附有RNA的纯化柱的杂质，第二缓冲液用于去除吸附有RNA的纯化柱的杂质和盐分，去RNA酶及去DNA酶的水用于溶解RNA。
另外上述中的从鼻息肉组织中提取RNA的试剂；将总RNA逆转录为cDNA的试剂；采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因进行实时荧光定量PCR反应的试剂分别独立包装。
在一可选实施方式中，所述鼻息肉组织为鼻腔病理活检获得的鼻息肉组织，或者所述鼻黏膜脱落细胞为刷取或粘取鼻息肉表面获取的鼻息肉细胞。其中采用刷取或粘取方式，避免了对患者造成创面，提高了患者检查的安全性，且操作更便捷，节约了人力成本和就医成本。
在一可选实施方式中，选用△Ct(Ct(ALOX15)-Ct(GAPDH))分析法分析扩增产物的数据结果，且与所述△Ct进行比较的界定值为1.675。所限定该界定值，能够使本公开提供的试剂盒在检测慢性鼻窦炎伴鼻息肉亚型时的准确率达到75％以上。
本公开实施方式还提供一种ALOX15基因作为生物标志物在制备用于检测慢性鼻窦炎伴鼻息肉亚型的产品中的应用。其中的所述产品可以为检测试剂、芯片或试剂盒。虽上述实施方式仅说明了试剂盒 的具体技术内容，但对于本领域技术人员来说，在公开本申请的技术方案的基础上，结合公知常识能够直接获取得到检测试剂和芯片产品的具体技术内容。
本公开实施方式提供了一种检测鼻腔脱落细胞中ALOX15基因表达量的方法，包括以下步骤：从鼻腔脱落细胞中提取RNA，将总RNA逆转录为cDNA，采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因分别采用ALOX15基因的特异性引物和内参基因的特异性引物进行实时荧光定量PCR扩增，基于扩增产物的检测结果计算ALOX15基因表达量。
本公开通过蛋白质组学和转录组学方法经大量的创造性实验筛选得到通过计算ALOX15基因表达量来检测伴有鼻息肉的慢性鼻窦炎亚型，且提供的针对ALOX15基因表达量的计算方法简便可靠，准确性高。目前现有的技术中尚未提供相应的任何报道。其中针对ALOX15基因为已知基因，基因ID为246，其DNA序列如SEQ ID NO:1所示，基因NM号为001140.4。
在一可选实施方式中，ALOX15基因的上游引物如SEQ ID NO:2所示，ALOX15基因的下游引物如SEQ ID NO:3所示。针对ALOX15基因的上游引物和下游引物的设计，敏感性更高，在进行检测ALOX15基因表达量时，使结果更准确，重复性更好。
在一可选实施方式中，所述内参基因为GAPDH，所述内参基因的上游引物如SEQ ID NO:4所示，所述下游引物如SEQ ID NO:5所示。针对该处特征的设计，结合上述ALOX15基因的上游引物和ALOX15基因的下游引物，获取合适的ΔCT值，以进行ALOX15基因表达量计算，且具有较高的准确率。
在一可选实施方式中，所述鼻腔脱落细胞采用毛刷于鼻息肉表面获取，且将获取鼻腔脱落细胞后的毛刷置于细胞裂解液中于4℃以下保存。本公开的方法基于该种方式避免了对患者造成创面，提高了患者检查的安全性，且操作更便捷，节约了人力成本和就医成本。
在一可选实施方式中，从鼻腔脱落细胞中提取RNA的方法，包括两种方法，其中第一种方法包括以下步骤：
步骤1：将所述鼻腔脱落细胞溶解于100～2000μL细胞裂解液中，并加入等体积的乙醇，混合均匀后加入至RNA纯化柱中，离心处理后，除去收集管中的滤液，将所述RNA纯化柱置于收集管中；
步骤2：向步骤1中获取的所述RNA纯化柱中加入300μL～700μL的第一缓冲液，离心处理后，除去第一滤液；继续向所述RNA纯化柱加入400μL～800μL第二缓冲液，离心处理后，除去第二滤液，取RNA纯化柱经洗脱得到RNA。
作为优选，所述从鼻腔脱落细胞中提取RNA的方法，还包括以下步骤：向除去所述第二滤液后的RNA纯化柱中添加10～100μL DNA酶反应液，经静置处理后，加入300μL～700μL所述第二缓冲液，离心处理后，除去第三滤液，取RNA纯化柱经洗脱后采用分光光度计测量RNA纯度，得到RNA；
所述DNA酶反应液的制备方法包括以下步骤：取DNA酶缓冲液、重组DNA酶，去RNA酶的双蒸水经混合得到DNA酶反应液。优选所述DNA酶反应液的制备方法包括以下步骤：取5μL 10×DNA酶缓冲液、4μL重组DNA酶，41μL去RNA酶的双蒸水经混合得到DNA酶反应液。
作为优选，当进行RNA提取时基因组含量较低或材料起始量较少时，所述从鼻腔脱落细胞中提取RNA的方法，还包括以下步骤：所述步骤1中，所述鼻腔脱落细胞溶解于细胞裂解液中后先加入至基因组DNA吸附柱中取滤液，再向所述滤液中加入等体积的乙醇。
作为优选，为了获取更高浓度的RNA，所述从鼻腔脱落细胞中提取RNA的方法，还包括以下步骤：取待洗脱的RNA纯化柱加入去RNA水解酶的蒸馏水或焦碳酸二乙酯处理水，室温静置后，经离心处理、洗脱所述RNA纯化柱，采用分光光度计测量RNA纯度，得到RNA。
其中步骤1采用细胞裂解液能够迅速破碎鼻腔脱落细胞并抑制鼻腔脱落细胞释放出的核酸酶的物质；采用基因组DNA吸附柱用于去除基因组DNA；步骤2中的RNA纯化柱用于富集RNA；其中收集管 用于收集去除基因组DNA后的溶液，用于去除吸附有RNA的纯化柱的杂质的第一缓冲液、用于去除RNA溶液中的杂质和盐分的第二缓冲液。
具体的，所述从鼻腔脱落细胞中提取RNA的第一种方法，包括以下步骤：
步骤1：将所述鼻腔脱落细胞溶解于300μL细胞裂解液中，并加入等体积的70％乙醇，使用移液枪将溶液混合均匀；立即将混合液加入至RNA纯化柱中，12000转/分钟，离心1min，除去滤液，将所述RNA纯化柱置于2mL收集管中；
步骤2：向步骤1中获取的所述RNA纯化柱中加入500μL的第一缓冲液，12000转/分钟，离心30s，除去第一滤液；继续向所述RNA纯化柱加入600μL第二缓冲液，12000转/分钟，离心30s，除去第二滤液；
步骤3：取5μL 10×DNA酶缓冲液、4μL重组DNA酶，41μL去RNA酶的双蒸水经混合得到DNA酶反应液，向除去所述第二滤液后的RNA纯化柱中添加50μL DNA酶反应液，室温静置15分钟，加入350μL所述第二缓冲液，12000转/分钟，离心30s，除去第三滤液；
步骤4：将步骤3中除去第三滤液的RNA纯化柱安置于1.5mL无RNA水解酶收集管，向RNA纯化柱加入50μL的去RNA水解酶的蒸馏水或0.1％焦碳酸二乙酯处理水，室温静置5分钟，12000转/分钟，离心2min，洗脱RNA纯化柱，采用分光光度计测量RNA溶液的OD260/OD280比值为1.7～2.1时，得到RNA。
或者所述从鼻腔脱落细胞中提取RNA的方法，包括以下步骤：
步骤1：取基因组DNA吸附柱置于2mL收集管内，将所述鼻腔脱落细胞溶解于100～2000μL细胞裂解液中后加入至基因组DNA吸附柱中，取滤液，向所述滤中加入等体积的70％乙醇，混合均匀后加入至RNA纯化柱中，12000转/分钟，离心1min，除去滤液，将所述RNA纯化柱置于2mL收集管中；
步骤2：向步骤1中获取的所述RNA纯化柱中加入500μL的第一缓冲液，12000转/分钟，离心30s，除去第一滤液；继续向所述RNA纯化柱加入600μL第二缓冲液，12000转/分钟，离心30s，除去第二滤液；
步骤3：将步骤2中除去第二滤液的RNA纯化柱安置于1.5mL无RNA水解酶收集管，向RNA纯化柱加入50μL的去RNA水解酶的蒸馏水或0.1％焦碳酸二乙酯处理水，室温静置5分钟，12000转/分钟，离心2min，洗脱RNA纯化柱，采用分光光度计测量RNA溶液的OD260/OD280比值为1.7～2.1时，得到RNA。
其中步骤1采用细胞裂解液能够迅速破碎鼻腔脱落细胞并抑制鼻腔脱落细胞释放出的核酸酶的物质；步骤1采用基因组DNA吸附柱用于去除基因组DNA；步骤2中的RNA纯化柱用于富集RNA；其中收集管用于收集去除基因组DNA后的溶液，用于去除吸附有RNA的纯化柱的杂质的第一缓冲液、用于去除RNA溶液中的杂质和盐分的第二缓冲液。
在一可选实施方式中，从鼻腔脱落细胞中提取RNA的第二种方法，包括以下步骤：向装有鼻腔脱落细胞的离心管中加入0.1mL～20mL RNA抽提液进行溶解、振荡后加入所述RNA抽提液体积的0.1～0.5倍的氯仿，震荡混匀，室温静置，将所述离心管离心，取上清液，加入所述氯仿体积的0.5～3倍的异丙醇，混匀后静置、离心，弃上清，保留第一沉淀，向所述第一沉淀中加入所述异丙醇体积的0.5～5倍的浓度65％～90％的乙醇，经洗涤后混匀、离心，弃上清，保留第二沉淀；盖紧所述离心管，再次离心，除去上清液，继续向所述离心管中加入0.01～5mL去RNA酶及去DNA酶的水溶解所述第二沉淀，采用分光光度计测量RNA纯度，得到RNA。
优选所述RNA抽提液为Trizol、RNAiso Blood、RNAiso Plus或其它含有苯酚、异硫氰酸胍、8－羟基喹啉、异硫氰酸胍和β-巯基乙醇中的任一种或几种的试剂。
