WO2012175013A1 - Système et méthode de diagnostic d'un corps humain ayant un état anormal - Google Patents

Système et méthode de diagnostic d'un corps humain ayant un état anormal Download PDF

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Publication number
WO2012175013A1
WO2012175013A1 PCT/CN2012/077175 CN2012077175W WO2012175013A1 WO 2012175013 A1 WO2012175013 A1 WO 2012175013A1 CN 2012077175 W CN2012077175 W CN 2012077175W WO 2012175013 A1 WO2012175013 A1 WO 2012175013A1
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Prior art keywords
nucleic acid
human body
probe
abnormal state
acid sequence
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PCT/CN2012/077175
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English (en)
Chinese (zh)
Inventor
李伟阳
曾玺
徐佳佳
蒋慧
杨玲
易赏
陈盛培
管彦芳
易鑫
胡学达
李英睿
张秀清
杨焕明
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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Publication date
Priority claimed from CN2011101746862A external-priority patent/CN102839211A/zh
Priority claimed from PCT/CN2011/082855 external-priority patent/WO2013075313A1/fr
Application filed by BGI Shenzhen Co Ltd filed Critical BGI Shenzhen Co Ltd
Priority to CN201280029759.1A priority Critical patent/CN103797130B/zh
Publication of WO2012175013A1 publication Critical patent/WO2012175013A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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

Definitions

  • the invention relates to the field of biomedicine. More specifically, the present invention relates to systems and methods for determining an abnormal state of a human body. Background technique
  • HBV is a global chronic viral infectious disease.
  • the hepatitis B virus infection rate in China is about 60%-70%, and the hepatitis B surface antigen carrying rate is about 7.18% of the total population.
  • the hepatitis B virus infection rate in China is about 60%-70%, and the hepatitis B surface antigen carrying rate is about 7.18% of the total population.
  • about 93 million people in the country carry hepatitis B virus, of which about 30 million are hepatitis B patients.
  • EBV is now known as the main cause of nasopharyngeal carcinoma.
  • Helicobacter pylori is the main cause of gastric cancer, and the detection rate of Helicobacter pylori in gastric mucosal biopsy specimens of patients with chronic gastritis can reach 80% ⁇ 90%, while patients with peptic ulcer are higher, more than 95%, even close to 100 %. Gastric cancer has been localized due to localized epithelial cells, so the detection rate is high.
  • pathogens including HBV, HCV, HIV, EBV, Helicobacter pylori, etc.
  • HBV HBV
  • HCV HIV
  • EBV EBV
  • Helicobacter pylori etc.
  • a non-invasive diagnostic and disease course monitoring method can be established, which can eliminate the damage of pathological tissue sampling of patients infected with pathogens, and can regularly track the infection degree and treatment effect of infected persons, improve the cure rate of infected patients and benefit Timely guidance of medication, for patients with advanced tumors caused by pathogens can also timely detect and judge the healing effect and the possibility of recurrence, can timely and effectively follow up the patient's condition and give the best advice.
  • an object of the present invention is to provide a method having an ability to effectively determine an abnormal state of a human body.
  • Another object of the present invention is to provide a system capable of effectively determining an abnormal state of a human body.
  • the present invention has been completed based on the following findings of the inventors: During the detection of human samples, for protein shields Detection, usually due to limitations of experimental conditions, can not get early state information of the human body. By utilizing the characteristics of the nucleic acid sequence, the human body state can be analyzed as early as possible by performing nucleic acid analysis on the ex vivo human sample.
  • the invention proposes a method of determining an abnormal state of a human body.
  • the method comprises: providing nucleic acid sequence information of a human sample, the nucleic acid sequence information of the human sample is obtained based on detecting the human sample; and nucleic acid sequence information based on the human sample , determining whether the human body has an abnormal state.
  • the method of this embodiment by analyzing the nucleic acid sequence information of the human sample, it is possible to determine whether or not the human body has an abnormal state based on the information contained in the nucleic acid sequence. Since the nucleic acid sequence information is consistent with the nucleic acid information of the in-situ state, it is possible to effectively determine whether the human body has an abnormal state.
  • the above method of determining the abnormal state of the human body may further have the following additional technical features:
  • the nucleic acid sequence information of the human sample is obtained based on nucleic acid sequence detection of the human sample.
