WO2018196879A1 - 一种样本核酸检测试剂盒、试剂及其应用 - Google Patents

一种样本核酸检测试剂盒、试剂及其应用 Download PDF

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WO2018196879A1
WO2018196879A1 PCT/CN2018/088121 CN2018088121W WO2018196879A1 WO 2018196879 A1 WO2018196879 A1 WO 2018196879A1 CN 2018088121 W CN2018088121 W CN 2018088121W WO 2018196879 A1 WO2018196879 A1 WO 2018196879A1
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nucleic acid
sample
pcr reaction
reaction solution
pcr
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French (fr)
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张太松
陈斐斐
张盼
王善辰
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Leadway HK Ltd
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Leadway HK Ltd
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Priority to EP18792022.8A priority patent/EP3636769B1/en
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
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    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • 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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis

Definitions

  • the invention belongs to the field of biological sciences and biotechnology, and particularly relates to a reagent, a method and a kit for detecting sample nucleic acids.
  • PCR polymerase chain reaction
  • PCR-based assays such as fluorescent PCR, DNA sequencing, and RNA sequencing
  • biological samples such as serum, plasma, whole blood, urine, vaginal secretions, saliva, and feces
  • Chelex-100 is a chemical ion chelating resin composed of a styrene and styrene ethylene copolymer, and the suspension is in an alkaline environment (pH 10 to 14) and 100 ° C. Lead to cell membrane rupture, and DNA is released from the nucleus after denaturation, and Chelex-100 can bind polyvalent cations with high selectivity, remove polyvalent metal ions in samples and buffers, and avoid degradation of nucleic acid template during boiling.
  • the Chelex-100 boiling method is mainly divided into three steps: concentration of the biological sample; addition of the lysate, boiling at high temperature; high-speed centrifugation, taking the supernatant, which is the extracted nucleic acid solution.
  • the lysate contains NaOH or KOH in addition to Chelex-100 to ensure that its pH is maintained at 10-14. This also results in the extracted nucleic acid solution maintaining a strong basicity. This strong basicity, if not neutralized, is directly mixed with the PCR reaction solution for amplification reaction, which results in inefficient PCR amplification reaction.
  • the Chinese patent application CN201110295395.9 is neutralized by adding a lysate for high-temperature boiling, and is neutralized by adding 200 mM Tris-HCl buffer (pH 6.8 to 7.5), thereby lowering the pH value in the extracted nucleic acid solution.
  • the neutralization solution needs to be manually added for neutralization, and the operation steps are somewhat cumbersome. If the operator forgets to join, the PCR amplification efficiency will be lowered, and the detection result will be affected.
  • nucleic acid extraction method is the alkaline lysis method.
  • Chinese patent application CN201210242862.6 utilizes a strong alkaline solution (such as NaOH or KOH solution) to lyse cells in a biological sample at a high temperature, release nucleic acid, and then add an acidic buffer for neutralization, and perform centrifugation to obtain the extracted nucleic acid. Solution.
  • the neutralization solution needs to be manually added for neutralization, and the operation steps are somewhat cumbersome. If the operator forgets to join, the PCR amplification efficiency will be lowered, and the detection result will be affected.
  • the Chelex-100 boiling method or the alkaline lysis method when the nucleic acid in the sample is extracted and detected by the prior art, there is a problem: the detection of HBV DNA which may exist in the clinical blood or plasma sample is taken as an example, and the utilization is strong.
  • the alkaline nucleic acid extract extracts the nucleic acid in the clinical blood or plasma sample to obtain the extracted nucleic acid solution, and then adds 2 to 5 ⁇ l of the extracted nucleic acid solution to about 35 ⁇ l of the PCR reaction solution for mixing, which is due to the addition.
  • the volume of the extracted nucleic acid solution is small, and has little effect on the mixed PCR reaction solution, but when the volume of the extracted nucleic acid solution added is large, for example, 20 ⁇ l of the extracted nucleic acid solution is added to 20 ⁇ l of PCR.
  • the volume ratio of the extracted nucleic acid solution is significantly increased, which affects the mixed PCR reaction solution, resulting in a high pH value, thereby affecting subsequent PCR amplification. Increase the reaction efficiency and reduce the detection sensitivity and accuracy of the sample nucleic acid.
  • the nucleic acid in the sample is extracted by the conventional weak alkaline nucleic acid lysate
  • the extracted nucleic acid solution added to the PCR reaction solution has a small volume (usually 2 to 5 ⁇ l)
  • the sample can be detected Nucleic acid, but the detection sensitivity is low; however, when the volume of the extracted nucleic acid solution added is large, for example, 20 ⁇ l of the extracted nucleic acid solution is added to 20 ⁇ l of the PCR reaction solution for mixing, and then the weak alkaline nucleic acid lysate is extracted.
  • the efficiency of the sample nucleic acid is poor, and the larger nucleic acid solution added is likely to contain more impurities and inhibitors of PCR amplification reaction, which may result in the PCR amplification reaction not being able to proceed normally, or may be subjected to PCR amplification. Reaction, but will reduce the detection sensitivity and accuracy of the sample nucleic acid.
  • the present invention provides a reagent, a method and a kit for detecting a sample nucleic acid, wherein the reagent, the method and the kit use a strongly basic nucleic acid extract to extract nucleic acid in a sample, and then The extracted sample nucleic acid is added to the PCR reaction solution for neutralization, and nucleic acid amplification and detection are simultaneously performed, so that it is not necessary to add a step of neutralizing the extracted sample nucleic acid when the sample nucleic acid is extracted, thereby greatly It saves detection time and is also convenient for semi-automatic or fully automated processing, improving the efficiency of PCR amplification reaction, increasing the sensitivity of sample nucleic acid detection and improving the accuracy of nucleic acid detection.
  • the nucleic acid in the sample is directly detected. Interference with other components, so some samples need to be pre-treated and preserved, and then the extracted sample nucleic acids are detected.
  • nucleic acid extract of the present invention is used for sample nucleic acid extraction.
  • the sputum contains a large amount of mucin and impurities, so the sample is pretreated during nucleic acid extraction, usually liquefied with NaOH, sodium citrate, 0.5% N-acetyl-L-cysteine (NALC) or two
  • NALC N-acetyl-L-cysteine
  • DTT thiothreitol
  • the nucleic acid extract of the present invention is used for sample nucleic acid extraction;
  • the fresh tissue is pretreated by washing with physiological saline, then mashing or chopping it, adding proteinase K
  • the nucleic acid extract of the present invention is used for sample nucleic acid extraction;
  • the pretreatment method of paraffin tissue section is first dewaxed with a dewaxing agent such as xylene, and after digestion with proteinase K, the nucleic acid extract of the present invention is further used.
  • Sample nucleic acid extraction usually liquefied with NaOH, sodium citrate, 0.5% N-ace
  • the present invention provides a sample nucleic acid detecting reagent comprising a nucleic acid extract and a PCR reaction solution, the nucleic acid extract comprising an alkali metal hydroxide 10 mM to 100 mM, Tris-HCl 1 mM to 25 mM, Chelex-100 2% to 5% (w/v), EDTA 0.1% to 0.4% (w/v), the pH of the nucleic acid extract is 10.5 to 12.5; and the PCR reaction solution includes Tris-HCl 5 mM to 75 mM.
  • the pH of the PCR reaction solution is 8.0 to 9.0.
  • alkali metal hydroxide is NaOH or KOH.
  • sample nucleic acid detecting reagent further includes a concentrate.
  • the concentrate comprises 2.5% to 4.5% (w/w) of NaCl and 10% to 13% (w/v) of PEG 6000.
  • the pH of the PCR reaction solution is 8.1 to 8.7.
  • the pH of the PCR reaction solution is 8.1 to 8.5.
  • sample nucleic acid detecting reagent is used in detecting HBV nucleic acid.
  • sample nucleic acid comprises HBV, human B27 gene, HPV 6/11, TB or HCMV nucleic acid.
  • the sample nucleic acid includes a HBV nucleic acid; in the nucleic acid extract, an alkali metal hydroxide concentration is 25 mM, a Tris-HCl concentration is 1 mM, and a Chelex-100 concentration is 2% (w/v).
  • the concentration of EDTA was 0.1% (w/v)
  • the pH of the nucleic acid extract was 11.5
  • the concentration of Tris-HCl was 10 mM in the PCR reaction solution
  • the pH of the PCR reaction solution was 8.1.
  • the invention also provides a sample nucleic acid detecting method, comprising: (1) extracting nucleic acid in a sample by using a nucleic acid extract to obtain a sample nucleic acid solution; (2) using the sample nucleic acid solution obtained in (1)
  • the PCR reaction solution is mixed together to obtain a mixed solution, and the mixed solution is used to detect nucleic acid in the sample, and the nucleic acid extract includes an alkali metal hydroxide 10 mM to 100 mM, Tris-HCl 1 mM to 25 mM, Chelex- 100% to 5% (w/v), EDTA 0.1% to 0.4% (w/v), the pH of the nucleic acid extract is 10.5 to 12.5; and the PCR reaction solution includes Tris-HCl 5 mM to 75 mM.
  • the pH of the PCR reaction solution is 8.0 to 9.0; and the pH of the mixed solution is 8.0 to 9.0.