在一优选实施方式中，所述从鼻腔脱落细胞中提取RNA的第二种方法，包括以下步骤：向装有鼻 腔脱落细胞的离心管中加入0.1～20mLRNA抽提液进行溶解、振荡后，室温静置3～7min；加入40μL～5mL氯仿，震荡混匀，室温静置3～7min，于3℃～5℃，10000～14000r/min离心10～20min；取上清液40μL～8mL，加入等体积的异丙醇，混匀后静置8～12min，于3℃～5℃，10000～14000r/min离心10～20min弃上清，保留第一沉淀；向所述第一沉淀中加入与异丙醇等体积的浓度65％～90％的乙醇，于3℃～5℃，7000～14000r/min离心10～20min，弃上清，保留第二沉淀；盖紧所述离心管，于3℃～5℃，7000～14000r/min离心1～3min，除去上清液，静置10～20min后继续向所述离心管中加入0.01～5mL去RNA酶及去DNA酶的水溶解所述第二沉淀，采用分光光度计测量RNA纯度，得到RNA。
具体的，从鼻腔脱落细胞中提取RNA的第二种方法，包括以下步骤：向装有鼻腔脱落细胞的离心管中加入1mLRNA抽提液进行溶解、振荡后，室温静置5min；加入200μL氯仿，震荡混匀，室温静置5min，于4℃，12000r/min离心15min；取上清液200μL，加入200μL异丙醇，混匀后静置10min，于4℃，12000r/min离心15min弃上清，保留第一沉淀；向所述第一沉淀中加入与异丙醇等体积的浓度75％的乙醇，于4℃，7500r/min离心15min，弃上清，保留第二沉淀；盖紧所述离心管，于4℃，7500r/min离心2min，除去上清液，静置15min后继续向所述离心管中加入50μL去RNA酶及去DNA酶的水溶解所述第二沉淀，采用分光光度计测量RNA溶液OD260/OD280比值为1.7～2.1，得到RNA。
作为优选，将总RNA逆转录为cDNA的方法包括以下步骤：取1～3μL的逆转录混合液、0～10μL的去RNA水解酶蒸馏水和提取的RNA于37℃温度条件下发生反转录反应15min，后于84℃温度条件下发生反转录酶的失活反应，得到逆转录产物cDNA。
在一可选实施方式中，将总RNA逆转录为cDNA的方法包括以下步骤：取2μL的所述逆转录混合液，8μL的所述去RNA水解酶蒸馏水，以及总量不超过500ng或体积不超过8μL的总RNA，所述去RNA水解酶蒸馏水补齐至10μL；轻柔混匀后进行逆转录反应,条件如下:在37℃的条件下，进行15分钟的反转录反应；在85℃的条件下，进行5秒的反转录酶的失活反应；产物4℃放置。
在一可选实施方式中，所述实时荧光定量PCR扩增包括以下步骤：
步骤1：制备实时荧光定量PCR反应液：包括1μL～25μL的PCR预混合液，0μL～10μL的双蒸水补齐总体积用水至10μL，0μL～2μL的机器荧光补偿及矫正剂，0.01～100μM的ALOX15基因的上游引物，0.01～100μM的ALOX15基因的下游引物，0.01～100μM的内参基因的上游引物，0.01～100μM的内参基因的下游引物，0.01μL～5μL的cDNA；
步骤2：采用两步法PCR扩增标准程序或三步法PCR扩增标准程序进行实时荧光定量PCR检测；
步骤3：计算ALOX15基因表达量。
具体的，制备实时荧光定量PCR反应液：包括5μL的PCR预混合液，2.8μL的双蒸水补齐总体积用水至10μL，0.2μL的机器荧光补偿及矫正剂，0.5μL的ALOX15基因的上游引物，0.5μL的ALOX15基因的下游引物，0.5μL的内参基因的上游引物，0.5μL的内参基因的下游引物，1ng/μL的所述cDNA。
在一可选实施方式中，所述两步法PCR扩增标准程序的反应条件包括以下步骤：第1阶段：在95℃的条件下预变性30秒；第2阶段PCR反应：在95℃的条件下，反应15秒，在60℃的条件下，反应60秒，退火延伸，如此进行40个循环；
所述三步法PCR扩增标准程序的反应条件包括以下步骤：第1阶段：在95℃的条件下预变性2分钟；第2阶段PCR反应：在95℃的条件下，反应1分钟，在55℃的条件下，反应1分钟，在72℃的条件下，反应1分钟，如此进行40个循环；最后72℃，7分钟退火延伸；
计算所述ALOX15基因表达量的方法为：计算ΔCT或2 -ΔΔCT来计算目的基因的表达量；其中ΔCT＝(CT(ALOX15)-CT(GAPDH))，-ΔΔCT＝-(ΔCT(处理标本CT(ALOX15)-CT(GAPDH))-健康对照组平均ΔCT)；健康对照组平均ΔCT＝∑每个对照组ΔCT(CT(ALOX15)-CT(GAPDH))/对照组 样本数。采用ΔCT法或2 -ΔΔCt法的相对定量法，选择了表达量相对恒定的内参基因，用内参基因的数量进行标准化，通过测定样品目的基因与内参基因的Ct值差异计算目的基因表达量，方法简便快速，检测精度高，可降低检测成本，节约检测时间。结果便于判读等优点。大大提升了实验效率。
具体的针对同一受试者ALOX15与内参基因的表达量差异，内参基因为体内表达较稳定的基因，通常不会随疾病等变化，因此与内参基因比较能够反映目标基因与内参基因的相对丰度，则可采用ΔCT法。针对不同受试者ALOX15与内参基因的表达量差异则可采用2 -ΔΔCt法。
一种上述检测鼻腔脱落细胞中ALOX15基因表达量的方法在制备用于检测伴有鼻息肉的慢性鼻窦炎亚型的试剂盒中的应用。
为了更清楚详细地介绍本公开实施方式所提供的用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒及ALOX15基因作为生物标志物的应用，以及本发明实施方式所提供的一种检测鼻腔脱落细胞中ALOX15基因表达量的方法及应用，下面将结合具体实施例进行描述。
实施例
实施例1：
一种用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒，包括以下试剂：
从鼻息肉组织中提取RNA的试剂：20mL RNA抽提液Trizol或RNAiso Blood或RNAiso Plus或其它含有苯酚、异硫氰酸胍，8－羟基喹啉、异硫氰酸胍、β-巯基乙醇等物质，能迅速破碎细胞并抑制细胞释放出的核酸酶的物质；2mL的氯仿；20mL的异丙醇；40mL的65～90％乙醇；5mL去RNA酶及去DNA酶的水；
将提取的RNA逆转录为cDNA的试剂：40μL的逆转录混合液(含有逆转录所需要的酶、RNA酶抑制剂、随机的6核苷酸引物、多聚胸腺嘧啶、T重复寡核苷酸、三磷酸脱氧核糖核苷酸混合物、缓冲液等)，160μL去RNA酶及去DNA酶的水；其中去RNA酶及去DNA酶的水用于补齐体系、溶解、稀释RNA；
采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因进行实时荧光定量PCR反应的试剂：25μL预混合液(含有PCR所需要的酶和缓冲液)、0～50μL的双蒸水(根据总体积用水补齐至50μL)，0～2μL的染料(用于进行机器的荧光补偿及矫正)，100μM的ALOX15基因的上游引物，100μM的ALOX15基因的下游引物，100μM的内参基因的上游引物，100μM的内参基因的下游引物，10μg阳性对照，10μg阴性对照，阳性对照是含有ALOX15的质粒，阴性对照是空质粒(质粒载体)。
实施例2：
一种用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒，包括以下试剂：
从鼻息肉组织中提取RNA的试剂：1mLRNA抽提液Trizol或RNAiso Blood或RNAiso Plus或其它含有苯酚、异硫氰酸胍，8－羟基喹啉、异硫氰酸胍、β-巯基乙醇等物质，能迅速破碎细胞并抑制细胞释放出的核酸酶的物质；0.2mL的氯仿；0.2mL的异丙醇；0.2mL的65～90％乙醇；0.05mL去RNA酶及去DNA酶的水；
将提取的RNA逆转录为cDNA的试剂：2μL的逆转录混合液(含有逆转录所需要的酶、RNA酶抑制剂、随机的6核苷酸引物、多聚胸腺嘧啶、T重复寡核苷酸、三磷酸脱氧核糖核苷酸混合物、缓冲液等)，7μL去RNA酶及去DNA酶的水；其中去RNA酶及去DNA酶的水用于补齐体系、溶解、稀释RNA；
采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因进行实时荧光定量PCR反应的试剂：5μL预混合液(含有PCR所需要的酶和缓冲液)、0～10μL的双蒸水(根据总体积用水补齐至10μL)，0～2μL的染料(用于进行机器的荧光补偿及矫正)，50μM的ALOX15基因的上游引物，50μM的ALOX15基因的下游引物，50μM的内参基因的上游引物，50μM的内参基因的下游引物，5μg 阳性对照，5μg阴性对照，阳性对照是含有ALOX15的质粒，阴性对照是空质粒(质粒载体)。
实施例3：
一种用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒，包括以下试剂：
从鼻息肉组织中提取RNA的试剂：0.1mL RNA抽提液Trizol或RNAiso Blood或RNAiso Plus或其它含有苯酚、异硫氰酸胍，8－羟基喹啉、异硫氰酸胍、β-巯基乙醇等物质，能迅速破碎细胞并抑制细胞释放出的核酸酶的物质；0.05mL的氯仿；0.015mL的异丙醇；0.0075mL的65～90％乙醇；0.01mL去RNA酶及去DNA酶的水；