  • the nucleic acid sequence information of the human sample can be easily obtained by the nucleic acid sequence detecting method, thereby improving the efficiency of determining the abnormal state of the human body.
  • the nucleic acid sequence detection is carried out by means of a second generation sequencing technique or a third generation sequencing technique. Thereby, the nucleic acid sequence of the human sample can be efficiently detected, and high-throughput deep sequencing can be realized.
  • an efficient and high-precision shield based on the second-generation sequencing technology and the third-generation sequencing technology can realize efficient and high-precision detection of nucleic acid sequence information of human samples, and can be very sensitive to human samples. Trace amounts of nucleic acid are detected.
  • the sample is cells, tissue, blood, body fluids, urine, feces, or a combination thereof of the human body.
  • the method of determining an abnormal state of a human body according to an embodiment of the present invention can be applied to various human body samples, and various abnormal states can be determined according to characteristics of different human body samples.
  • the human sample is plasma or serum.
  • plasma and serum samples of the human body can be obtained by a conventional method, and nucleic acid sequence analysis can be performed thereon, and the abnormal state of the human body can be directly determined.
  • the nucleic acid sequence information comprises sequence information of free nucleic acids in the human sample.
  • the abnormal state of the human body can be determined based on the sequence information of the free nucleic acid. Further, after the sequence information of the free nucleic acid is compared with the normal nucleic acid sequence of the human body or the nucleic acid sequence of the pathogen, a plurality of abnormal states of the human body can be obtained.
  • the sequence information of the free nucleic acid in the human sample is obtained by performing sequencing detection after removing the cells in the human sample.
  • the accuracy and accuracy of the sequencing detection of the free nucleic acid can be improved, so that it is possible to further effectively determine that the human body has an abnormal state.
  • the abnormal state is selected from at least one of the occurrence of a disease, a stage of development of the disease, a therapeutic effect of the disease, and a prognosis.
  • the disease is at least one of a neoplastic disease, an immune disease, and a hereditary disease.
  • the method of determining the abnormal state of the human body can effectively determine whether the human body has a neoplastic disease, an immune disease, or a hereditary disease.
  • the neoplastic disease is selected from the group consisting of lung cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, breast cancer, At least one of bladder cancer, kidney cancer, ovarian cancer, cervical cancer, thyroid cancer, nasopharyngeal cancer, and brain gelatin.
  • the nucleic acid sequence information comprises a nucleic acid fragment sequence selected from at least one of the following: HBV, HPV, EBV, H. pylori
  • the human body is determined to have cervical cancer, liver cancer, nasopharyngeal cancer, At least one type of gastric cancer.
  • the method of determining the abnormal state of the human body can effectively determine whether the human body has liver cancer, cervical cancer, nasopharyngeal cancer or gastric cancer based on the detected nucleic acid sequence information.
  • the probe prior to sequencing the nucleic acid, may be used to remove the nucleic acid containing the specific sequence, and then the nucleic acid remaining after the removal is subjected to sequencing detection.
  • the nucleic acid having a specific sequence can be removed by the probe, so that the accuracy and accuracy of sequence detection of the remaining material can be improved.
  • a probe for removing a nucleic acid of a specific sequence may bind to a consensus sequence in the human genome, or an antibody or protein which can bind to a methylation site in the human genome. Thereby, the accuracy and accuracy of the detection can be further improved.
  • a nucleic acid containing a specific sequence can be captured by a probe, and then the nucleic acid containing the specific sequence is subjected to sequencing detection.
  • the nucleic acid subjected to nucleic acid sequence analysis can be screened in advance by the probe, and the efficiency of the method for determining the abnormal state of the human body can be further improved.
  • the probe is specific for at least one selected from the group consisting of HBV, HPV, EBV, Helicobacter pylori, thereby enabling effective determination of whether the human body has liver cancer, cervical cancer, nasal Pharyngeal or gastric cancer.
  • the invention also provides a nucleic acid probe set.
  • the set of nucleic acid probes comprises a plurality of probes and has the following characteristics:
  • the entire nucleic acid sequence of the nucleic acid probe set covers 70% to 100% of the genomic sequence corresponding to at least one virus selected from the group consisting of HBV, HPV, EBV and H. pylori.