  • alkali metal hydroxide is NaOH or KOH.
  • the sample is concentrated with a concentrate.
  • the concentrate comprises 2.5% to 4.5% (w/w) of NaCl and 10% to 13% (w/v) of PEG 6000.
  • the pH of the PCR reaction solution is 8.1 to 8.7.
  • the pH of the PCR reaction solution is 8.1 to 8.5.
  • sample nucleic acid comprises HBV, human B27 gene, HPV 6/11, TB or HCMV nucleic acid.
  • the sample nucleic acid includes a HBV nucleic acid; in the nucleic acid extract, an alkali metal hydroxide concentration is 25 mM, a Tris-HCl concentration is 1 mM, and a Chelex-100 concentration is 2% (w/v).
  • the concentration of EDTA was 0.1% (w/v)
  • the pH of the nucleic acid extract was 11.5
  • the concentration of Tris-HCl was 10 mM in the PCR reaction solution
  • the pH of the PCR reaction solution was 8.1.
  • the invention also provides a sample nucleic acid detecting kit, the nucleic acid detecting kit comprising a nucleic acid extract and an PCR reaction solution, the nucleic acid extract comprising an alkali metal hydroxide 10 mM to 100 mM, Tris-HCl 1 mM to 25 mM, Chelex -100 2% to 5% (w/v), EDTA 0.1% to 0.4% (w/v), the pH of the nucleic acid extract is 10.5 to 12.5; and the PCR reaction solution includes Tris-HCl 5 mM to 75 mM The pH of the PCR reaction solution is 8.0 to 9.0.
  • alkali metal hydroxide is NaOH or KOH.
  • the nucleic acid detection kit further includes a concentrate.
  • the concentrate comprises 2.5% to 4.5% (w/w) of NaCl and 10% to 13% (w/v) of PEG 6000.
  • the pH of the PCR reaction solution is 8.1 to 8.7.
  • the pH of the PCR reaction solution is 8.1 to 8.5.
  • sample nucleic acid comprises HBV, human B27 gene, HPV 6/11, TB or HCMV nucleic acid.
  • the sample nucleic acid includes a HBV nucleic acid; in the nucleic acid extract, an alkali metal hydroxide concentration is 25 mM, a Tris-HCl concentration is 1 mM, and a Chelex-100 concentration is 2% (w/v).
  • the concentration of EDTA was 0.1% (w/v)
  • the pH of the nucleic acid extract was 11.5
  • the concentration of Tris-HCl was 10 mM in the PCR reaction solution
  • the pH of the PCR reaction solution was 8.1.
  • the present invention also provides the use of the nucleic acid detection kit for detecting HBV, human B27 gene, HPV 6/11 type, Mycobacterium tuberculosis or human cytomegalovirus in a sample.
  • the beneficial effect first adding a certain amount of sample to the strongly alkaline nucleic acid extract, extracting the nucleic acid in the sample, and then adding the extracted sample nucleic acid solution to the PCR reaction solution (the pH value is higher than the nucleic acid extract liquid) Neutralization in the low pH), as well as sample nucleic acid amplification and detection, thus eliminating the need to neutralize the extracted sample nucleic acid solution during sample nucleic acid extraction, thereby effectively saving detection Time, reduce the chances of increasing manual errors due to numerous steps, facilitate semi-automatic or fully automated processing, and this will increase the efficiency of PCR amplification of sample nucleic acids and increase the sensitivity and accuracy of detection of sample nucleic acids.
  • the present invention improves the nucleic acid extract and the PCR reaction solution in the prior art, and utilizes the nucleic acid extract of the present invention (pH 10.5 ⁇ ) 12.5) extracting the nucleic acid in the clinical blood or plasma sample to obtain the extracted sample nucleic acid solution, and adding the extracted sample nucleic acid solution to the PCR reaction solution (pH 8.0 to 9.0) of the present invention for mixing.
  • the extracted sample nucleic acid solution is 2 ⁇ 5 ⁇ l or 20 ⁇ l, the pH of the mixed PCR reaction solution will not be affected, that is, it will remain in the proper pH range, so as not to affect the subsequent PCR amplification.
  • the reaction efficiency also increases the sensitivity and accuracy of the detection of the sample nucleic acid.
  • the nucleic acid extract of the present invention can improve the extraction efficiency of the sample nucleic acid and reduce the possible impurities and PCR reaction in the extracted sample nucleic acid solution, compared to the extraction of the nucleic acid in the sample by using the conventional weak alkaline nucleic acid lysate. Inhibitor.
  • the nucleic acid extract of the present invention when used in combination with the PCR reaction solution of the present invention, when the volume of the extracted sample nucleic acid solution added to the PCR reaction solution is small (usually 2 to 5 ⁇ l), the sample can be detected.
  • the nucleic acid in the medium has a high detection sensitivity; when the volume of the extracted sample nucleic acid solution added is large, if 20 ⁇ l of the extracted sample nucleic acid solution is added to 20 ⁇ l of the PCR reaction solution for mixing, it is also highly sensitive The sample nucleic acid is detected with high accuracy.
  • Figure 1 shows the results of fluorescent PCR detection of different concentrations of HBV positive samples when the pH of the PCR reaction solution is 8.1.
  • Figure 2 shows the results of fluorescent PCR detection of different concentrations of HBV positive samples when the pH of the PCR reaction solution is 8.9.
  • Figure 3 shows the results of fluorescent PCR detection when the concentration of HBV positive samples is 20 IU/ml, and the pH of the PCR reaction solution is changed, when the pH values are 8.1, 8.3, 8.5, 8.7 and 8.9, respectively.
  • the present invention In order to extract the nucleic acid in the sample, the present invention first adds a certain amount of the sample to the strongly alkaline nucleic acid extract, lyses the cells or viruses in the sample, and releases the nucleic acid carried by them, thereby being extracted.
  • the pH of the sample nucleic acid solution extracted is relatively high, it will affect the subsequent PCR amplification reaction of the nucleic acid, and even cause the nucleic acid amplification reaction to fail to start.
  • the inventors added the extracted sample nucleic acid solution to the lower pH PCR reaction solution for neutralization, and also performed sample nucleic acid amplification and detection, thereby eliminating the sample.
  • the step of neutralizing the extracted sample nucleic acid during nucleic acid extraction can effectively save the detection time, reduce the chance of increasing manual errors due to the numerous steps, facilitate semi-automatic or fully automated processing, and increase the sample nucleic acid.
  • PCR amplification reaction efficiency increases the sensitivity and accuracy of detection of sample nucleic acids.
  • the nucleic acid extract for extracting the nucleic acid in the sample may be selected from a high concentration of a strong alkali solution or a strong alkaline solution containing Chelex-100, and the nucleic acid extract has a pH of ⁇ 10, preferably 10.5 ⁇ . 12.5, the nucleic acid in the sample can be effectively extracted.
  • the high concentration strong alkali solution may be selected from an alkali metal hydroxide solution of 0.05M to 0.6M, preferably a 0.05M to 0.6M NaOH solution or a KOH solution; and a strong alkaline solution containing Chelex-100 includes a base.
  • the metal hydroxide is 10 mM to 100 mM, Tris-HCl 1 mM to 25 mM, Chelex-100 2% to 5%, and EDTA 0.1% to 0.4%, and the alkali metal hydroxide is preferably NaOH or KOH.
  • the concentrated liquid may include 2.5% to 4.5% of NaCl and 10% to 13% of PEG 6000; as a second specific example of the concentrated liquid, the concentrated liquid may include PEG8000 20%, NaCl 13.5%.
  • the extracted sample nucleic acid solution is added to a lower pH PCR reaction solution in a suitable volume ratio, and the extracted sample nucleic acid solution is The pH is lowered to a level suitable for performing a PCR amplification reaction.
  • the pH of the PCR reaction solution is preferably 8.0 to 9.0, and the value is too high or too low, which affects the efficiency of the PCR amplification reaction and reduces the detection sensitivity and accuracy.
  • the PCR reaction solution contains Tris-HCl, KCl, dNTPs and Mg 2+, etc., wherein Tris-HCl can be replaced by a buffer system such as Tris-Acetate or Tris-Borate, and when the PCR reaction solution is mixed with the extracted sample nucleic acid solution, it is ensured.
  • the concentration of Tris-HCl is 5 mM to 75 mM.
  • the PCR reaction solution may further include a DNA polymerase for performing a nucleic acid amplification reaction such as a Taq enzyme, a fluorescent dye such as a primer and SYBR Green, or a TaqMan probe, a two-hybrid probe, a molecular beacon probe, or an MGB probe. Fluorescent probe.
  • a DNA polymerase, a fluorescent dye or a fluorescent probe can also be added to the PCR reaction solution when a nucleic acid amplification reaction is required.
  • an anti-Taq enzyme antibody is added to the PCR reaction solution, and the antibody binds to the active site of the Taq enzyme, thereby making the Taq enzyme unable to function. Only in the denaturing phase, the anti-Taq enzyme antibody is inactivated at high temperature and cannot bind to the active site of the Taq enzyme, so that the Taq enzyme can catalyze to synthesize a new DNA strand.