将总RNA逆转录为cDNA的试剂：1μL的逆转录混合液(含有逆转录所需要的酶、RNA酶抑制剂、随机的6核苷酸引物、多聚胸腺嘧啶、T重复寡核苷酸、三磷酸脱氧核糖核苷酸混合物、缓冲液等)，0～10μL去RNA酶及去DNA酶的水；其中去RNA酶及去DNA酶的水用于补齐体系、溶解、稀释RNA；
采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因进行实时荧光定量PCR反应的试剂：1μL预混合液(含有PCR所需要的酶和缓冲液)、0～10μL的双蒸水(根据总体积用水补齐至10μL)，0～2μL的染料(用于进行机器的荧光补偿及矫正)，1μM的ALOX15基因的上游引物，1μM的ALOX15基因的下游引物，1μM的内参基因的上游引物，1μM的内参基因的下游引物，1μg阳性对照，1μg阴性对照，阳性对照是含有ALOX15的质粒，阴性对照是空质粒(质粒载体)。
实施例4：
一种用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒，包括以下试剂和工具：
从鼻息肉组织中提取RNA的试剂：100μL的细胞裂解液(RL缓冲液加入50×二硫苏糖醇(DTT))、用于去除基因组DNA的基因组DNA吸附柱、用于收集去除基因组DNA后溶液的收集管、用于富集RNA的RNA纯化柱、0.1mL用于去除吸附有RNA的纯化柱的杂质的第一缓冲液、0.1mL用于去除RNA溶液中的杂质和盐分的第二缓冲液、用于收集RNA的离心管、0.01mL用于溶解RNA的去RNA酶及去DNA酶的水；
将总RNA逆转录为cDNA的试剂：1μL的逆转录混合液(含有逆转录所需要的酶、RNA酶抑制剂、随机的6核苷酸引物、多聚胸腺嘧啶、T重复寡核苷酸、三磷酸脱氧核糖核苷酸混合物、缓冲液等)，0～10μL去RNA酶及去DNA酶的水；其中去RNA酶及去DNA酶的水用于补齐体系、溶解、稀释RNA；
采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因进行实时荧光定量PCR反应的试剂：25μL预混合液(含有PCR所需要的酶和缓冲液)、0～10μL的双蒸水(根据总体积用水补齐至10μL)，0～2μL的染料(用于进行机器的荧光补偿及矫正)，0.01μM的ALOX15基因的上游引物，0.01μM的ALOX15基因的下游引物，0.01μM的内参基因的上游引物，0.01μM的内参基因的下游引物，1μg阳性对照，1μg阴性对照，阳性对照是含有ALOX15的质粒，阴性对照是空质粒(质粒载体)。
实施例5：
一种用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒，包括以下试剂和工具：
从鼻息肉组织中提取RNA的试剂：0.3mL的细胞裂解液(RL缓冲液加入50×二硫苏糖醇(DTT))、用于富集RNA的RNA纯化柱、0.5mL用于去除吸附有RNA的纯化柱的杂质的第一缓冲液、0.6mL用于去除RNA溶液中的杂质和盐分的第二缓冲液、4μL的去除基因组DNA的重组DNA酶、5μL的去除基因组DNA的DNA酶缓冲液、41μL去RNA酶的双蒸水、用于收集RNA的离心管、0.05mL用于溶解RNA的去RNA酶及去DNA酶的水；
将总RNA逆转录为cDNA的试剂：2μL的逆转录混合液(含有逆转录所需要的酶、RNA酶抑制剂、随机的6核苷酸引物、多聚胸腺嘧啶、T重复寡核苷酸、三磷酸脱氧核糖核苷酸混合物、缓冲液等)，0～10μL去RNA酶及去DNA酶的水；其中去RNA酶及去DNA酶的水用于补齐体系、溶解、稀释RNA；
采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因进行实时荧光定量PCR反应的试 剂：5μL预混合液(含有PCR所需要的酶和缓冲液)、0～10μL的双蒸水(根据总体积用水补齐至10μL)，0～2μL的染料(用于进行机器的荧光补偿及矫正)，10μmol/L的ALOX15基因的上游引物，10μmol/L的ALOX15基因的下游引物，10μmol/L的内参基因的上游引物，10μmol/L的内参基因的下游引物，1μg阳性对照，1μg阴性对照，阳性对照是含有ALOX15的质粒，阴性对照是空质粒(质粒载体)。
实施例6：
一种用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒，包括以下试剂和工具：
从鼻息肉组织中提取RNA的试剂：2mL的细胞裂解液(RL缓冲液加入50×二硫苏糖醇(DTT))、用于去除基因组DNA的基因组DNA吸附柱、用于收集去除基因组DNA后溶液的收集管、用于富集RNA的RNA纯化柱、0.7mL用于去除吸附有RNA的纯化柱的杂质的第一缓冲液、0.7mL用于去除RNA溶液中的杂质和盐分的第二缓冲液、用于收集RNA的离心管、1mL用于溶解RNA的去RNA酶及去DNA酶的水；
将总RNA逆转录为cDNA的试剂：2μL的逆转录混合液(含有逆转录所需要的酶、RNA酶抑制剂、随机的6核苷酸引物、多聚胸腺嘧啶、T重复寡核苷酸、三磷酸脱氧核糖核苷酸混合物、缓冲液等)，0～8μL去RNA酶及去DNA酶的水(根据RNA量用水补齐至8μL)；其中去RNA酶及去DNA酶的水用于补齐体系、溶解、稀释RNA；
采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因进行实时荧光定量PCR反应的试剂：5μL预混合液(含有PCR所需要的酶和缓冲液)、0-10μL的双蒸水(根据总体积用水补齐至10μL)，0～2μL的染料(用于进行机器的荧光补偿及矫正)，1μM的ALOX15基因的上游引物，1μM的ALOX15基因的下游引物，1μM的内参基因的上游引物，1μM的内参基因的下游引物，1μg阳性对照，1μg阴性对照。
上述实施例4～6中，所用的第一缓冲液RWA buffer的生产厂家为Takara公司，货号9767；第二缓冲液RWB buffer的生产厂家为Takara公司，货号9767。
本公开实施例1～6提供的试剂盒均能够实现慢性鼻窦炎伴鼻息肉亚型的检测，现提供如下用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒的具体效果检测实验：
鼻息肉亚型检测效果实验：
实验例一：
1、实验方法：
(1)、样本的收集与处理：
随机选取78名CRSwNP患者采用生理盐水冲洗鼻腔后，在鼻内镜下取鼻息肉。将鼻息肉切割成直径约0.5厘米的组织，浸泡于RNA稳定和储存溶液(RNAlater)中，4℃短期保存，后转移入低于-20℃长期保存。
(2)、RNA的提取：
步骤1：将浸泡于RNA稳定和储存溶液(RNAlater)中的组织称重，称取约0.01g重量的组织放于加磁珠的离心管里，置于液氮中，匀浆机上进行砸磨(3000r，5min)(或者手工研磨)。向装有组织细胞的试管中加入1mL Trizol进行溶解，收集到离心管中，充分振荡，室温静置5分钟；然后加入上述RNA提取试剂组的200μL氯仿(三氯甲烷)，剧烈震荡混匀，室温静置5分钟。
步骤2：12，000转/分钟，4℃，离心15分钟。
步骤3：取上清液，实际获得其体积约200μL，加入离心管，加入上述RNA提取试剂组的等量(约200μL)异丙醇。混匀后静置10分钟，12000转/分钟，4℃，离心15分钟。弃上清，保留沉淀。
步骤4：加入上述RNA提取试剂组的(与异丙醇等量)约200μL的75％乙醇(约150μL无水乙 醇及50μL去DNA酶及RNA酶水的混合物)清洗沉淀，混匀。7500转/分钟，4℃，离心15分钟。弃上清，保留沉淀。
步骤5：盖紧离心管，7500转/分钟，4℃，离心2分钟。
步骤6：开盖，弃上清，静置于通风橱15分钟。
步骤7：加入上述RNA提取试剂组的0.02mL无RNA水解酶和无DNA水解酶(RNase-free和DNase-free)的水溶解沉淀。
步骤8：利用分光光度计测量RNA浓度，且OD260/OD280比值在1.7-2.1之间。
(3)、逆转录制备cDNA：在冰上配制逆转录(RT)反应液，具体包括以下试剂：2μL的逆转录混合液(含有逆转录所需要的酶、RNA酶抑制剂、随机的6核苷酸引物、多聚胸腺嘧啶、T重复寡核苷酸、三磷酸脱氧核糖核苷酸混合物、缓冲液等)，不超过500ng或不超过8μL的总RNA、0～8μL去RNA酶及去DNA酶的水(根据RNA量用水补齐至8μL)；取提取的总RNA和上述的逆转录反应液加入反应体系中进行逆转录反应得到cDNA模板，其中，反应体系可按需求相应放大，10μL反应体系可最大使用500ng的总RNA；
逆转录反应条件如下:
37℃15分钟(反转录反应)
84℃5秒(反转录酶的失活反应)
产物4℃放置。