  • the nucleic acid probe set of the present invention has 1 to 20,000 nucleic acid probes; preferably, the nucleic acid probe set has 1000 to 5000 nucleic acid probes; more preferably, the nucleic acid probe set has 2,500 Nucleic acid probes.
  • the abundance of the biotinylated dNTP in the nucleic acid probe set is 1:4.
  • the nucleic acid probes are concentrated with partial overlap between the probes.
  • the length of the probe in the set of nucleic acid probes is from 100 to 500 bp; preferably, the length of the probe is from 200 to 300 bp; Preferably, the probe is 250 bp long.
  • the probe is obtained by PCR amplification using a viral genome as a template.
  • the amplification template is a hepatitis B virus (HBV) genome, a hepatitis C virus (HCV) genome, and an AIDS virus.
  • nucleic acid chip having a surface-immobilized set of nucleic acid probes of the present invention is also provided.
  • nucleic acid probe set and the nucleic acid chip of the invention for detecting the manner in which the virus is integrated in the sample to be tested; preferably, the integration mode is selected from the group consisting of: rearrangement , ectopic, insert, replace, or a combination thereof.
  • a method of preparing a nucleic acid probe of the invention comprising the steps of: a. obtaining a probe source sample;
  • the dNTP of the PCR amplification system is biotin-labeled dNTP, in order to obtain a PCR amplification product with biotin label;
  • the biotin-labeled PCR amplification product obtained in the step (b) is interrupted to obtain a PCR amplification product of the fragmented biotin label, which is a probe.
  • the sample of step (a) has the following characteristics:
  • the sample is a virus sample containing nucleic acid
  • the sample is virions, serum, blood, tissue samples, exfoliated cells, epithelial cells, or a combination thereof; and/or the sample is selected from the group consisting of hepatitis B virus (HBV), hepatitis C virus (HCV), and HIV ( HIV), papillomavirus (HPV), or a combination thereof; and/or
  • the samples are type B HBV and / or type C HBV.
  • step (b) has the following characteristics:
  • step (b) The amplification described in step (b) is to amplify the full length of the viral DNA in the sample.
  • the ratio of the labeled dNTP to the unlabeled dNTP in the step (b) is 1: 2-8; the preferred ratio is 1: 3-6; more preferably, the ratio is 1:4.
  • the interruption in step (c) is interrupted by ultrasound.
  • step (d) is further included: purifying and/or quantifying the probe obtained in step (c).
  • the probe prepared according to the method for producing a nucleic acid probe of the present invention has a length of 100 to 500 bp; preferably, the probe has a length of 200 to 300 bp, more preferably, a probe The length is 250 bp.
  • the present invention also provides a method for detecting a method of genetic integration of a virus in a sample to be tested, comprising the steps of:
  • step ( ⁇ ) performing library construction on the sample obtained in step ( i );
  • step (iii) hybridizing the probe of the present invention to the library obtained in step (ii) to capture a nucleic acid sequence associated with integration of the viral gene;
  • step (i) amplifying the nucleic acid sequence captured in step (iii) to obtain an amplification product related to viral integration; (V) sequencing the amplification product obtained in step (iv) to obtain nucleic acid information related to virus integration .
  • step (i) has the following characteristics:
  • the sample to be tested is tissue, blood, exfoliated cells, epithelial cells; and/or
  • the sample to be tested is derived from a human or non-human mammal, preferably from a human; and/or
  • the sample to be tested is derived from an HBV infected person or a liver cancer patient.
  • step (iii) has the following characteristics:
  • the probe is a denatured single-stranded DNA; and/or Adding a linker blocking molecule and a tag blocking molecule to the hybridization solution; and/or
  • linker blocking molecule The sequence of the linker blocking molecule is set forth in SEQ ID NO: 8.
  • the sequence of the tag blocking molecule is shown in SEQ ID NO: 9 and SEQ ID NO: 10.
  • step (V) the amplification product is hybridized with a sequencing probe immobilized on a solid phase carrier, and subjected to solid phase bridge PCR amplification to form a sequencing cluster;
  • the sequencing clusters were sequenced by the "Synthesis-Side Sequencing" method to obtain nucleic acid information related to the way of virus integration.