  • the nucleic acid in the sample is extracted by the nucleic acid extract of the present invention
  • a specific DNA or RNA fragment in the sample nucleic acid is selected in advance, and then the DNA or RNA is targeted for the DNA or RNA.
  • the fragment is designed with at least one pair of specific primers. If fluorescent PCR amplification is performed, it is optional to add a fluorescent dye such as SYBR Green, or to design at least one fluorescent probe for the selected specific DNA or RNA fragment, so that amplification can be performed according to the amplification. The fluorescence intensity produced when the specific DNA or RNA fragment in the sample is detected.
  • the sample nucleic acid detecting reagent in this embodiment includes a nucleic acid extract and a PCR reaction solution, and the composition thereof is as follows:
  • the PCR reaction solution further includes a fluorescent dye such as SYBR Green, or a fluorescent probe such as TaqMan, preferably a TaqMan fluorescent probe.
  • the PCR reaction solution may further include an enzyme mixture.
  • the enzyme mixture and the PCR reaction solution may be stored separately, and the enzyme mixture is added to the PCR reaction solution when a nucleic acid amplification reaction is required.
  • the enzyme mixture mainly comprises a DNA polymerase
  • the DNA polymerase may be selected from Taq DNA polymerase (abbreviated as Taq enzyme), Vent DNA polymerase, Deep DNA polymerase, Pfu DNA polymerase, Tgo DNA polymerase, KOD1 DNA polymerase and the like are preferably Taq enzymes.
  • the enzyme mixture mainly includes MMLV reverse transcriptase, Taq enzyme and RNase inhibitor.
  • the enzyme mixture may further include an anti-Taq enzyme antibody regardless of whether the nucleic acid in the test sample is DNA or RNA.
  • sample nucleic acid detecting reagent further comprises a concentrate: 2.5% to 4.5% (w/v) of NaCl, and 10% to 13% (w/v) of PEG6000.
  • hepatitis B virus HBV
  • HCMV human cytomegalovirus
  • the NaOH in the above nucleic acid extract may be replaced with other alkali metal hydroxide such as KOH.
  • Example 2 Sample nucleic acid extraction step
  • sample nucleic acid extraction is carried out by using the nucleic acid detecting reagent in Example 1, and can be classified into the following two sample nucleic acid extraction methods depending on the sample:
  • the specific nucleic acid extraction steps are slightly different depending on the sample used for the detection and the object to be detected.
  • the nucleic acid extraction step is the protocol 1 of the present embodiment
  • the nucleic acid extraction step is the present implementation.
  • Example 1 when detecting human papillomavirus type 6/11 (HPV 6/11) in a genital endocrine sample, the nucleic acid extraction step is the protocol 2 of the present embodiment; when detecting the tuberculosis in the sputum sample In the case of mycobacteria (TB), the nucleic acid extraction step is the protocol 1 of the present embodiment; when the human cytomegalovirus (HCMV) in the urine sample is detected, the nucleic acid extraction step is the first embodiment of the present embodiment.
  • the protocol 2 of the present embodiment when detecting human papillomavirus type 6/11 (HPV 6/11) in a genital endocrine sample, the nucleic acid extraction step is the protocol 2 of the present embodiment; when detecting the tuberculosis in the sputum sample In the case of mycobacteria (TB), the nucleic acid extraction step is the protocol 1 of the present embodiment; when the human cytomegalovirus (HCMV) in the urine sample is detected, the
  • the PCR reaction solution was dispensed into the PCR reaction tube in portions per part, and transferred to the sample processing area. 4 to 20 ⁇ l of the sample nucleic acid solution extracted in Example 2 were respectively added to the corresponding reaction tubes, the reaction tube was tightly capped, and transferred to a detection zone for PCR amplification. Each reaction tube was placed in a PCR machine in a certain order, and a PCR program was detected and fluorescence was detected for fluorescence PCR amplification.
  • hepatitis B virus HBV
  • 40 ⁇ l of a PCR detection system was prepared, in which 20 ⁇ l of the extracted sample nucleic acid solution and 20 ⁇ l of the PCR reaction solution were extracted.
  • TB Mycobacterium tuberculosis
  • HCMV human cytomegalovirus
  • the sample type is serum or plasma, and the detection of hepatitis B virus (HBV) is taken as an example.
  • HBV hepatitis B virus
  • Control method 50 ⁇ l of the sample to be tested was added with 50 ⁇ l of nucleic acid extract (NaCl 2M, Tris-HCl (pH 8.1) 50 mM, mercaptoethanol 1% (v/v), Chelex-100 5% (w/v), pH 8.59) The mixture was vortexed for 15 sec, in a 100 ° C water bath or a dry bath for 10 min, and then centrifuged at 12,000 rpm for 10 min, and 4 ⁇ l of the supernatant and 20 ⁇ l of the supernatant were separately taken for PCR amplification.
  • nucleic acid extract NaCl 2M, Tris-HCl (pH 8.1) 50 mM, mercaptoethanol 1% (v/v), Chelex-100 5% (w/v), pH 8.59
  • the PCR reaction system includes a PCR reaction solution (containing TAKARA PCR buffer, dNTPs, Mg 2+ , a primer and a TaqMan probe, pH 9.05) and a Taq enzyme.
  • the detection sensitivity of this control method was 500 IU/ml.
  • Table 1 shows the results of HBV detection in serum or plasma samples, where "+” indicates a positive result and "-" indicates a negative result.
  • the experimental results showed that among the 20 samples, the nucleic acid detection reagent, the nucleic acid extraction method and the fluorescent PCR detection used in the present invention detected 11 HBV positive and 9 HBV negative.
  • the control method when 4 ⁇ l of the supernatant was used for the detection, 10 cases of HBV were detected and 10 cases of HBV were negative.
  • One of the control samples was inconsistent with the clinical sample information; when 20 ⁇ l of the supernatant was used for detection
  • the detection results of the 20 test samples were all negative, because the pH of the nucleic acid extraction reagent in the control method was not strongly alkaline relative to the method of the present invention, and the nucleic acid extraction efficiency was not high.
  • the sample amount is increased to 20 ul, it is also possible to simultaneously increase the substance which inhibits PCR amplification.
  • Example 5 Effect of pH of PCR reaction solution on sensitivity of HBV detection
  • the HBV positive samples were detected: the high concentration HBV positive samples were serially diluted to obtain a series of low concentration HBV positive samples.
  • concentrations ranged from 1000 IU/ml, 500 IU/ml, 250 IU/ml, 100 IU/ml, 50 IU/ml and 20 IU/ml, respectively.
  • the pH of the PCR reaction solution was adjusted to 8.1, and the results of fluorescent PCR detection for these HBV positive samples are shown in FIG. The results showed that the six low-concentration HBV-positive samples all had obvious sigmoid amplification curves and were stably detected.
  • the pH of the PCR reaction solution was adjusted to 8.1, 8.3, 8.5, 8.7 and 8.9, respectively, and the results of the fluorescent PCR assay are shown in FIG.
  • the results showed that the amplification efficiency of the HBV positive samples with a concentration of 20 IU/ml increased significantly with the decrease of the pH of the PCR reaction solution.
  • the pH value was 8.1, the amplification efficiency was the best.
  • the results also showed that when the pH values of the PCR reaction solutions were 8.1, 8.3, 8.5 and 8.7, respectively, positive samples of 20 IU/ml could be detected, but the amplification efficiency, S-type expansion at pH values of 8.1, 8.3 and 8.5, respectively.
  • the linear and fluorescence values of the increasing curve were significantly better than the pH of 8.7; the amplification efficiency of pH 8.1, the linearity and fluorescence of the S-type amplification curve were significantly better than the pH values of 8.3 and 8.5. It can be seen that under the same conditions of other detection conditions, changing the pH value can have a very large influence on the amplification efficiency and the linear and fluorescence values of the S-type amplification curve, and the amplification efficiency and the fluorescence of the S-type amplification curve.
  • the increase in value increases the sensitivity of the assay, so the detection sensitivity at pH 8.1, 8.3, and 8.5 is significantly better than at pH 8.7; the sensitivity at pH 8.1 is significantly better than at pH 8.3 and 8.5. . This means that while the pH value is gradually decreasing, the detection sensitivity is gradually increased.
  • the present inventors unexpectedly found that adjusting the pH of the PCR reaction solution, that is, from 8.9 to 8.7, from 8.7 to 8.3 to 8.5, and from 8.3 to 8.1, unexpectedly The amplification effect, which significantly improves the detection accuracy.
  • the sample type is a clinical test of whole blood, taking the human B27 gene test as an example.
  • Control method 500 ⁇ l whole blood sample was pretreated with red blood cell lysate (NH 4 Cl 15 mM, NaHCO 3 1 mM, EDTA-2Na 0.1 mM), after centrifugation, the supernatant was removed, and the precipitate was retained; 100 ⁇ l of nucleic acid was added to the precipitate.
  • red blood cell lysate NH 4 Cl 15 mM, NaHCO 3 1 mM, EDTA-2Na 0.1 mM
  • the PCR reaction system includes a PCR reaction solution (containing TAKARA PCR buffer, dNTPs, Mg 2+ , a primer and a TaqMan probe, pH 9.05) and a Taq enzyme.
  • Table 2 shows the results of human B27 gene assay in whole blood samples, where "+” indicates a positive result and "-" indicates a negative result.