(4)制备实时荧光定量PCR反应液：包括5μL预混合液(含有PCR所需要的酶和缓冲液)、0.2μL的机器荧光补偿及矫正剂、1ng/μL cDNA或阳性对照或阴性对照、0.5μL ALOX15基因的上游引物、0.5μL ALOX15基因的下游引物、0.5μL内参基因的上游引物、0.5μL内参基因的下游引物和2.8μL的双蒸水；
(5)、实时荧光定量PCR检测：
反应条件：
第1阶段：预变性：95℃，30秒。
第2阶段：PCR反应：95℃，15秒；60℃，1分钟退火延伸，共进行40个循环；第2阶段中：熔解曲线：60℃逐渐升温至95℃，速率为0.1℃/秒，采集荧光；
反应结束后确认实时荧光定量PCR的扩增曲线如图1至图4所示，以及熔解曲线如图5至图8所示，读取ALOX15及GAPDH的Ct值，选用△Ct分析法(ALOX15的Ct值减GAPDH的Ct值)进行分析，采用GAPDH作为内参基因。
(6)、数据分析：
步骤1：实验质控的判断：阳性对照Ct值<20且阴性对照Ct值>38视为实验有效，否则实验无效。
步骤2：分型的判断：目的基因的Ct值减内参基因的Ct值，根据ROC曲线，ALOX15的Ct值的最佳界值为1.675，若ALOX15的Ct值≥1.675，则为非嗜酸性粒细胞型慢性鼻窦炎伴鼻息肉；若ALOX15的Ct值＜1.675，则为典型的嗜酸性粒细胞型慢性鼻窦炎伴鼻息肉。
2、实验结果：选取的78名患者基于上述提供的试剂盒的鼻息肉亚型检测结果如表1所示：
表1 本公开提供的试剂盒及组织病理方式获取鼻息肉组织的鼻息肉亚型检测结果
受试者编号 △Ct值 依据界值判断的结果 病理结果
1 3.029 非嗜酸性粒细胞型 非嗜酸性粒细胞型
2 2.616 非嗜酸性粒细胞型 非嗜酸性粒细胞型
3 3.188 非嗜酸性粒细胞型 非嗜酸性粒细胞型
4 6.052 非嗜酸性粒细胞型 非嗜酸性粒细胞型
5 1.695 非嗜酸性粒细胞型 非嗜酸性粒细胞型
6 1.313 嗜酸性粒细胞型 嗜酸性粒细胞型
7 5.437 非嗜酸性粒细胞型 非嗜酸性粒细胞型
8 1.475 嗜酸性粒细胞型 嗜酸性粒细胞型
9 7.778 非嗜酸性粒细胞型 非嗜酸性粒细胞型
10 4.292 非嗜酸性粒细胞型 非嗜酸性粒细胞型
11 2.284 非嗜酸性粒细胞型 非嗜酸性粒细胞型
12 4.198 非嗜酸性粒细胞型 非嗜酸性粒细胞型
13 3.383 非嗜酸性粒细胞型 非嗜酸性粒细胞型
14 3.684 非嗜酸性粒细胞型 非嗜酸性粒细胞型
15 3.302 非嗜酸性粒细胞型 非嗜酸性粒细胞型
16 4.144 非嗜酸性粒细胞型 非嗜酸性粒细胞型
17 3.714 非嗜酸性粒细胞型 非嗜酸性粒细胞型
18 3.556 非嗜酸性粒细胞型 非嗜酸性粒细胞型
19 4.136 非嗜酸性粒细胞型 非嗜酸性粒细胞型
20 2.186 非嗜酸性粒细胞型 非嗜酸性粒细胞型
21 4.062 非嗜酸性粒细胞型 非嗜酸性粒细胞型
22 4.420 非嗜酸性粒细胞型 非嗜酸性粒细胞型
23 0.313 嗜酸性粒细胞型 嗜酸性粒细胞型
24 2.999 非嗜酸性粒细胞型 非嗜酸性粒细胞型
25 2.892 非嗜酸性粒细胞型 非嗜酸性粒细胞型
26 3.048 非嗜酸性粒细胞型 非嗜酸性粒细胞型
27 2.152 非嗜酸性粒细胞型 非嗜酸性粒细胞型
28 0.387 嗜酸性粒细胞型 嗜酸性粒细胞型
29 1.547 嗜酸性粒细胞型 嗜酸性粒细胞型
30 2.401 非嗜酸性粒细胞型 嗜酸性粒细胞型
31 1.655 嗜酸性粒细胞型 嗜酸性粒细胞型
32 0.547 嗜酸性粒细胞型 嗜酸性粒细胞型
33 0.282 嗜酸性粒细胞型 嗜酸性粒细胞型
34 1.829 非嗜酸性粒细胞型 嗜酸性粒细胞型
35 -0.895 嗜酸性粒细胞型 嗜酸性粒细胞型
36 2.152 非嗜酸性粒细胞型 嗜酸性粒细胞型
37 3.272 非嗜酸性粒细胞型 嗜酸性粒细胞型
38 2.537 非嗜酸性粒细胞型 嗜酸性粒细胞型
39 3.968 非嗜酸性粒细胞型 嗜酸性粒细胞型
40 3.158 非嗜酸性粒细胞型 嗜酸性粒细胞型
41 0.183 嗜酸性粒细胞型 嗜酸性粒细胞型
42 -0.933 嗜酸性粒细胞型 非嗜酸性粒细胞型
43 -0.021 嗜酸性粒细胞型 嗜酸性粒细胞型
44 0.296 嗜酸性粒细胞型 嗜酸性粒细胞型
45 -1.137 嗜酸性粒细胞型 嗜酸性粒细胞型
46 3.202 非嗜酸性粒细胞型 嗜酸性粒细胞型
47 0.442 嗜酸性粒细胞型 嗜酸性粒细胞型
48 0.765 嗜酸性粒细胞型 嗜酸性粒细胞型
49 2.253 非嗜酸性粒细胞型 嗜酸性粒细胞型
50 0.769 嗜酸性粒细胞型 嗜酸性粒细胞型
51 2.422 非嗜酸性粒细胞型 嗜酸性粒细胞型
52 0.749 嗜酸性粒细胞型 嗜酸性粒细胞型
53 -3.834 嗜酸性粒细胞型 嗜酸性粒细胞型
54 1.930 非嗜酸性粒细胞型 嗜酸性粒细胞型
55 0.767 嗜酸性粒细胞型 嗜酸性粒细胞型
56 0.778 嗜酸性粒细胞型 嗜酸性粒细胞型
57 0.061 嗜酸性粒细胞型 嗜酸性粒细胞型
58 4.640 非嗜酸性粒细胞型 嗜酸性粒细胞型
59 -0.547 嗜酸性粒细胞型 嗜酸性粒细胞型
60 0.019 嗜酸性粒细胞型 嗜酸性粒细胞型
61 0.355 嗜酸性粒细胞型 嗜酸性粒细胞型
62 0.251 嗜酸性粒细胞型 嗜酸性粒细胞型
63 0.589 嗜酸性粒细胞型 嗜酸性粒细胞型
64 3.664 非嗜酸性粒细胞型 嗜酸性粒细胞型
65 2.454 非嗜酸性粒细胞型 嗜酸性粒细胞型
66 0.224 嗜酸性粒细胞型 嗜酸性粒细胞型
67 3.117 非嗜酸性粒细胞型 嗜酸性粒细胞型
68 1.637 嗜酸性粒细胞型 嗜酸性粒细胞型
69 1.489 嗜酸性粒细胞型 嗜酸性粒细胞型
70 2.073 非嗜酸性粒细胞型 嗜酸性粒细胞型
71 1.317 嗜酸性粒细胞型 嗜酸性粒细胞型
72 0.206 嗜酸性粒细胞型 嗜酸性粒细胞型
73 -1.086 嗜酸性粒细胞型 嗜酸性粒细胞型
74 -0.012 嗜酸性粒细胞型 嗜酸性粒细胞型
75 1.609 嗜酸性粒细胞型 嗜酸性粒细胞型
76 -3.187 嗜酸性粒细胞型 嗜酸性粒细胞型
77 0.732 嗜酸性粒细胞型 嗜酸性粒细胞型
78 1.152 嗜酸性粒细胞型 嗜酸性粒细胞型
上述78例样本的ROC曲线图如图9所示，采用本试剂盒对慢性鼻窦炎伴鼻息肉分型的预测，准确率为78.2％。
实验例二：
1、实验方法：
(1)、样本的收集与处理：
随机选取47名CRSwNP患者用生理盐水冲洗鼻腔后，在鼻内镜下用毛刷(Copan公司产)于鼻息肉表面按压30s，旋转3-4圈，刷取息肉表面，将毛刷置于裂解液中，4℃短期保存(不超过24小时)，或转移入低于-20℃长期保存。
(2)、RNA的提取：
步骤1：向装有脱落细胞的试管中加入1mL Trizol进行溶解，充分振荡，室温静置5分钟，然后加入200μL氯仿(三氯甲烷)，剧烈震荡混匀，室温静置5分钟；
步骤2：12，000转/分钟，4℃，离心15分钟；
步骤3：取上清液，实际获得其体积约200μL，加入离心管，加入与氯仿等量(约200μL)的异丙醇。混匀后静置10分钟，12000转/分钟，4℃，离心15分钟。弃上清，保留沉淀；
步骤4：加入上述RNA提取试剂组的等量(与异丙醇等量)约200μL的75％乙醇(约150μL无水乙醇及50μL去DNA酶及RNA酶水的混合物)清洗沉淀，混匀，7500转/分钟，4℃，离心15分钟，弃上清，保留沉淀；
步骤5：盖紧离心管，7500转/分钟，4℃，离心2分钟；
步骤6：开盖，弃上清，静置于通风橱15分钟；
步骤7：加入上述RNA提取试剂组的0.02mL无RNA水解酶和无DNA水解酶(RNase-free和DNase-free)的水溶解沉淀；
步骤8：利用分光光度计测量RNA浓度，OD260/OD280比值在1.7～2.1为好；
(3)、逆转录制备cDNA：同实验例一；
(4)、制备实时荧光定量PCR反应液：同实验例一；
(5)、实时荧光定量PCR检测：同实验例一；反应结束后确认实时荧光定量PCR的扩增曲线如图10至图12所示，以及熔解曲线如图13至图15所示；
(6)、数据分析：目的基因的Ct值减内参基因的Ct值，根据前期试验结果，ALOX15的Ct值的最佳界值为1.675，若ALOX15的Ct值≥1.675，则为非嗜酸性粒细胞型慢性鼻窦炎伴鼻息肉；若ALOX15的Ct值＜1.675，则为典型的嗜酸性粒细胞型慢性鼻窦炎伴鼻息肉；
2、实验结果：计算结果如表2所示。
表2 本公开提供的试剂盒采用刷取鼻息肉表面获取鼻黏膜脱落细胞的鼻息肉亚型检测结果
受试者编号 △Ct值 依据界值判断的病理结果 病理结果
1 3.413 非嗜酸性粒细胞型 非嗜酸性粒细胞型
2 0.491 嗜酸性粒细胞型 嗜酸性粒细胞型
3 2.77 非嗜酸性粒细胞型 非嗜酸性粒细胞型
4 0.711 嗜酸性粒细胞型 非嗜酸性粒细胞型
5 7.17 非嗜酸性粒细胞型 非嗜酸性粒细胞型
6 1.818 非嗜酸性粒细胞型 非嗜酸性粒细胞型
7 2.952 非嗜酸性粒细胞型 非嗜酸性粒细胞型
8 0.167 嗜酸性粒细胞型 嗜酸性粒细胞型
9 2.014 非嗜酸性粒细胞型 非嗜酸性粒细胞型