  • the library in the step (ii), is constructed as: end-repairing the interrupted genomic DNA, adding a linker, and amplifying the fragment having the linker, and obtaining the expanded with the linker
  • the addition mixture is the sample library.
  • the linker has the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2;
  • the constructed library has the tag sequence as shown in SEQ ID NO: 3 and SEQ ID NO: 4.
  • the invention also provides a kit useful in the methods of the invention described above, the kit comprising:
  • the kit further comprises a reagent selected from the group consisting of:
  • the invention also provides a kit useful in the methods of the invention described above, the kit comprising:
  • the contained components are disposed in different containers, whereby it can be conveniently used.
  • the kit further comprises a reagent selected from the group consisting of: a reagent required for performing PCR amplification, a reagent required for performing a blocking reaction, a reagent required for performing a hybridization reaction, The reagents, or combinations thereof, required to perform the sequencing reaction.
  • the invention also provides a system for determining an abnormal state of a human body.
  • the system includes: a nucleic acid sequence information receiver that receives nucleic acid sequence information of a human sample; and a nucleic acid sequence information analyzer, the nucleic acid sequence information analyzer and the nucleic acid The sequence information receiver is connected, and based on the nucleic acid sequence information of the human sample, determines whether the human body has an abnormal state.
  • the nucleic acid sequence information analyzer prestores at least one selected from the group consisting of: a genomic sequence of a human normal state, a genomic sequence of a pathogen, and a genomic sequence of a normal population.
  • the nucleic acid sequence can be effectively analyzed, and the efficiency of the system for determining the abnormal state of the human body is improved.
  • the pathogen is at least one selected from the group consisting of HBV, HPV, EBV, and H. pylori. Thereby, it is possible to effectively determine whether the human body has liver cancer, cervical cancer, nasopharyngeal cancer or gastric cancer.
  • nucleic acid sequence detecting device further comprising a nucleic acid sequence detecting device, wherein the nucleic acid sequence detecting device is connected to the nucleic acid sequence information receiver, configured to perform nucleic acid sequence detection on the human sample to obtain the nucleic acid sequence information and transmit To the nucleic acid sequence information receiver.
  • the nucleic acid can be directly detected by the sequence and delivered to the nucleic acid sequence receiver, thereby performing nucleic acid sequence analysis to determine whether the human body has an abnormal state, thereby improving the efficiency of determining the abnormal state of the human body.
  • the nucleic acid sequence detecting device is by means of a second generation sequencing technique or a third generation sequencing technique.
  • the nucleic acid sequence of the human sample can be efficiently detected, and high-throughput deep sequencing can be realized.
  • the inventors of the present invention have found that an efficient and high-precision shield based on the second-generation sequencing technology and the third-generation sequencing technology can realize efficient and high-precision detection of nucleic acid sequence information of human samples, and can be very sensitive to human samples. Trace amounts of nucleic acid are detected.
  • nucleic acid capture device connected to the nucleic acid sequence detecting device, wherein the free nucleic acid capture device is provided with a probe, the probe being adapted to capture a nucleic acid containing a specific sequence, and delivering the nucleic acid containing the specific sequence to the nucleic acid sequence detecting device for nucleic acid sequence detection; or the probe is adapted to remove a nucleic acid containing a specific sequence, and the nucleic acid to be removed Delivery to the nucleic acid sequence detecting device for nucleic acid sequence detection.
  • a nucleic acid containing a specific sequence is captured using a probe, and then the nucleic acid containing the specific sequence is subjected to sequencing detection.
  • the nucleic acid subjected to nucleic acid sequence analysis can be screened in advance by the probe, and the efficiency of the method for determining the abnormal state of the human body can be further improved.
  • the probe is specific for at least one selected from the group consisting of HBV, HPV, EBV, H. pylori, which is derived from the set of nucleic acid probes of the invention described above, having All the advantages are not described here.
  • a probe for removing a nucleic acid of a specific sequence may bind to a consensus sequence in the human genome, or an antibody or protein which can bind to a methylation site in the human genome. Thereby, the accuracy and accuracy of the detection can be further improved.