  • the experimental results showed that among the 13 test samples, 6 kinds of human B27 gene were positive and 7 cases of human B27 gene were negative by using the nucleic acid detection reagent, nucleic acid extraction method and fluorescent PCR detection of the present invention; Five cases of human B27 gene were positive, 8 cases of human B27 gene were negative, and one of the control tests was inconsistent with the clinical sample information.
  • the sample type is a clinical test for genital tract secretions, taking human papillomavirus type 6/11 (HPV 6/11) as an example.
  • HPV 6/11 human papillomavirus type 6/11
  • Control method 50 ⁇ l of the sample to be tested was added with 50 ⁇ l of nucleic acid extract (NaCl 2M, Tris-HCl (pH 8.0) 50 mM, mercaptoethanol 1% (v/v), Chelex-100 5% (w/v), pH 8.59) Shake well for 15 sec, 100 ° C water bath or dry bath for 10 min, then centrifuge at 10,000 rpm for 5 min, and take 4 ⁇ l of the supernatant for PCR amplification.
  • the PCR reaction system includes a PCR reaction solution (containing TAKARA PCR buffer, dNTPs, Mg 2+ , a primer and a TaqMan probe, pH 9.05) and a Taq enzyme.
  • Table 3 shows the results of HPV testing in samples of genital tract secretions, where "+” indicates a positive result and "-" indicates a negative result.
  • the experimental results showed that among the 15 test samples, 10 nucleic acid positive samples and 5 HPV negative samples were detected by the nucleic acid detection reagent, nucleic acid extraction method and fluorescent PCR detection used in the present invention; 8 positive samples were detected by the control method. 7 cases were negative, and 2 cases of the control were inconsistent with the clinical sample information.
  • Example 8 Clinical sample TB detection
  • the sample type is a clinical test of sputum, taking the detection of Mycobacterium tuberculosis (TB) as an example.
  • TB Mycobacterium tuberculosis
  • nucleic acid detection reagents, nucleic acid extraction methods and fluorescent PCR detection of Example 1, Example 2 and Example 3, 10 clinically confirmed positive samples were detected.
  • Control method 500 ⁇ l sputum sample, adding 5 ml of 1 M NaOH solution, liquefying for 30 min, shaking at 14,000 for 5 min, removing the supernatant, and retaining the precipitate; adding 50 ⁇ l of nucleic acid extract (SDS 0.1% (w/v) to the precipitate, NP40 1% (v/v), Tween-20 1% (v/v), Chelex-100 15% (w/v), pH 5.02), mix by shaking for 15 sec, 100 ° C water bath or dry bath for 30 min, then 12,000 After centrifugation at rpm for 10 min, 10 ⁇ l of the supernatant was taken for PCR amplification.
  • the PCR reaction system includes a PCR reaction solution (containing TAKARA PCR buffer, dNTPs, Mg 2+ , a primer and a TaqMan probe, pH 9.05) and a Taq enzyme.
  • Table 4 shows the results of TB detection in sputum samples, where "+” indicates a positive result and "-" indicates a negative result.
  • the experimental results showed that among the 10 test samples, 5 TB positive and 5 TB negative were detected by the nucleic acid detection reagent, nucleic acid extraction method and fluorescent PCR detection used in the present invention; 4 cases were positive by the control method. Six cases were negative, and one sample of the control was inconsistent with the clinical sample information.
  • Example 9 Clinical sample HCMV detection
  • the sample type is a clinical test of urine, taking the human cytomegalovirus test (HCMV) test as an example.
  • HCMV human cytomegalovirus test
  • Control method 250 ⁇ l of the urine sample to be tested was collected, centrifuged at 14,000 for 5 min, the supernatant was removed, and the precipitate was retained; 250 ⁇ l of nucleic acid extract (Tris-HCl (pH 8.0) 100 mM, Chelex-100 5%, EDTA (pH) was added to the precipitate. 8.0) 10 mM, NaCl 50 mM, SDS 2% (w/v), pH 8.2), vortexed for 15 sec, centrifuged in a 100 ° C water bath or dry bath for 10 min and then centrifuged at 13,000 rpm for 10 min, and 4 ⁇ l of the supernatant was taken for PCR amplification.
  • the PCR reaction system includes a PCR reaction solution (containing TAKARA PCR buffer, dNTPs, Mg 2+ , a primer and a TaqMan probe, pH 9.05) and a Taq enzyme.
  • Table 5 shows the results of HCMV detection in urine samples, where "+” indicates a positive result and "-" indicates a negative result.