10 1.83 非嗜酸性粒细胞型 嗜酸性粒细胞型
11 2.015 非嗜酸性粒细胞型 嗜酸性粒细胞型
12 3.665 非嗜酸性粒细胞型 非嗜酸性粒细胞型
13 1.757 非嗜酸性粒细胞型 非嗜酸性粒细胞型
14 0.218 嗜酸性粒细胞型 非嗜酸性粒细胞型
15 2.329 非嗜酸性粒细胞型 嗜酸性粒细胞型
16 1.107 嗜酸性粒细胞型 嗜酸性粒细胞型
17 0.321 嗜酸性粒细胞型 嗜酸性粒细胞型
18 0.898 嗜酸性粒细胞型 嗜酸性粒细胞型
19 0.394 嗜酸性粒细胞型 嗜酸性粒细胞型
20 0.483 嗜酸性粒细胞型 非嗜酸性粒细胞型
21 0.619 嗜酸性粒细胞型 非嗜酸性粒细胞型
22 0.869 嗜酸性粒细胞型 非嗜酸性粒细胞型
23 1.55 嗜酸性粒细胞型 嗜酸性粒细胞型
24 1.02 嗜酸性粒细胞型 非嗜酸性粒细胞型
25 -0.121 嗜酸性粒细胞型 嗜酸性粒细胞型
26 -0.709 嗜酸性粒细胞型 嗜酸性粒细胞型
27 -0.303 嗜酸性粒细胞型 嗜酸性粒细胞型
28 -0.196 嗜酸性粒细胞型 嗜酸性粒细胞型
29 -1.375 嗜酸性粒细胞型 嗜酸性粒细胞型
30 -2.017 嗜酸性粒细胞型 嗜酸性粒细胞型
31 -0.42 嗜酸性粒细胞型 嗜酸性粒细胞型
32 -2.01 嗜酸性粒细胞型 嗜酸性粒细胞型
33 -1.25 嗜酸性粒细胞型 嗜酸性粒细胞型
34 -1.26 嗜酸性粒细胞型 嗜酸性粒细胞型
35 -0.26 嗜酸性粒细胞型 嗜酸性粒细胞型
36 -2.312 嗜酸性粒细胞型 嗜酸性粒细胞型
37 -1.309 嗜酸性粒细胞型 嗜酸性粒细胞型
38 -0.786 嗜酸性粒细胞型 嗜酸性粒细胞型
39 -1.363 嗜酸性粒细胞型 嗜酸性粒细胞型
40 -0.375 嗜酸性粒细胞型 嗜酸性粒细胞型
41 -0.072 嗜酸性粒细胞型 嗜酸性粒细胞型
42 -0.088 嗜酸性粒细胞型 嗜酸性粒细胞型
43 -0.789 嗜酸性粒细胞型 嗜酸性粒细胞型
44 -0.891 嗜酸性粒细胞型 嗜酸性粒细胞型
45 -0.581 嗜酸性粒细胞型 嗜酸性粒细胞型
46 -1.423 嗜酸性粒细胞型 嗜酸性粒细胞型
47 -1.128 嗜酸性粒细胞型 嗜酸性粒细胞型
由表2提供的数据结果获取到，本公开实验例二中的试剂盒对慢性鼻窦炎伴鼻息肉亚型检测的准确率为80.9％。
实验例三:
1、试验方法：
(1)、样本的收集与处理：
某患者用生理盐水冲洗鼻腔后，在鼻内镜下用毛刷(Copan公司产)于鼻息肉表面按压30s，旋转3-4圈，刷取息肉表面，将毛刷置于裂解液中，4℃短期保存(不超过24小时)，或转移入低于-20℃长期保存。
(2)RNA的提取：
步骤1：将基因组DNA吸附柱(基因组DNA Eraser Spin Column)安放到2mL的收集管(Collection Tube)上；
步骤2：将含脱落细胞的裂解液(细胞裂解液)转移入到基因组DNA吸附柱中；
步骤3：12，000转/分钟，离心1分钟；
步骤4：弃基因组DNA吸附柱，保留2mL收集管中的滤液；
步骤5：向上述步骤4中加入300μL的70％乙醇(此时可能会出现沉淀)，使用移液枪将溶液混合均匀；
步骤6：立即将混合液(含沉淀)全部转入到RNA纯化柱(RNA Spin Column)(含2mL收集管)中；
步骤7：12，000转/分钟，离心1分钟，弃滤液。将RNA纯化柱放回到2mL收集管中；
步骤8：将500μL的第一缓冲液(Buffer RWA)加入至RNA纯化柱中，12，000转/分钟，离心30秒钟，弃滤液；
步骤9：将600μL的第二缓冲液(Buffer RWB)加入至RNA纯化柱中，12，000转/分钟，离心30秒钟，弃滤液。
步骤10：将RNA纯化柱安置于1.5mL的无RNA水解酶收集管(RNase Free Colletion Tube)上，在RNA纯化柱膜中央处加入50μL的无RNA水解酶的蒸馏水(RNase Free dH2O)或0.1％焦碳酸二乙酯(DEPC)处理水，室温静置5分钟；
步骤11：12，000转/分钟离心，去RNA酶及去DNA酶的水洗脱RNA2分钟；
步骤12：利用分光光度计测量RNA浓度，OD260/OD280比值为2.0。
(3)、逆转录制备cDNA：同实验例一；
(4)、制备实时荧光定量PCR反应液：
步骤1：配置SYBR Green 1预混合液45μL；和ROX:1.8μL，混匀后分为3份，分别为A.11.7μL；B.11.7μL；C.23.4μL，分别向A中加入1ng/μL阳性对照得到A溶液，B中加入1ng/μL阴性对照得到B溶液，C中加入2ng获得的cDNA得到C溶液(SYBR Green 1预混合液和ROX均为Takara公司产品，货号RR820A)；
步骤2：配置8组平行孔；
第1、2平行孔：A溶液、ALOX15基因的特异性引物、3.8μL灭菌双蒸水；
第3、4平行孔：B溶液、ALOX15基因的特异性引物、3.8μL灭菌双蒸水；
第5、6平行孔：C溶液、ALOX15基因的特异性引物、3.8μL灭菌双蒸水；
第7、8平行孔：C溶液、GAPDH基因的特异性引物、3.8μL灭菌双蒸水；
步骤3：采用透明胶膜封板，离心，进行PCR操作。
步骤4：两步法PCR扩增标准程序：
(5)、实时荧光定量PCR检测：
反应条件：
第1阶段：预变性：95℃，30秒。
第2阶段：PCR反应：95℃，15秒；60℃，1分钟退火延伸，共进行40个循环。；
反应结束后确认实时荧光定量PCR的扩增曲线如图16所示，以及熔解曲线如图17所示，读取ALOX15及GAPDH的Ct值，选用△Ct分析法(ALOX15的Ct值减GAPDH的Ct值)进行分析，采 用GAPDH作为内参基因。
(6)、数据分析：同实验例一；
2、实验结果：阳性对照孔平均Ct：16.2；阴性对照孔平均Ct：39.7；样品ALOX15平均Ct：19.4；样品GAPDH平均Ct：16.4；差异值：19.4-16.4为3，大于1.675，为非嗜酸性粒细胞型慢性鼻窦炎伴鼻息肉。
实验例四：
本公开实施例1～6提供的试剂盒均能够实现慢性鼻窦炎伴鼻息肉亚型的检测，现以实施例5为例进行如下用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒的效果检测实验：
(1)样本的收集与处理：
某患者用生理盐水冲洗鼻腔后，在鼻内镜下取息肉。将息肉切割成直径约0.5厘米的组织，浸泡于RNA稳定和储存溶液(RNAlater)中，4℃短期保存，后转移入低于-20℃长期保存。
(2)RNA的提取：
步骤1：将浸泡于RNA稳定和储存溶液(RNAlater)中的组织称重，称取约0.01g重量的组织放于加磁珠的离心管里，置于液氮中，匀浆机上进行砸磨(3000r，5min)(或者手工研磨)；加入0.3mL细胞裂解液，12，000转/分钟，离心15分钟；
步骤2：吸取上清，加入与上清液等体积的70％乙醇(70％无水乙醇及30％DEPC或去RNA酶及DNA酶的水)，使用移液枪将溶液混合均匀；
步骤3：立即将混合液(含沉淀)全部转入到RNA纯化柱(含2mL收集管)中；
步骤4：12，000转/分钟，离心1分钟，弃滤液。将RNA纯化放回到2mL收集管中；
步骤5：将500μL的第一缓冲液(Buffer RWA)加入至RNA纯化柱中，12，000转/分钟，离心30秒钟，弃滤液；
步骤6：将600μL的第二缓冲液(Buffer RWB)加入至RNA纯化柱中，12，000转/分钟，离心30秒钟，弃滤液；
步骤7：DNA酶I(DNase I)反应液的配制：取5μL 10×DNA酶I缓冲液，4μL重组DNA酶I(Recombinant DNase I，(无RNA酶，5U/μL)，41μL无RNA酶的双蒸水到新的1.5mL管(无RNA酶)中，混合均匀；
步骤8：向RNA纯化柱膜中央加入50μL DNase I反应液，室温静置15分钟；
步骤9：向RNA纯化柱膜中央加入350μL的第二缓冲液，12，000转/分钟，离心30秒钟，弃滤液；
步骤10：重复步骤6；
步骤11：将RNA纯化柱重新安置于2mL收集管上，12，000转/分钟，离心2分钟；
步骤12：将RNA纯化柱安置于1.5mL的无RNA水解酶收集管(RNase Free Colletion Tube)上，在RNA纯化柱膜中央处加入50μL的无RNA水解酶的蒸馏水(RNase Free dH2O)或0.1％焦碳酸二乙酯(DEPC)处理水，室温静置5分钟；
步骤13：12，000转/分钟离心，去RNA酶及去DNA酶的水洗脱RNA2分钟；
步骤14：利用分光光度计测量RNA浓度，OD260/OD280比值为2.0；
(3)逆转录制备cDNA：同实验例一；
(4)制备实时荧光定量PCR反应液：同实验例三；
(5)、实时荧光定量PCR检测：
反应条件采用三步法PCR扩增标准程序：
第1阶段：预变性：95℃，2分钟；
第2阶段：PCR反应：95℃，1分钟；55℃，1分钟,72℃1分钟，共进行40个循环，最后72℃，7分钟退火延伸；