  • FIG. 1 is a schematic flow chart of a method for determining an abnormal state of a human body according to an embodiment of the present invention
  • FIG. 2 is a flow chart showing a method for determining an abnormal state of a human body according to another embodiment of the present invention
  • FIG. 4 is a schematic diagram of a system for determining an abnormal state of a human body according to an embodiment of the present invention
  • FIG. 5 is a schematic diagram of a system according to another embodiment of the present invention
  • FIG. FIG. 6 is a schematic diagram of a system for determining an abnormal state of a human body according to still another embodiment of the present invention
  • FIG. 7 is a partial genome PCR for HBV according to still another embodiment of the present invention. The result of the electrophoresis detection after amplification;
  • FIG. 4 is a schematic diagram of a system for determining an abnormal state of a human body according to an embodiment of the present invention
  • FIG. 5 is a schematic diagram of a system according to another embodiment of the present invention
  • FIG. 8 is an electrophoresis detection result after breaking the full length product of HB V according to still another embodiment of the present invention
  • FIG. 9 is a diagram of the hybridization of the library according to still another embodiment of the present invention.
  • the fragment size detection result of the library and
  • FIG. 10 is another hybridization method according to another embodiment of the present invention. Fragment size detection result library.
  • connection and “connected” should be understood broadly, and may be, for example, a fixed connection, an integral connection, or a Disassembling the connection; it may be a mechanical connection or an electrical connection, or it may be the internal connection of the two components; it may be directly connected or indirectly connected through an intermediate medium, and those skilled in the art may understand the above according to the specific situation. The specific meaning of the term.
  • the present invention has been completed based on the following findings of the present invention:
  • the detection of a protein shield is usually unable to directly correspond to the information of its in-situ state due to limitations of experimental conditions, and the nucleic acid sequence is due to itself.
  • the sexual shield is relatively stable, so the results of nucleic acid analysis on the isolated human sample can directly correspond to the information of the in-situ state, and thus can effectively analyze the state of the human body.
  • a method for determining an abnormal state of a human body includes the following steps: Step 100: providing nucleic acid sequence information of a human sample;
  • Step 200 Determine whether the human body has an abnormal state based on the nucleic acid sequence information of the human sample.
  • the method of this embodiment by analyzing the nucleic acid sequence information of the human sample, it is possible to determine whether or not the human body has an abnormal state based on the information contained in the nucleic acid sequence. Since the nucleic acid sequence information is consistent with the nucleic acid information in the in-situ state, it is possible to effectively determine whether the human body has an abnormal state.
  • nucleic acid may be any inclusion. a polymer of deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA, Degree is not subject to any special restrictions.
  • nucleic acid sequence information means all information contained in a nucleic acid sequence, including but not limited to, a base sequence of a nucleic acid, whether or not it is modified.
  • human body sample is not particularly limited, and may be applied to the type of human body sample of the embodiment of the present invention, including but not limited to cells, tissues, blood, body fluids, urine, excretions or A more specific example of the combination includes plasma or serum.
  • plasma is used as a human sample. The inventors of the present application found that plasma was selected as a research sample, the background noise was small, and the detection result was high in accuracy. Those skilled in the art can select the type of sample to be tested and analyzed as needed.
  • the method of determining an abnormal state of a human body according to an embodiment of the present invention can be applied to various human body samples, and can determine a plurality of abnormal states by taking advantage of the characteristics of different human body samples.
  • abnormal state refers to a state in which the human body is different from the normal state, including but not limited to a physiological state, a mental state, such as a pathological state.
  • the abnormal state is selected from at least one of the occurrence of a disease, a stage of development of the disease, a therapeutic effect of the disease, and a prognosis.
  • the disease is at least one of a neoplastic disease, an immunological disease, and a hereditary disease.
  • the method for determining the abnormal state of the human body in the embodiment of the present invention can effectively determine whether the human body has a neoplastic disease, an immune disease, or a hereditary disease.
  • the neoplastic disease is selected from the group consisting of lung cancer, liver cancer, gastric cancer, esophageal cancer, colorectal cancer, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, ovarian cancer, cervical cancer, thyroid cancer, nasopharyngeal cancer.
  • the nucleic acid sequence information comprises a nucleic acid fragment sequence selected from at least one of the following: HBV, HPV, EBV, H.
  • the human body is determined to have cervical cancer, liver cancer, nasopharyngeal cancer, At least one type of gastric cancer.