  • the experimental results showed that among the 10 test samples, 5 nucleic acid positive samples and 5 HCMV negative samples were detected by the nucleic acid detection reagent, nucleic acid extraction method and fluorescent PCR detection used in the present invention; 5 positive samples were detected by the control method, 5 The cases were negative; the two test results were consistent.
  • nucleic acid detection reagents nucleic acid extraction method and fluorescent PCR detection of Example 1, Example 2 and Example 3, HBV, human B27 gene, HPV, TB, HCMV positive standard samples were detected: the positive standard samples were diluted, respectively. Diluted according to Table 6, respectively, and each concentration was repeated 8 times. The test results are shown in Table 6, in which "+” indicates a positive result and "-" indicates a lower limit of the reagent detection.
  • the HBV detection sensitivity of the present invention is 20 IU/ml
  • the human B27 gene detection sensitivity is 5 ng/reaction
  • the HPV6/11 detection sensitivity is 250 copies/ml
  • the TB detection sensitivity is 5 bacteria/ml
  • the HCMV detection sensitivity is 500 copies/ Ml.

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Abstract

本发明提供一种检测样本核酸的试剂、方法和试剂盒，采用强碱性的核酸提取液(pH值为10.5～12.5)提取样本中的核酸，然后直接将提取出的核酸加入到 PCR 反应液(pH 值为 8.0～9.0)中进行中和，同时进行核酸扩增和检测。本发明可以检测血液、血浆、全血、生殖道分泌物、痰液或尿液等不同类型样本中的核酸。本发明通过在 PCR 反应液中对提取出的样本核酸进行中和，而不是在核酸提取时进行中和，可有效提高核酸提取效率和 PCR 扩增效率，显著提高样本核酸检测的准确性和灵敏度。

Description

一种样本核酸检测试剂盒、试剂及其应用 技术领域
本发明属于生物科学和生物技术领域，特别涉及一种检测样本核酸的试剂、方法和试剂盒。
背景技术
随着分子生物学技术的快速发展，它已经渗透到生物学、医学、植物学、遗传学和动物学等各个方面。作为分子生物学技术的一项重大突破，聚合酶链式反应(PCR)技术具有高灵敏度、高特异性和省时快速等特点，已广泛地运用于人类和动物的疾病诊断(如产前诊断、新生儿筛查以及遗传代谢疾病检测)、法医侦检、亲缘关系分析、种子纯度鉴定、分子标记辅助育种、基因定位和转基因生物检测等等。
运用PCR技术进行检测主要涉及到三个方面：核酸提取、核酸扩增和扩增产物检测。核酸提取的质量与数量通常直接决定着PCR反应的成败。因此，大量基于PCR技术的检验方法(如荧光PCR、DNA测序和RNA测序等)需要在扩增前提取与纯化生物样本(如血清、血浆、全血、尿液、阴道分泌物、唾液、粪便、棉拭子、上皮脱落细胞、新鲜组织和石蜡组织切片等)中的DNA和/或RNA核酸分子。
长期以来，生物样本中核酸的提取和纯化一直是耗时、繁琐的过程，严重减慢了检测速度。尤其是对极其微量的生物样本而言，常规的核酸提取方法对样本中微量DNA的提取回收率低是导致后续PCR扩增效果差的主要原因。因此，建立一种快速的DNA提取方法对提高PCR检测效率是至关重要的。
根据核酸的不同性质，有非常多的提取方法，其中一种比较常用的方法就是基于Chelex-100的煮沸法，即Chelex-100煮沸法。在这种煮沸法中，Chelex-100是一种苯乙烯和苯二乙烯共聚体组成的化学离子螯合树脂，其悬液在碱性环境(pH 10～14)和100℃的条件下，可导致细胞膜破裂，并使DNA变性后从细胞核中释放出来，并且Chelex-100可高选择性地结合多价阳离子，去除样本和缓冲液中的多价金属离子，避免煮沸过程中核酸模板发生降解，此外它还能够有效除去非核酸有机物，具有经济、简便、高效等优点，可以减少提取过程中核酸的损失，目前已被广泛用于微量血液、组织斑块、精斑、毛发和骨骼等法医物证检材的核酸提取中。
Chelex-100煮沸法主要分为三个步骤：生物样本浓缩；加入裂解液，高温煮沸；高速离 心，取上清液，即为所提取的核酸溶液。在煮沸法中，裂解液除了含有Chelex-100之外，还含有NaOH或KOH，从而确保它的pH值保持在10～14。这也因此导致所提取的核酸溶液维持较强的碱性。这种较强的碱性如果不加以中和的话，直接与PCR反应液混合在一起进行扩增反应，那么会导致PCR扩增反应效率低下。为了解决这个问题，中国专利申请CN201110295395.9在加入裂解液进行高温煮沸后，加入200mM Tris-HCl缓冲液(pH 6.8～7.5)进行中和，从而降低所提取的核酸溶液中的pH值。然而，当利用这种方法提取生物样本中的核酸时，需要手工加入中和液进行中和，操作步骤有些繁琐，如果操作人员忘记加入的话，将会降低PCR扩增效率，影响检测结果。
此外，还有一种常用的核酸提取方法就是碱裂解法。中国专利申请CN201210242862.6利用强碱溶液(如NaOH或KOH溶液)在高温下让生物样本中的细胞发生裂解，释放出核酸，然后加入酸性缓冲液进行中和，进行离心，获得所提取的核酸溶液。同样地，当利用这种方法提取生物样本中的核酸时，需要手工加入中和液进行中和，操作步骤有些繁琐，如果操作人员忘记加入的话，将会降低PCR扩增效率，影响检测结果。
不论采用Chelex-100煮沸法还是碱裂解法，利用现有技术提取和检测样本中的核酸时，都会存在一个问题：以检测临床血液或血浆样本中可能存在的HBV DNA为例进行说明，利用强碱性的核酸提取液对临床血液或血浆样本中的核酸进行提取，获得提取出的核酸溶液，随后加入2～5μl提取出的核酸溶液到35μl左右的PCR反应液中进行混合，这是由于加入的提取出的核酸溶液的体积较小，对混合后的PCR反应液的影响不大，但是当加入的提取出的核酸溶液的体积较大时，如将加入20μl提取出的核酸溶液到20μl PCR反应液中进行混合时，虽然总体积仍然是40μl，但是提取出的核酸溶液所占的体积比例显著增加，这会影响混合后的PCR反应液，导致pH值偏高，从而影响后续的PCR扩增反应效率，降低样本核酸的检测灵敏度和准确性。
此外，利用常规的弱碱性核酸裂解液提取样本中的核酸后，当往PCR反应液中加入的提取出的核酸溶液体积较小(通常为2～5μl)时，虽能检测出样本中的核酸，但是检测灵敏度较低；然而当加入的提取出的核酸溶液体积较大时，如将加入20μl提取出的核酸溶液到20μl PCR反应液中进行混合，这时因弱碱性核酸裂解液提取样本核酸的效率较差，而且所加入的体积较大的核酸溶液很可能含有较多的杂质和PCR扩增反应抑制剂，会导致PCR扩增反应往往无法正常进行，或者虽能进行PCR扩增反应，但会降低样本核酸的检测灵敏度和准确性。
发明内容
针对现有技术的不足之处，本发明提供一种检测样本核酸的试剂、方法和试剂盒，所述试剂、方法和试剂盒均是利用强碱性的核酸提取液提取样本中的核酸，然后将提取出的样本核酸加入PCR反应液中进行中和，同时进行核酸扩增和检测，这样就不需在样本核酸提取时再增加一个对提取出的样本核酸进行中和的步骤，从而极大地节省检测时间，而且也便于进行半自动化或全自动化处理，提高PCR扩增反应效率，增加样本核酸检测的灵敏度和提高核酸检测的准确性。
由于血清、血浆、全血、痰液、尿液、生殖道分泌物、泪液、粪便、脊髓液新鲜组织、石蜡组织切片等样本中的成分非常复杂，直接对样本中的核酸进行检测，会受到其他成分的干扰，因此需要对一些样本进行前处理和保存，然后对提取出的样本核酸进行检测。
针对不同的样本，它们的前处理方法各有不同，比如，全血含有红细胞，故在核酸提取时，可利用常见的红细胞裂解液进行处理，然后再用本发明的核酸提取液进行样本核酸提取；痰液含有大量的粘蛋白和杂质，故在核酸提取时，对该样本进行前处理，通常用NaOH液化、柠檬酸钠、0.5％N-乙酰-L-半胱氨酸(NALC)或二硫苏糖醇(DTT)去除粘蛋白，然后再用本发明的核酸提取液进行样本核酸提取；新鲜组织的预处理方法是先用生理盐水清洗，然后将其捣碎或剪碎，加蛋白酶K消化后，再用本发明的核酸提取液进行样本核酸提取；石蜡组织切片的预处理方法是先用二甲苯等脱蜡剂脱蜡，加蛋白酶K消化后，再用本发明的核酸提取液进行样本核酸提取。
本发明提供一种样本核酸检测试剂，所述样本核酸检测试剂包括核酸提取液和PCR反应液，所述核酸提取液包括碱金属氢氧化物10mM～100mM，Tris-HCl 1mM～25mM，Chelex-100 2％～5％(w/v)，EDTA 0.1％～0.4％(w/v)，所述核酸提取液的pH值为10.5～12.5；所述PCR反应液包括Tris-HCl 5mM～75mM，所述PCR反应液的pH值为8.0～9.0。