反应结束后确认实时荧光定量PCR的扩增曲线如图18所示，以及熔解曲线如图19所示，读取ALOX15及GAPDH的Ct值，选用△Ct分析法(ALOX15的Ct值减GAPDH的Ct值)进行分析，采用GAPDH作为内参基因。
(6)数据分析：同实验例一；
2、实验结果：阳性对照孔平均Ct：16.7；阴性对照孔平均Ct：38.4；样品ALOX15平均Ct：19.2；样品GAPDH平均Ct：16.4；差异值：为2.8，大于1.675，为非嗜酸性粒细胞型慢性鼻窦炎伴鼻息肉。
综上所述，本公开提供一种用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒，筛选采用ALOX15基因作为生物标志物，将其应用到试剂盒中，以实现采用试剂盒进行检测慢性鼻窦炎伴鼻息肉亚型的方法，通过试剂盒能够快速、准确、全面的针对患者鼻息肉亚型进行准确鉴别，以尽早根据鼻息肉的炎症亚型进行针对化治疗，有效指导针对慢性鼻窦炎伴鼻息肉患者的药物治疗方式及手术方式的确定，准确预估药物治疗的反应、判断预后效果。
实施例7
样本的收集与处理：
某患者用生理盐水冲洗鼻腔后，在鼻内镜下用毛刷(Copan公司产)于鼻息肉表面按压30s,旋转3～4圈，刷取息肉表面，将毛刷置于由裂解液，4℃短期保存(不超过24小时)，或转移入低于-20℃长期保存。
一种检测鼻腔脱落细胞中ALOX15基因表达量的方法，包括以下步骤：
步骤一：从鼻腔脱落细胞中提取RNA：
步骤1：取基因组DNA吸附柱置于2mL收集管内，将所述鼻腔脱落细胞溶解于300μL细胞裂解液中后加入至基因组DNA吸附柱中，取滤液，向所述滤中加入等体积的70％乙醇，混合均匀后加入至RNA纯化柱中，12000转/分钟，离心1min，除去滤液，将所述RNA纯化柱置于2mL收集管中；
步骤2：向步骤1中获取的所述RNA纯化柱中加入500μL的第一缓冲液，12000转/分钟，离心30s，除去第一滤液；继续向所述RNA纯化柱加入600μL第二缓冲液，12000转/分钟，离心30s，除去第二滤液；
步骤3：将步骤2中除去第二滤液的RNA纯化柱安置于1.5mL无RNA水解酶收集管，向RNA纯化柱加入50μL的去RNA水解酶的蒸馏水或0.1％焦碳酸二乙酯处理水，室温静置5分钟，12000转/分钟，离心2min，洗脱RNA纯化柱，采用分光光度计测量RNA溶液的OD260/OD280比值为2.0，得到RNA；
步骤二：逆转录制备cDNA，包括以下步骤：取2μL的所述逆转录混合液，0～8μL的所述去RNA水解酶蒸馏水(根据RNA量用水补齐至8μL)，以及总量不超过500ng或体积不超过8μL的总RNA，所述去RNA水解酶蒸馏水补齐至10μL；轻柔混匀后进行逆转录反应,条件如下:在37℃的条件下，进行15分钟的反转录反应；在85℃的条件下，进行5秒的反转录酶的失活反应；产物4℃放置。
步骤三：实时荧光定量PCR扩增检测，包括以下步骤：
步骤1：制备实时荧光定量PCR反应液：包括1μL的PCR预混合液，0-10μL的双蒸水(根据总体积用水补齐至10μL)，0.2μL的机器荧光补偿及矫正剂，1μM的ALOX15基因的上游引物，1μM的ALOX15基因的下游引物，1μM的内参基因的上游引物，1μM的内参基因的下游引物，0.01μL的所述cDNA，1μg的阳性对照，1μg的阴性对照，阳性对照是含有ALOX15的质粒，阴性对照是空质粒(质粒载体)；
步骤2：采用两步法PCR扩增标准程序：所述两步法PCR扩增标准程序的反应条件包括以下步骤： 第1阶段：在95℃的条件下预变性30秒；第2阶段PCR反应：在95℃的条件下，反应15秒，在60℃的条件下，反应60秒，退火延伸，如此进行40个循环。
步骤四：计算所述ALOX15基因表达量：
反应结束后确认实时荧光定量PCR的扩增曲线和熔解曲线,读取ALOX15及GAPDH的Ct值，选用△Ct分析法(ALOX15的Ct值减GAPDH的Ct值)进行分析，采用GAPDH作为内参基因；阳性对照Ct值<20且阴性对照Ct值>38视为实验有效，否则实验无效；
采用ΔCT方法比较ALOX15与内参基因的表达量差异：得ALOX15的平均CT值为20.9，GAPDH的平均CT值为18.9，ΔCT值为2.0。
实施例8：
样本的收集与处理：
某患者用生理盐水冲洗鼻腔后，在鼻内镜下用毛刷(Copan公司产)于鼻息肉表面按压30s,旋转3～4圈，刷取息肉表面，将毛刷置于由裂解液，4℃短期保存(不超过24小时)，或转移入低于-20℃长期保存。
一种检测鼻腔脱落细胞中ALOX15基因表达量的方法，包括以下步骤：
步骤一：从鼻腔脱落细胞中提取RNA：
步骤1：取基因组DNA吸附柱置于2mL收集管内，将所述鼻腔脱落细胞溶解于100μL细胞裂解液中后加入至基因组DNA吸附柱中，于12000转/min的条件下离心60s，取滤液，向所述滤中加入等体积的70％乙醇，混合均匀后加入至RNA纯化柱中，12000转/分钟，离心1min，除去滤液，将所述RNA纯化柱置于2mL收集管中；
步骤2：向步骤1中获取的所述RNA纯化柱中加入300μL的第一缓冲液，12000转/分钟，离心30s，除去第一滤液；继续向所述RNA纯化柱加入400μL第二缓冲液，12000转/分钟，离心30s，除去第二滤液；
步骤3：将步骤2中除去第二滤液的RNA纯化柱安置于1.5mL无RNA水解酶收集管，向RNA纯化柱加入50μL的去RNA水解酶的蒸馏水或0.1％焦碳酸二乙酯处理水，室温静置5分钟，12000转/分钟，离心2min，洗脱RNA纯化柱，采用分光光度计测量RNA溶液的OD260/OD280比值为2.0，得到RNA；
步骤二：逆转录制备cDNA，包括以下步骤：取1μL的所述逆转录混合液，0～10μL的所述去RNA水解酶蒸馏水，以及总量不超过500ng或体积不超过8μL的总RNA，所述去RNA水解酶蒸馏水补齐至10μL；轻柔混匀后进行逆转录反应,条件如下:在37℃的条件下，进行15分钟的反转录反应；在85℃的条件下，进行5秒的反转录酶的失活反应；产物4℃放置。
步骤三：实时荧光定量PCR扩增检测，包括以下步骤：
步骤1：制备实时荧光定量PCR反应液：包括25μL的PCR预混合液，0～10μL的双蒸水(根据总体积用水补齐至10μL)，0～2μL的染料(用于进行机器的荧光补偿及矫正)，0.01μM的ALOX15基因的上游引物，0.01μM的ALOX15基因的下游引物，0.01μM的内参基因的上游引物，0.01μM的内参基因的下游引物，5μL的cDNA，1μg阳性对照，1μg阴性对照，阳性对照是含有ALOX15的质粒，阴性对照是空质粒(质粒载体)；
步骤2：采用两步法PCR扩增标准程序：所述两步法PCR扩增标准程序的反应条件包括以下步骤：第1阶段：在95℃的条件下预变性30秒；第2阶段PCR反应：在95℃的条件下，反应15秒，在60℃的条件下，反应60秒，退火延伸，如此进行40个循环。
步骤四：计算所述ALOX15基因表达量：
反应结束后确认实时荧光定量PCR的扩增曲线和熔解曲线,读取ALOX15及GAPDH的Ct值，选 用△Ct分析法(ALOX15的Ct值减GAPDH的Ct值)进行分析，采用GAPDH作为内参基因；阳性对照Ct值<20且阴性对照Ct值>38视为实验有效，否则实验无效；
采用ΔCT方法比较ALOX15与内参基因的表达量差异：得ALOX15的平均CT值为19.5，GAPDH的平均CT值为18.0，ΔCT值为1.5。
实施例9：
样本的收集与处理：
某患者用生理盐水冲洗鼻腔后，在鼻内镜下用毛刷(Copan公司产)于鼻息肉表面按压30s,旋转3～4圈，刷取息肉表面，将毛刷置于由裂解液，4℃短期保存(不超过24小时)，或转移入低于-20℃长期保存。
一种检测鼻腔脱落细胞中ALOX15基因表达量的方法，包括以下步骤：
步骤一：从鼻腔脱落细胞中提取RNA：
步骤1：取基因组DNA吸附柱置于2mL收集管内，将所述鼻腔脱落细胞溶解于2000μL细胞裂解液中后加入至基因组DNA吸附柱中，于12000转/min的条件下离心60s，取滤液，向所述滤中加入等体积的70％乙醇，混合均匀后加入至RNA纯化柱中，12000转/分钟，离心1min，除去滤液，将所述RNA纯化柱置于2mL收集管中；
步骤2：向步骤1中获取的所述RNA纯化柱中加入700μL的第一缓冲液，12000转/分钟，离心30s，除去第一滤液；继续向所述RNA纯化柱加入800μL第二缓冲液，12000转/分钟，离心30s，除去第二滤液；
步骤3：将步骤2中除去第二滤液的RNA纯化柱安置于1.5mL无RNA水解酶收集管，向RNA纯化柱加入50μL的去RNA水解酶的蒸馏水或0.1％焦碳酸二乙酯处理水，室温静置5分钟，12000转/分钟，离心2min，洗脱RNA纯化柱，采用分光光度计测量RNA溶液的OD260/OD280比值为2.0，得到RNA；
步骤二：逆转录制备cDNA，包括以下步骤：取3μL的所述逆转录混合液，0～8μL去RNA酶及去DNA酶的水(根据RNA量用水补齐至8μL)，以及总量不超过500ng或体积不超过8μL的总RNA，所述去RNA水解酶蒸馏水补齐至10μL；轻柔混匀后进行逆转录反应,条件如下:在37℃的条件下，进行15分钟的反转录反应；在85℃的条件下，进行5秒的反转录酶的失活反应；产物4℃放置。