进一步地，所述碱金属氢氧化物为NaOH或KOH。
进一步地，所述样本核酸检测试剂还包括浓缩液。
进一步地，所述浓缩液包括NaCl 2.5％～4.5％(w/w)，PEG6000 10％～13％(w/v)。
进一步地，所述PCR反应液的pH值为8.1～8.7。
进一步地，所述PCR反应液的pH值为8.1～8.5。
进一步地，所述PCR反应液的pH值为8.1。
进一步地，所述样本核酸检测试剂在检测HBV核酸中的应用。
进一步地，所述样本核酸包括HBV、人B27基因、HPV 6/11、TB或HCMV核酸。
进一步地，所述样本核酸包括HBV核酸；在所述核酸提取液中，碱金属氢氧化物浓度为25mM、Tris-HCl的浓度为1mM、Chelex-100的浓度为2％(w/v)、EDTA的浓度为0.1％(w/v)，所述核酸提取液的pH值为11.5；在所述PCR反应液中，Tris-HCl的浓度为10mM，所述PCR反应液的pH值为8.1。
本发明还提供一种样本核酸检测方法，所述样本核酸检测方法包括(1)利用核酸提取液提取样本中的核酸，获得样本核酸溶液；(2)将(1)中获得的样本核酸溶液与PCR反应液混合在一起得到混合液，利用所述混合液进行扩增反应，检测样本中的核酸，所述核酸提取液包括碱金属氢氧化物10mM～100mM，Tris-HCl 1mM～25mM，Chelex-100 2％～5％(w/v)，EDTA 0.1％～0.4％(w/v)，所述核酸提取液的pH值为10.5～12.5；所述PCR反应液包括Tris-HCl 5mM～75mM，所述PCR反应液的pH值为8.0～9.0；所述混合液的pH值为8.0～9.0。
进一步地，所述碱金属氢氧化物为NaOH或KOH。
进一步地，在步骤(1)之前，利用浓缩液对样本进行浓缩。
进一步地，所述浓缩液包括NaCl 2.5％～4.5％(w/w)，PEG6000 10％～13％(w/v)。
进一步地，所述PCR反应液的pH值为8.1～8.7。
进一步地，所述PCR反应液的pH值为8.1～8.5。
进一步地，所述PCR反应液的pH值为8.1。
进一步地，所述样本核酸包括HBV、人B27基因、HPV 6/11、TB或HCMV核酸。
进一步地，所述样本核酸包括HBV核酸；在所述核酸提取液中，碱金属氢氧化物浓度为25mM、Tris-HCl的浓度为1mM、Chelex-100的浓度为2％(w/v)、EDTA的浓度为0.1％(w/v)，所述核酸提取液的pH值为11.5；在所述PCR反应液中，Tris-HCl的浓度为10mM，所述PCR反应液的pH值为8.1。
本发明还提供一种样本核酸检测试剂盒，所述核酸检测试剂盒包括核酸提取液和PCR反应液，所述核酸提取液包括碱金属氢氧化物10mM～100mM，Tris-HCl 1mM～25mM，Chelex-100 2％～5％(w/v)，EDTA 0.1％～0.4％(w/v)，所述核酸提取液的pH值为10.5～12.5；所述PCR反应液包括Tris-HCl 5mM～75mM，所述PCR反应液的pH值为8.0～9.0。
进一步地，所述碱金属氢氧化物为NaOH或KOH。
进一步地，所述核酸检测试剂盒还包括浓缩液。
进一步地，所述浓缩液包括NaCl 2.5％～4.5％(w/w)，PEG6000 10％～13％(w/v)。
进一步地，所述PCR反应液的pH值为8.1～8.7。
进一步地，所述PCR反应液的pH值为8.1～8.5。
进一步地，所述PCR反应液的pH值为8.1。
进一步地，所述样本核酸包括HBV、人B27基因、HPV 6/11、TB或HCMV核酸。
进一步地，所述样本核酸包括HBV核酸；在所述核酸提取液中，碱金属氢氧化物浓度为25mM、Tris-HCl的浓度为1mM、Chelex-100的浓度为2％(w/v)、EDTA的浓度为0.1％(w/v)，所述核酸提取液的pH值为11.5；在所述PCR反应液中，Tris-HCl的浓度为10mM，所述PCR反应液的pH值为8.1。
本发明还提供所述核酸检测试剂盒在检测样本中的HBV、人B27基因、HPV 6/11型、结核分枝杆菌或人巨细胞病毒中的应用。
有益效果：先将一定量的样本加入到强碱性的核酸提取液中，将样本中的核酸提取出，然后将提取出的样本核酸溶液加入到PCR反应液(其pH值比核酸提取液中的pH值低)中进行中和，同时还可进行样本核酸扩增和检测，这样就可省去在样本核酸提取时对提取出的样本核酸溶液进行中和这一步骤，从而有效地节省检测时间，降低因步骤繁多增加人工出错的机会，便于进行半自动化或全自动化处理，而且这还会提高样本核酸的PCR扩增反应效率，增加样本核酸的检测灵敏度和准确性。以检测临床血液或血浆样本中可能存在的HBV DNA为例进行说明，本发明通过对现有技术中的核酸提取液和PCR反应液进行改进，利用本发明的核酸提取液(pH值为10.5～12.5)对临床血液或血浆样本中的核酸进行提取，获得提取出的样本核酸溶液，将提取出的样本核酸溶液加入到本发明的PCR反应液(pH值为8.0～9.0)中进行混合，这时不论加入的提取出的样本核酸溶液为2～5μl还是20μl，都不会影响混合后的PCR反应液的pH值，即仍然保持在合适的pH值范围，从而不会影响后续的PCR扩增反应效率，同时还会增加样本核酸的检测灵敏度和准确性。此外，相比于利用常规的弱碱性核酸裂解液提取样本中的核酸，利用本发明的核酸提取液可提高样本核酸的提取效率，降低提取出的样本核酸溶液中可能存在的杂质和PCR反应抑制剂。而且当将本发明的核酸提取液与本发明的PCR反应液配合使用时，往PCR反应液中加入的提取出的样本核酸溶液体积较小(通常为2～5μl)时，也能检测出样本中的核酸，而且具有较高的检测灵敏度；当加入的提取出的样本核酸溶液体积较大时，如将加入20μl提取出的样本核酸溶液到20μl PCR反应液中进行混合，也能够高灵敏地和高准确性地检测出样本核酸。
附图说明
图1为PCR反应液pH值为8.1时，不同浓度HBV阳性样本的荧光PCR检测结果。
图2为PCR反应液pH值为8.9时，不同浓度HBV阳性样本的荧光PCR检测结果。
图3为HBV阳性样本浓度为20IU/ml，改变PCR反应液pH值，当pH值分别为8.1、8.3、8.5、8.7和8.9时的荧光PCR检测结果。
具体实施方式
为了提取样本中的核酸，本发明先将一定量的样本加入到强碱性的核酸提取液中，让样本中的细胞或病毒发生裂解，将它们携带的核酸释放出来，从而被提取出来，这时提取出的样本核酸溶液pH值比较高，会影响后续的核酸PCR扩增反应，甚至会导致核酸扩增反应无法启动。为此，为了降低这种影响，发明人将提取出的样本核酸溶液加入到pH值较低的PCR反应液中进行中和，同时还可进行样本核酸扩增和检测，这样就可省去样本核酸提取时对提取出的样本核酸进行中和这一步骤，可有效地节省检测时间，降低因步骤繁多增加人工出错的机会，便于进行半自动化或全自动化处理，而且这还会提高样本核酸的PCR扩增反应效率，增加样本核酸的检测灵敏度和准确性。
在本发明中，用于提取样本中的核酸的核酸提取液可选自高浓度的强碱溶液或含Chelex-100的强碱性溶液，而且核酸提取液的pH值≥10，优选为10.5～12.5，可有效提取出样本中的核酸。其中，高浓度的强碱溶液可选自为0.05M～0.6M的碱金属氢氧化物溶液，优选为0.05M～0.6M的NaOH溶液或KOH溶液；含Chelex-100的强碱性溶液包括碱金属氢氧化物10mM～100mM，Tris-HCl 1mM～25mM，Chelex-100 2％～5％，EDTA 0.1％～0.4％，所述碱金属氢氧化物优选为NaOH或KOH。
另外，当样本中的所要检测的靶核酸分子浓度比较低时，可优选在核酸提取之前将样本加入浓缩液中进行浓缩，然后再利用本发明的核酸提取液提取浓缩后的样本中的核酸。其中，作为浓缩液的第一个具体的例子，浓缩液可包括NaCl 2.5％～4.5％，PEG6000 10％～13％；作为浓缩液的第二个具体的例子，浓缩液可包括PEG8000 20％，NaCl 13.5％。
当利用本发明的核酸提取液将样本中的核酸提取出后，将提取出的样本核酸溶液以合适的体积用量比例添加到较低pH值的PCR反应液中，将提取出的样本核酸溶液的pH值降低到适合开展PCR扩增反应的水平。在本发明中，PCR反应液的pH值优选为8.0～9.0，其值太高或太 低，都会影响PCR扩增反应的效率，降低检测灵敏度和准确性。PCR反应液含有Tris-HCl，KCl，dNTPs和Mg 2+等，其中Tris-HCl可用Tris-Acetate、Tris-Borate等缓冲系统替换，而且当PCR反应液与提取出的样本核酸溶液混合后，确保Tris-HCl的浓度为5mM～75mM。此外，PCR反应液还可包括Taq酶等用于进行核酸扩增反应的DNA聚合酶、引物和SYBR Green等荧光染料或者TaqMan探针、双杂交探针、分子信标探针或MGB探针等荧光探针。当然，也可在需要进行核酸扩增反应时，才将DNA聚合酶、荧光染料或荧光探针加入到PCR反应液中。
在本发明中，为了进一步降低PCR扩增时可能产生的非特异性扩增，在PCR反应液中加入抗Taq酶抗体，该抗体可结合Taq酶的活性位点，从而使得Taq酶不能发挥作用，只有在变性阶段，抗Taq酶抗体在高温下失活而不能结合到Taq酶的活性位点上，从而使得Taq酶能够发挥催化作用，合成出新的DNA链。
当利用本发明的核酸提取液将样本中的核酸提取出后，利用PCR扩增反应检测样本中的核酸时，事先要选择该样本核酸中的特定DNA或RNA片段，然后针对这种DNA或RNA片段设计至少一对特异性引物，若进行荧光PCR扩增时，可选择加入SYBR Green等荧光染料，或者针对所选择的特定DNA或RNA片段，设计至少一条荧光探针，这样就可根据扩增时产生的荧光强度，检测样本中的特定DNA或RNA片段。
以下实施例进一步说明本发明。这些实施例不是用来限制本发明范围，而是提供对本发明的进一步理解。
实施例1：核酸检测试剂
本实施例中的样本核酸检测试剂包括核酸提取液和PCR反应液，其组成如下所示：
核酸提取液：NaOH 10mM～100mM，Tris-HCl 1mM～25mM，Chelex-100 2％～5％(w/v)，EDTA 0.1％～0.4％(w/v)，pH＝10.5～12.5。
PCR反应液(pH＝8.0～9.0)，包括Tris-HCl 5mM～75mM，此外还包括dNTPs、KCl、Mg 2+和一对引物。当采用荧光PCR检测时，PCR反应液还包括SYBR Green等荧光染料，或TaqMan等荧光探针，优选为TaqMan荧光探针。PCR反应液还可包括酶混合液。当然，也可将酶混合液和PCR反应液独自保存，在需要进行核酸扩增反应时，才将酶混合液加入到PCR反应液中。当检测样本中的核酸是DNA时，酶混合液主要包括一种DNA聚合酶，所述DNA聚合酶可选自Taq DNA聚合酶(简称为Taq酶)、Vent DNA聚合酶、Deep DNA聚合酶、Pfu DNA聚合酶、Tgo DNA聚合酶和KOD1DNA聚合酶等，优选为Taq酶。当检测样本中的核酸是RNA时，酶混合液 主要包括MMLV逆转录酶、Taq酶和RNase抑制剂。当所选择的DNA聚合酶是Taq酶时，不论检测样本中的核酸是DNA或RNA，酶混合液还可包括抗Taq酶抗体。
进一步地，所述样本核酸检测试剂还包括浓缩液：NaCl 2.5％～4.5％(w/v)、PEG6000 10％～13％(w/v)。
当检测血清或血浆样本中的乙型肝炎病毒(HBV)时，浓缩液：NaCl 2.5％(w/v)、PEG600010％(w/v)；核酸提取液：NaOH 25mM、Tris-HCl 1mM、Chelex-100 2％(w/v)、EDTA 0.1％(w/v)，pH＝11.5；PCR反应液，pH 8.1，其中PCR反应液包括Tris-HCl 10mM，此外还包括KCl，dNTPs，Mg 2+，BSA，引物和TaqMan探针，优选地还包括Taq酶。
当检测全血样本中的人B27基因时，浓缩液：NaCl 2.5％(w/v)、PEG6000 13％(w/v)；核酸提取液：NaOH 25mM、Tris-HCl 10mM、Chelex-100 3％(w/v)、EDTA 0.1％(w/v)，pH＝12.0；PCR反应液，pH 8.8，其中PCR反应液包括Tris-HCl 5mM，此外还包括KCl，dNTPs，Mg 2+，BSA，引物和TaqMan探针，优选地还包括Taq酶。
当检测生殖道内分泌物样本中的人乳头瘤病毒6/11型(HPV 6/11)时，核酸提取液：NaOH10mM、Tris-HCl 10mM、Chelex-100 2％(w/v)、EDTA 0.1％(w/v)，pH＝10.5；PCR反应液，pH9.0，其中PCR反应液包括Tris-HCl 75mM，此外还包括KCl，dNTPs，Mg 2+，BSA，引物和TaqMan探针，优选地还包括Taq酶溶液。
当检测痰液样本中的结核分枝杆菌(TB)时，浓缩液：NaCl 2.5％(w/v)、PEG6000 10％(w/v)；核酸提取液：NaOH 25mM、Tris-HCl 25mM；Chelex-100 2％(w/v)；EDTA 0.2％(w/v)，pH＝11.5；PCR反应液，pH 8.0，其中PCR反应液包括Tris-HCl 50mM，此外还包括KCl，dNTPs，Mg 2+，BSA，引物和TaqMan探针，优选地还包括Taq酶。
当检测尿液样本中的人巨细胞病毒(HCMV)时，浓缩液：NaCl 4.5％(w/v)、PEG6000 13％(w/v)；核酸提取液：NaOH 100mM、Tris-HCl 10mM；Chelex-100 5％(w/v)、EDTA 0.4％(w/v)，pH＝12.5；PCR反应液，pH 8.9，其中PCR反应液包括Tris-HCl 10mM，此外还包括KCl，dNTPs，Mg 2+，BSA，引物和TaqMan探针，优选地还包括Taq酶。
其中上述核酸提取液中的NaOH可用KOH等其他碱金属氢氧化物替换。
实施例2：样本核酸提取步骤
利用实施例1中的核酸检测试剂进行如下的样本核酸提取，根据样本的不同可分为以下两种样本核酸提取方法：
方案1：
1.取待测样本200μl，加入200μl浓缩液，振荡混匀后12,000rpm离心10min；
2.弃上清液，沉淀中加入50μl核酸提取液；
3.振荡混匀，100℃水浴或干浴10min，然后12,000rpm离心10min，取上清液，即为所提取出的样本核酸溶液，供PCR扩增用，或-20℃±5℃储存备用。
方案2：
1.取待测样本100μl，振荡混匀后，12,000rpm离心10min，弃上清液，往沉淀中加入50μl核酸提取液；
2.振荡混匀，100℃水浴或干浴10min，然后12,000rpm离心10min，取上清液，即为所提取出的样本核酸溶液，供PCR扩增用，或-20℃±5℃储存备用。
在实际情形中，根据检测所用的样本和检测对象的不同，具体的核酸提取步骤略有不同。比如，当检测血清或血浆样本中的乙型肝炎病毒(HBV)时，其核酸提取步骤为本实施例的方案1；当检测全血样本中的人B27基因时，其核酸提取步骤为本实施例的方案1；当检测生殖道内分泌物样本中的人乳头瘤病毒6/11型(HPV 6/11)时，其核酸提取步骤为本实施例的方案2；当检测痰液样本中的结核分枝杆菌(TB)时，其核酸提取步骤为本实施例的方案1；当检测尿液样本中的人巨细胞病毒(HCMV)时，其核酸提取步骤为本实施例的方案1。
实施例3：荧光PCR检测
PCR检测体系40μl，将PCR反应液按每人份量分装到PCR反应管中，转移至样本处理区。对应的反应管中分别加入实施例2中所提取出的样本核酸溶液4～20μl，盖紧反应管，转移至检测区进行PCR扩增。将各反应管按一定顺序放入PCR仪上，设置检测PCR程序和检测荧光，进行荧光PCR扩增。
当检测血清或血浆样本中的乙型肝炎病毒(HBV)时，PCR检测体系40μl，其中所提取出的样本核酸溶液20μl，PCR反应液20μl。
当检测全血样本中的人B27基因时，PCR检测体系40μl，其中所提取出的样本核酸溶液4μl，PCR反应液36μl。
当检测生殖道内分泌物样本中的人乳头瘤病毒6/11型(HPV 6/11)时，PCR检测体系40μl， 其中所提取出的样本核酸溶液4μl，PCR反应液36μl。
当检测痰液样本中的结核分枝杆菌(TB)时，PCR检测体系40μl，其中所提取出的样本核酸溶液10μl，PCR反应液30μl。
当检测尿液样本中的人巨细胞病毒(HCMV)时，PCR检测体系40μl，其中所提取出的样本核酸溶液4μl，PCR反应液36μl。
实施例4：临床样本HBV检测
样本类型为血清或血浆，以检测乙型肝炎病毒(HBV)为例进行说明。按实施例1、实施例2和实施例3的核酸检测试剂、核酸提取方法和荧光PCR检测，对20例临床已确认阴阳性的临床样本进行检测。
对照方法：取待测样本50μl加入50μl核酸提取液(NaCl 2M、Tris-HCl(pH 8.1)50mM、巯基乙醇1％(v/v)、Chelex-100 5％(w/v)，pH 8.59)，振荡混匀15sec，100℃水浴或干浴10min，然后12,000rpm离心10min，分别取上清液4μl和20μl用于PCR扩增。PCR反应体系包括PCR反应液(含TAKARA PCR buffer,dNTPs，Mg 2+，引物和TaqMan探针，pH 9.05)和Taq酶。这种对照方法的检测灵敏度为500IU/ml。
表1是血清或血浆样本中的HBV检测结果，其中“+”表示阳性结果，“-”表示阴性结果。
表1 血清或血浆样本中的HBV检测结果
Figure PCTCN2018088121-appb-000001
Figure PCTCN2018088121-appb-000002
实验结果显示，在这20例检测样本中，采用本发明所采用的核酸检测试剂、核酸提取方法和荧光PCR检测，共检测出11例HBV阳性，9例HBV阴性。对对照方法而言，当取4μl上清液用于检测时，检测出10例HBV阳性，10例HBV阴性，对照检测有1例与临床样本信息不一致；当取20μl上清液用于检测时，这20例检测样本的检测结果全为阴性，这是因为相对于本发明方法，对照方法中的核酸提取试剂的pH值是非强碱性的，核酸提取效率不高。另外，当样本量增加到20ul时，也可能同时增加了抑制PCR扩增的物质。
实施例5：PCR反应液pH值对HBV检测灵敏度的影响
按实施例1、实施例2和实施例3的核酸检测试剂、核酸提取方法和荧光PCR检测，检测HBV阳性样本：分别将高浓度HBV阳性样本进行梯度稀释，获得一系列低浓度HBV阳性样本，浓度从高到低分别为1000IU/ml、500IU/ml、250IU/ml、100IU/ml、50IU/ml和20IU/ml。
调节PCR反应液的pH值到8.1，针对这些HBV阳性样本的荧光PCR检测结果如图1所示。结果显示，这6个低浓度HBV阳性样本都具有明显的S形扩增曲线，都能稳定地检出。
调节PCR反应液的pH值到8.9时，针对这些HBV阳性样本的荧光PCR检测结果如图2所示。结果显示，在这6个低浓度HBV阳性样本中，浓度为1000IU/ml、500IU/ml和250IU/ml的阳性样本能够稳定检出，而浓度为100IU/ml、50IU/ml和20IU/ml的阳性样本不能够稳定检出。可知，本发明的PCR反应液pH值为8.9时，它的检测灵敏度为250IU/ml，也要优于对照方法的500IU/ml。
此外，针对浓度为20IU/ml的HBV阳性样本，调节PCR反应液的pH值，使之分别为8.1、 8.3、8.5、8.7和8.9，荧光PCR检测结果如图3所示。结果显示，随着PCR反应液pH值下降，对浓度为20IU/ml的HBV阳性样本的扩增效率显著增加，当pH值为8.1时，扩增效率最佳。结果还显示，PCR反应液的pH值分别为8.1、8.3、8.5和8.7时，均能检测20IU/ml的阳性样本，但是pH值分别为8.1、8.3和8.5时的扩增效率、S型扩增曲线的线性及荧光值显著优于pH值为8.7的；pH值为8.1的扩增效率、S型扩增曲线的线性及荧光值显著优于pH值为8.3和8.5的。由此可见，在其他检测条件相同的情况下，改变pH值能够对扩增效率和S型扩增曲线的线性及荧光值造成非常大的影响，而扩增效率和S型扩增曲线的荧光值提高能增加检测的灵敏度，因此pH值为8.1、8.3和8.5时的检测灵敏度明显优于pH值为8.7时的；pH值为8.1时的检测灵敏度明显优于pH值为8.3和8.5时的。这意味着pH值逐渐降低的同时，检测灵敏度逐渐增大。