步骤三：实时荧光定量PCR扩增检测，包括以下步骤：
步骤1：制备实时荧光定量PCR反应液：包括5μL预混合液(含有PCR所需要的酶和缓冲液)、0-10μL的双蒸水(根据总体积用水补齐至10μL)，0～2μL的染料(用于进行机器的荧光补偿及矫正)，1μM的ALOX15基因的上游引物，1μM的ALOX15基因的下游引物，1μM的内参基因的上游引物，1μM的内参基因的下游引物，2μL的cDNA，1μg阳性对照，1μg阴性对照；
步骤2：采用两步法PCR扩增标准程序：所述两步法PCR扩增标准程序的反应条件包括以下步骤：第1阶段：在95℃的条件下预变性30秒；第2阶段PCR反应：在95℃的条件下，反应15秒，在60℃的条件下，反应60秒，退火延伸，如此进行40个循环。
步骤四：计算所述ALOX15基因表达量：
反应结束后确认实时荧光定量PCR的扩增曲线和熔解曲线,读取ALOX15及GAPDH的Ct值，选用△Ct分析法(ALOX15的Ct值减GAPDH的Ct值)进行分析，采用GAPDH作为内参基因；阳性对照Ct值<20且阴性对照Ct值>38视为实验有效，否则实验无效；
采用ΔCT方法比较ALOX15与内参基因的表达量差异：得ALOX15的平均CT值为20.1，GAPDH的平均CT值为17.9，ΔCT值为2.2。
本公开上述实施例7～9中，优选的所用的第一缓冲液RWA buffer的生产厂家为Takara公司，货号 9767；第二缓冲液RWB buffer的生产厂家为Takara公司，货号9767。但本申请保护范围并局限于上述的第一缓冲液和第二缓冲液。本领域技术人员可根据实际应用需要进行选择。
实施例10：
样本的收集与处理：
78例受试者用生理盐水冲洗鼻腔后，在鼻内镜下用毛刷(Copan公司产)于鼻息肉表面按压30s，旋转3-4圈，刷取息肉表面，将毛刷置于后续所述裂解液中，4℃短期保存(不超过24小时)，或转移入低于-20℃长期保存。
一种检测鼻腔脱落细胞中ALOX15基因表达量的方法，包括以下步骤：
步骤一：从鼻腔脱落细胞中提取RNA：向装有鼻腔脱落细胞的离心管中加入1mLRNA抽提液进行溶解、振荡后，室温静置5min；加入200μL氯仿，震荡混匀，室温静置5min，于4℃，12000r/min离心15min；取上清液200μL，加入200μL异丙醇，混匀后静置10min，于4℃，12000r/min离心15min弃上清，保留第一沉淀；向所述第一沉淀中加入与异丙醇等体积的浓度75％的乙醇，于4℃，7500r/min离心15min，弃上清，保留第二沉淀；盖紧所述离心管，于4℃，7500r/min离心2min，除去上清液，静置15min后继续向所述离心管中加入50μL去RNA酶及去DNA酶的水溶解所述第二沉淀，采用分光光度计测量RNA溶液OD260/OD280比值为1.8，得到RNA；
步骤二：逆转录制备cDNA步骤同实施例7。
步骤三：实时荧光定量PCR扩增检测步骤同实施例7。
步骤四：计算所述ALOX15基因表达量：
反应结束后确认实时荧光定量PCR的扩增曲线和熔解曲线,读取ALOX15及GAPDH的Ct值，选用△Ct分析法(ALOX15的Ct值减GAPDH的Ct值)进行分析，采用GAPDH作为内参基因；阳性对照Ct值<20且阴性对照Ct值>38视为实验有效，否则实验无效；
步骤1：计算健康对照组平均ΔCT：本例中受试者1～10为健康对照组，计算方法如下：平均ΔCT(CT(ALOX15)-CT(GAPDH))＝(1.754+1.655+1.829+2.2+1.308+1.449+1.234+1.343+1.49+1.695/10＝1.5975；
步骤2：计算受试者相对表达量：
以1.5975为基准，求出每个受试者相对表达量(表示本基因在受试者中相对健康对照组平均值的表达高低，数值代表变化倍数，例如1.5相当于该受试者的表达量是健康对照组平均值的1.5倍)，计算结果如表3所示。
计算公式为2 -ΔΔCT，-ΔΔCT＝-(ΔCT(受试者CT(ALOX15)-CT(GAPDH))-平均ΔCT(健康对照组平均ΔCT))。
表3 实施例8提供的方法的ALOX15基因表达量计算结果
Figure PCTCN2019093281-appb-000001
Figure PCTCN2019093281-appb-000002
Figure PCTCN2019093281-appb-000003
实施例11：
样本的收集与处理：
获取细胞前嘱患者用生理盐水冲洗鼻腔，在鼻内镜下用毛刷(Copan公司产)于鼻息肉表面按压30s,旋转3-4圈，刷取息肉表面，将毛刷置于裂解液中，4℃短期保存(不超过24小时)，或转移入低于-20℃长期保存。
一种检测鼻腔脱落细胞中ALOX15基因表达量的方法，包括以下步骤：
步骤一：从鼻腔脱落细胞中提取RNA步骤：向装有鼻腔脱落细胞的离心管中加入20mLRNA抽提液进行溶解、振荡后，室温静置7min；加入10mL氯仿，震荡混匀，室温静置7min，于5℃，14000r/min离心20min；取上清液20mL，加入20mL的异丙醇，混匀后静置12min，于5℃，14000r/min离心20min弃上清，保留第一沉淀；向所述第一沉淀中加入40mL的浓度90％的乙醇，于5℃，14000r/min离心3min，弃上清，保留第二沉淀；盖紧所述离心管，于5℃，14000r/min离心3min，除去上清液，静置20min后继续向所述离心管中加入5mL去RNA酶及去DNA酶的水溶解所述第二沉淀，采用分光光度计测量RNA溶液OD260/OD280比值为2.1，得到RNA；
步骤二：逆转录制备cDNA步骤同实施例7。
步骤三：实时荧光定量PCR扩增检测步骤中的制备实时荧光定量PCR反应液同实施例7，针对PCR扩增标准程序采用三步法，所述三步法PCR扩增标准程序的反应条件包括以下步骤：第1阶段：在95℃的条件下预变性2分钟；第2阶段PCR反应：在95℃的条件下，反应1分钟，在55℃的条件 下，反应1分钟，在72℃的条件下，反应1分钟，如此进行40个循环；最后72℃，7分钟退火延伸。
步骤四：计算所述ALOX15基因表达量：
反应结束后确认实时荧光定量PCR的扩增曲线和熔解曲线,读取ALOX15及GAPDH的Ct值，选用△Ct分析法(ALOX15的Ct值减GAPDH的Ct值)进行分析，采用GAPDH作为内参基因；阳性对照Ct值<20且阴性对照Ct值>38视为实验有效，否则实验无效；
阳性对照孔平均Ct：16.7；阴性对照孔平均Ct：38.4；样品ALOX15平均Ct：21.5；样品GAPDH平均Ct：19.4；差异值：为2.1。表示该患者ALOX15表达为GAPDH的0.23倍(1/2 2.1)。
实施例12：
样本的收集与处理：
获取细胞前嘱患者用生理盐水冲洗鼻腔，在鼻内镜下用毛刷(Copan公司产)于鼻息肉表面按压30s,旋转3-4圈，刷取息肉表面，将毛刷置于裂解液中，4℃短期保存(不超过24小时)，或转移入低于-20℃长期保存。
一种检测鼻腔脱落细胞中ALOX15基因表达量的方法，包括以下步骤：
步骤一：从鼻腔脱落细胞中提取RNA步骤：向装有鼻腔脱落细胞的离心管中加入0.1mLRNA抽提液进行溶解、振荡后，室温静置7min；加入0.03mL氯仿，震荡混匀，室温静置7min，于5℃，14000r/min离心20min；取上清液20mL，加入0.015mL的异丙醇，混匀后静置12min，于5℃，14000r/min离心20min弃上清，保留第一沉淀；向所述第一沉淀中加入0.0075mL的浓度90％的乙醇，于5℃，14000r/min离心3min，弃上清，保留第二沉淀；盖紧所述离心管，于5℃，14000r/min离心3min，除去上清液，静置20min后继续向所述离心管中加入0.01mL去RNA酶及去DNA酶的水溶解所述第二沉淀，采用分光光度计测量RNA溶液OD260/OD280比值为2.1，得到RNA；
步骤二：逆转录制备cDNA步骤同实施例7。
步骤三：实时荧光定量PCR扩增检测步骤中的制备实时荧光定量PCR反应液同实施例7，针对PCR扩增标准程序采用三步法，所述三步法PCR扩增标准程序的反应条件包括以下步骤：第1阶段：在95℃的条件下预变性2分钟；第2阶段PCR反应：在95℃的条件下，反应1分钟，在55℃的条件下，反应1分钟，在72℃的条件下，反应1分钟，如此进行40个循环；最后72℃，7分钟退火延伸。
步骤四：计算所述ALOX15基因表达量：
反应结束后确认实时荧光定量PCR的扩增曲线和熔解曲线,读取ALOX15及GAPDH的Ct值，选用△Ct分析法(ALOX15的Ct值减GAPDH的Ct值)进行分析，采用GAPDH作为内参基因；阳性对照Ct值<20且阴性对照Ct值>38视为实验有效，否则实验无效；
阳性对照孔平均Ct：15.9；阴性对照孔平均Ct：38.7；样品ALOX15平均Ct：17.8；样品GAPDH平均Ct：16.4；差异值：17.8-16.4为1.4。表示该患者ALOX15表达为GAPDH的0.51倍(1/2 1.4)。