在检测血清或血浆中的HBV DNA时，本发明意外地发现调整PCR反应液的pH值，即从8.9调整到8.7，从8.7调整到8.3～8.5，从8.3调整到8.1时，取得了意想不到的扩增效果，从而显著提高了检测准确性。
实施例6：临床样本人B27基因检测
样本类型为全血的临床检测，以人B27基因检测为例。按实施例1、实施例2和实施例3的核酸检测试剂、核酸提取方法和荧光PCR检测，对13例临床已确认阴阳性的临床样本进行检测。
对照方法：先将500μl全血样本用红细胞裂解液(NH 4Cl 15mM，NaHCO 3 1mM，EDTA-2Na 0.1mM)进行预处理，离心后，去除上清液，保留沉淀；往沉淀中加入100μl核酸提取液(Tris-HCl(pH 8.0)100mM,Chelex-100 5％,EDTA(pH 8.0)10mM,NaCl 50mM,SDS 2％(w/v)，pH 8.2)，振荡混匀15sec，100℃水浴或干浴10min，然后12,000rpm离心10min，取上清液4μl用于PCR扩增。PCR反应体系包括PCR反应液(含TAKARA PCR buffer,dNTPs，Mg 2+，引物和TaqMan探针，pH 9.05)和Taq酶。
表2是全血样本中的人B27基因检测结果，其中“+”表示阳性结果，“-”表示阴性结果。
表2 全血样本中的人B27基因检测结果
样本编号 本发明检测结果 对照方法检测结果 临床样本信息
1 + + +
2 + + +
3 + + +
4 + + +
5 + - +
6 + + +
7 - - -
8 - - -
9 - - -
10 - - -
11 - - -
12 - - -
13 - - -
实验结果显示，在这13例检测样本中，采用本发明所采用的核酸检测试剂、核酸提取方法和荧光PCR检测，共检测出6例人B27基因阳性，7例人B27基因阴性；对照方法检测出5例人B27基因阳性，8例人B27基因阴性，对照检测有1例与临床样本信息不一致。
实施例7：临床样本HPV6/11检测
样本类型为生殖道分泌物的临床检测，以人乳头瘤病毒6/11型(HPV 6/11)检测为例。按实施例1、实施例2和实施例3的核酸检测试剂、核酸提取方法和荧光PCR检测，对15例临床已确认阴阳性的临床样本进行检测。
对照方法：取待测样本50μl加入50μl核酸提取液(NaCl 2M、Tris-HCl(pH 8.0)50mM、巯基乙醇1％(v/v)、Chelex-100 5％(w/v)，pH 8.59)，振荡混匀15sec，100℃水浴或干浴10min，然后10,000rpm离心5min，取上清液4μl用于PCR扩增。PCR反应体系包括PCR反应液(含TAKARA PCR buffer,dNTPs，Mg 2+，引物和TaqMan探针，pH 9.05)和Taq酶。
表3是生殖道分泌物样本中的HPV检测结果，其中“+”表示阳性结果，“-”表示阴性结果。
表3 生殖道分泌物样本中的HPV基因检测结果
样本编号 本发明检测结果 对照方法检测结果 临床样本信息
1 + + +
2 + - +
3 + + +
4 + - +
5 + + +
6 + + +
7 + + +
8 + + +
9 + + +
10 + + +
11 - - -
12 - - -
13 - - -
14 - - -
15 - - -
实验结果显示：在这15例检测样本中，采用本发明所采用的核酸检测试剂、核酸提取方法和荧光PCR检测，共检测出10例HPV阳性，5例HPV阴性；对照方法检出8例阳性，7例阴性，对照有2例结果与临床样本信息不一致。
实施例8：临床样本TB检测
样本类型为痰液的临床检测，以结核分枝杆菌(TB)检测为例。按实施例1、实施例2和实施例3的核酸检测试剂、核酸提取方法和荧光PCR检测，对10例临床上已确认阴阳性的临床样本进行检测。
对照方法：500μl痰液样本，加入5ml 1M NaOH溶液，振摇液化30min后，14,000离心5min，去除上清液，保留沉淀；往沉淀中加入50μl核酸提取液(SDS 0.1％(w/v),NP40 1％(v/v),Tween-20 1％(v/v),Chelex-100 15％(w/v)，pH 5.02)，振荡混匀15sec，100℃水浴或干浴30min，然后12,000rpm离心10min，取上清液10μl用于PCR扩增。PCR反应体系包括PCR反应液(含TAKARA PCR buffer,dNTPs，Mg 2+，引物和TaqMan探针，pH 9.05)和Taq酶。
表4是痰液样本中的TB检测结果，其中“+”表示阳性结果，“-”表示阴性结果。
表4 痰液样本中的TB检测结果
样本编号 本发明检测结果 对照方法检测结果 临床样本信息
1 + + +
2 + - +
3 + + +
4 + + +
5 + + +
6 - - -
7 - - -
8 - - -
9 - - -
10 - - -
实验结果显示：在这10例检测样本中，采用本发明所采用的核酸检测试剂、核酸提取方法和荧光PCR检测，共检测出5例TB阳性，5例TB阴性；对照方法检测4例阳性，6例阴性，对照有1例样本结果与临床样本信息不一致。
实施例9：临床样本HCMV检测
样本类型为尿液的临床检测，以人巨细胞病毒检(HCMV)测为例。按实施例1、实施例2和实施例3的核酸检测试剂、核酸提取方法和荧光PCR检测，对10例临床已确认阴阳性的临床样本进行检测。
对照方法：收集待测尿液样本250μl，14,000离心5min，去除上清液，保留沉淀；往沉淀中加入250μl核酸提取液(Tris-HCl(pH 8.0)100mM,Chelex-100 5％,EDTA(pH 8.0)10mM,NaCl 50mM,SDS 2％(w/v)，pH 8.2)，振荡混匀15sec，100℃水浴或干浴10min然后13,000rpm离心10min，取上清液4μl用于PCR扩增。PCR反应体系包括PCR反应液(含TAKARA PCR buffer,dNTPs，Mg 2+，引物和TaqMan探针，pH 9.05)和Taq酶。
表5是尿液样本中的HCMV检测结果，其中“+”表示阳性结果，“-”表示阴性结果。
表4 尿液样本中的HCMV检测结果
样本编号 本发明检测结果 对照方法检测结果 临床样本信息
1 + + +
2 + + +
3 + + +
4 + + +
5 + + +
6 - - -
7 - - -
8 - - -
9 - - -
10 - - -
实验结果显示：10例检测样本中，采用本发明所采用的核酸检测试剂、核酸提取方法和荧光PCR检测，共检测出5例HCMV阳性，5例HCMV阴性；对照方法检测出5例阳性，5例阴性；两种检测结果相一致。
实施例10：灵敏度检测结果
按实施例1、实施例2和实施例3的核酸检测试剂、核酸提取方法和荧光PCR检测，检测HBV、人B27基因、HPV、TB、HCMV阳性标样本：分别将阳性标样本进行稀释，浓度分别按照表6稀释，每个浓度重复8次。测试结果如表6所示，其中“+”表示阳性结果，“-”表示低于试剂检测下限。
表6 灵敏度检测结果
Figure PCTCN2018088121-appb-000003
以上结果显示本发明的HBV检测灵敏度为20IU/ml，人B27基因检测灵敏度为5ng/反应，HPV6/11检测灵敏度为250copies/ml，TB检测灵敏度为5个菌/ml，HCMV检测灵敏度为500copies/ml。

Claims (12)

  1. 一种样本核酸检测试剂，所述样本核酸检测试剂包括核酸提取液和PCR反应液，其特征在于，所述核酸提取液包括碱金属氢氧化物10mM～100mM，Tris-HCl 1mM～25mM，Chelex-100 2％～5％(w/v)，EDTA 0.1％～0.4％(w/v)，所述核酸提取液的pH值为10.5～12.5；所述PCR反应液包括Tris-HCl 5mM～75mM，所述PCR反应液的pH值为8.0～9.0。
  2. 如权利要求1所述的样本核酸检测试剂，其特征在于，所述碱金属氢氧化物为NaOH或KOH。
  3. 如权利要求1所述的样本核酸检测试剂，其特征在于，所述样本核酸检测试剂还包括浓缩液，所述浓缩液包括NaCl 2.5％～4.5％(w/w)，PEG6000 10％～13％(w/v)。
  4. 如权利要求1所述的样本核酸检测试剂，其特征在于，所述PCR反应液的pH值为8.1～8.7。
  5. 如权利要求4所述的样本核酸检测试剂，其特征在于，所述PCR反应液的pH值为8.1～8.5。
  6. 如权利要求5所述的样本核酸检测试剂，其特征在于，所述PCR反应液的pH值为8.1。
  7. 如权利要求1～6之一所述的样本核酸检测试剂在检测HBV核酸中的应用。
  8. 一种样本核酸检测试剂盒，所述样本核酸检测试剂盒包括核酸提取液和PCR反应液，其特征在于，所述核酸提取液包括碱金属氢氧化物10mM～100mM，Tris-HCl 1mM～25mM，Chelex-100 2％～5％(w/v)，EDTA 0.1％～0.4％(w/v)，所述核酸提取液的pH值为10.5～12.5；所述PCR反应液包括Tris-HCl 5mM～75mM，所述PCR反应液的pH值为8.0～9.0。
  9. 如权利要求8所述的样本核酸检测试剂盒，其特征在于，所述碱金属氢氧化物为NaOH或KOH。
  10. 如权利要求8所述的样本核酸检测试剂盒，其特征在于，所述样本核酸检测试剂盒还包括浓缩液，所述浓缩液包括NaCl 2.5％～4.5％(w/w)，PEG6000 10％～13％(w/v)。
  11. 如权利要求8所述的样本核酸检测试剂盒，其特征在于，所述PCR反应液的pH值为8.1～8.7。
  12. 如权利要求8所述的样本核酸检测试剂盒，其特征在于，所述PCR反应液的pH值为8.1～8.5。
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