工业实用性：
本公开提供一种用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒，通过蛋白质组学和转录组学方法筛选采用ALOX15基因作为生物标志物，将其应用到试剂盒中，以实现采用试剂盒进行检测慢性鼻窦炎伴鼻息肉亚型的方法，使最终获取的试剂盒包括ALOX15基因的特异性引物。在具有该特异性引物的基础上，本公开的试剂盒能够快速对鼻息肉亚型进行鉴别，且相比较传统的病理检测方法准确性更高，此试剂盒可以同时对样品进行大批量、快速检测，节约了人力成本和就医成本。且系统化试剂盒鉴别准确率较高，可以全面的反应组织病理学特点。解决现有技术中人为误差的影响，避免了组织切片反应了组织局部特征造成误诊的弊端。通过试剂盒进行快速、准确、全面的鼻息肉亚型鉴别对临床诊疗至关重要，以尽早根据鼻息肉的炎症亚型进行针对化治疗，有效指导针对慢性鼻窦炎伴鼻息肉患者的药物治疗方式及手术方式的确定，准确预估药物治疗的反应、判断预后效果。
本公开所提供的试剂盒能够通过采用刷取或粘取的方式从鼻息肉的表面获取鼻息肉细胞来进行检测，从而确定患者的慢性鼻窦炎伴鼻息肉亚型，避免了对患者造成创面，提高了患者检查的安全性，且操作更便捷，节约了人力成本和就医成本。
本公开提供的检测鼻腔脱落细胞中ALOX15基因表达量的方法，以有效筛选的ALOX15基因作为生物标志物，提供对其基因表达量的检测方法，实现对鼻腔脱落细胞中ALOX15基因表达量的计算，能够有效获取ALOX15基因表达量，提供的方法简单快捷，敏感性高，重复性好，适宜广泛推广应用。
本公开提供的检测鼻腔脱落细胞中ALOX15基因表达量的方法，其中ALOX15基因能编码脂氧合酶蛋白质，编码的酶作用于各种多不饱和脂肪酸底物以产生各种生物活性脂质介质，例如类花生酸，肝素，脂氧素等。将花生四烯酸转化为12-氢过氧化二十碳四烯酸/12-HPETE和15-氢过氧化二十碳四烯酸/15-HPETE。还将亚油酸转化为13-氢过氧十八碳二烯酸。也可以作用于(12S)-过氧化氢二十一碳四烯酸/(12S)-HPETE以产生肝霉素A3。编码的酶及其反应产物能够调节炎症和免疫反应，与ALOX15相关的疾病包括脑室周围白质软化等。其通过氧化巨噬细胞中膜结合的磷脂酰乙醇胺帮助清除炎症期间的凋亡细胞，并且抑制与炎性单核细胞清除凋亡细胞相关的自身免疫应答。目前现有技术中尚未公开有ALOX15基因，更未发现该基因的用途。其中公开提供的针对鼻腔脱落细胞中ALOX15基因表达量的方法可用于检测鼻腔脱落细胞中ALOX15表达情况，可用于进一步为鼻腔伴有鼻息肉的慢性鼻窦炎亚型的检测基因筛选技术提供了基础，为临床指导和药物治疗提供了可靠的基础。保证了用于检测伴有鼻息肉的慢性鼻窦炎亚型的试剂盒在临床应用的可行性。
本公开提供的检测鼻腔脱落细胞中ALOX15基因表达量的方法，根据实际需求采用ΔCt或2 -ΔΔCt法的相对定量法，选择了表达量相对恒定的内参基因，用内参基因的数量进行标准化，通过测定样品目的基因与内参基因的Ct值差异计算目的基因表达量，方法简便快速，检测精度高，可降低检测成本，节约检测时间。结果便于判读等优点。大大提升了实验效率。

Claims (20)

  1. 一种用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒，其中，所述试剂盒包括ALOX15基因的特异性引物。
  2. 根据权利要求1所述的用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒，其中，所述ALOX15基因的上游引物如SEQ ID NO:2所示，所述ALOX15基因的下游引物如SEQ ID NO:3所示。
  3. 根据权利要求1或2任一项所述的用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒，其中，所述试剂盒进一步还包括内参基因的特异性引物。
  4. 根据权利要求3所述的用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒，其中，所述内参基因为GAPDH，所述GAPDH的上游引物如SEQ ID NO:4所示，所述GAPDH的下游引物如SEQ ID NO:5所示。
  5. 根据前述权利要求任一项所述的用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒，其中，所述试剂盒进一步还包括：从鼻息肉组织或从鼻黏膜脱落细胞中提取RNA的试剂；将总RNA逆转录为cDNA的试剂；采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因进行实时荧光定量PCR反应的试剂。
  6. 根据权利要求5所述的用于检测慢性鼻窦炎伴鼻息肉亚型的试剂盒，其中，
    所述将总RNA逆转录为cDNA的试剂包括：逆转录混合液和去RNA酶及去DNA酶的水；
    采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因进行实时荧光定量PCR反应的试剂包括：PCR预混合液、双蒸水、机器荧光补偿及矫正剂、ALOX15基因的上游引物、ALOX15基因的下游引物、内参基因的上游引物和内参基因的下游引物。
  7. ALOX15基因作为生物标志物用于检测慢性鼻窦炎伴鼻息肉亚型中的用途。
  8. 根据权利要求1-6中任一项所述的试剂盒用于检测慢性鼻窦炎伴鼻息肉亚型中的用途。
  9. 检测ALOX15的试剂用于检测慢性鼻窦炎伴鼻息肉亚型的用途。
  10. 根据权利要求9所述的用途，其中，所述检测ALOX15的试剂包括ALOX15基因的特异性引物，例如所述ALOX15基因的特异性引物的上游引物如SEQ ID NO:2所示，所述ALOX15基因的下游引物如SEQ ID NO:3所示。
  11. 根据权利要求9所述的用途，其中，检测ALOX15的试剂选自由针对ALOX15的引物、抗体、适配体、探针或其组合组成的组。
  12. 一种检测鼻腔脱落细胞中ALOX15基因表达量的方法，包括以下步骤：
    从鼻腔脱落细胞中提取RNA，
    将总RNA逆转录为cDNA，
    采用定量聚合酶链反应将cDNA中的ALOX15基因和内参基因分别采用ALOX15基因的特异性引物和内参基因的特异性引物进行实时荧光定量PCR扩增，以及
    基于扩增产物的检测结果计算ALOX15基因表达量。
  13. 根据权利要求12所述的检测鼻腔脱落细胞中ALOX15基因表达量的方法，其中，
    ALOX15基因的上游引物如SEQ ID NO:2所示，ALOX15基因的下游引物如SEQ ID NO:3所示；
    所述内参基因为GAPDH，所述内参基因的上游引物如SEQ ID NO:4所示，所述下游引物如SEQ ID NO:5所示。
  14. 根据权利要求12或13所述的检测鼻腔脱落细胞中ALOX15基因表达量的方法，其中，所述鼻腔脱落细胞采用毛刷于鼻息肉表面获取，且将获取鼻腔脱落细胞后的毛刷置于细胞裂解液中于4℃以下保存。
  15. 一种检测伴有鼻息肉的慢性鼻窦炎亚型方法，包括通过根据权利要求12-14中任一项所述的方法检测鼻腔脱落细胞中ALOX15基因表达量。
  16. 一种检测伴有鼻息肉的慢性鼻窦炎亚型方法，包括检测检测鼻腔脱落细胞中的ALOX15基因表达量，例如使用权利要求1-6中任一项所述的试剂盒检测所述腔脱落细胞中的ALOX15基因表达量。
  17. 根据权利要求1-6中任一项所述的试剂盒、权利要求7所述的用途、权利要求8所述用途、权利要求9-11中任一项所述的用途、权利要求15所述的方法或权利要求16所述的方法，其中，通过荧光PCR检测鼻腔脱落细胞中ALOX15基因表达量。
  18. 根据权利要求17所述的试剂盒、用途或方法，其中，根据△Ct(Ct(ALOX15)-Ct(GAPDH))确定慢性鼻窦炎伴鼻息肉亚型，Ct(ALOX15)为ALOX15基因的Ct值，Ct(GAPDH)为内参基因GAPDH的Ct值。
  19. 根据权利要求18所述的试剂盒、用途或方法，其中，所述△Ct大于或等于1.675代表非嗜酸性粒细胞型慢性鼻窦炎伴鼻息肉，而△Ct小于1.675代表嗜酸性粒细胞型慢性鼻窦炎伴鼻息肉。
  20. 根据权利要求1-6中任一项所述的试剂盒、权利要求7所述的用途、权利要求8所述用途、权利要求9-11中任一项所述的用途、权利要求15所述的方法或权利要求16所述的方法，其中，所述慢性鼻窦炎伴鼻息肉亚型为非嗜酸性粒细胞型慢性鼻窦炎伴鼻息肉或嗜酸性粒细胞型慢性鼻窦炎伴鼻息肉。
PCT/CN2019/093281 2018-07-03 2019-06-27 检测慢性鼻窦炎伴鼻息肉亚型的方法和试剂盒及alox15基因作为生物标志物的应用 Ceased WO2020007227A1 (zh)

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