  • the method of determining the abnormal state of the human body can effectively determine whether the human body has liver cancer, cervical cancer, nasopharyngeal cancer or gastric cancer based on the detected nucleic acid sequence information.
  • the source of the nucleic acid sequence information of the human sample is not particularly limited.
  • the nucleic acid sequence information of the human sample is obtained based on the detection of the human sample.
  • the method for detecting the nucleic acid sequence information of the human body sample by detecting the human body sample is not particularly limited, and may be obtained by directly performing nucleic acid sequence sequencing analysis on the human body sample, or may be obtained by other methods such as shield spectrum or the like.
  • nucleic acid sequence information of a human sample can be obtained based on direct nucleic acid sequence detection of a human sample. That is, as shown in FIG.
  • nucleic acid sequence detection can be performed by means of a second generation sequencing technique or a third generation sequencing technique.
  • the second-generation sequencing technology represented by the side-synthesis sequencing method and the third-generation sequencing method represented by single-molecule sequencing can efficiently detect nucleic acid sequences of human samples and achieve high-throughput depth. Sequencing.
  • an efficient and high-precision shield based on the second-generation sequencing technology and the third-generation sequencing technology can realize efficient and high-precision detection of nucleic acid sequence information of human samples, and can be very sensitive to human samples. Trace amounts of nucleic acid are detected to further improve the efficiency of determining abnormal conditions in the human body.
  • high-throughput refers to the simultaneous detection of a large number of nucleic acids
  • depth means that the nucleic acid can be repeatedly detected multiple times, for example, in Example 1, 100 rounds of sequencing can be performed. Of course, depending on the sample, the number of repetitions can also be selected as needed.
  • Second generation sequencing technologies currently available include, but are not limited to, Illumina/HiSeq 2000 ⁇ Roche/454 ⁇ ABI/SOLiD.
  • the sequencing method employed is Illumina/HiSeq 2000.
  • a method of analyzing nucleic acid sequence information to determine an abnormal state is not particularly limited. After the nucleic acid sequence information is obtained, the normal genomic information of the human body or the genomic information of the pathogen may be compared, and after the comparison result is obtained, it is determined whether the human body has an abnormal state. It is also possible to determine the abnormal state of the human body based on the content of the nucleic acid sequence in the sample. According to an embodiment of the present invention, it is possible to determine whether or not an abnormal state exists in a human body by analyzing the content of free nucleic acid in a human sample.
  • the inventors of the present invention found that the content of free nucleic acid in the peripheral blood of a cancer patient is much higher than that of normal human peripheral blood (at least about 10 times). According to a further embodiment of the present invention, it is possible to analyze whether there is a mutation site or a modified site such as a methylation site in the free nucleic acid sequence in the human sample, thereby judging whether the individual has some specific abnormal state. Specifically, the operation can be performed by using the usual comparison software. According to an example of the present invention, the SOAP software package is used for the comparison analysis, thereby efficiently analyzing the nucleic acid sequence information and obtaining accurate and accurate information. result.
  • the type of nucleic acid which can be used in the method for determining an abnormal state of a human body in the embodiment of the present invention is not particularly limited, and according to an embodiment of the present invention, the nucleic acid sequence information used includes a human body sample. Sequence information of free nucleic acids. Thereby, the abnormal state of the human body can be determined based on the sequence information of the free nucleic acid. Further, after the sequence information of the free nucleic acid is aligned with the normal nucleic acid sequence of the human body or the nucleic acid sequence of the pathogen, various abnormal states of the human body can be obtained. For ease of understanding, the free nucleic acid is described in detail below.
  • free nucleic acid refers to a nucleic acid in a free state outside the cell, which may be DNA,
  • RNA or other type of nucleic acid The inventors of the present invention found that under normal conditions, a small amount of nucleic acid enters into peripheral blood due to metabolism and becomes a free nucleic acid, and in an abnormal state, such as a cancer patient, the content of free nucleic acid is much higher than that under normal conditions. Free nucleic acid content.
  • the amount of free nucleic acid (such as free radical DNA) depends on the biological characteristics of the tumor, that is, the degree of malignancy of the tumor cells, the degree of invasion, whether metastasis, disease progression, and the like.
  • sequence information of free nucleic acid in a human sample is obtained by performing sequencing detection after removing cells in a human sample. Thereby, the accuracy and accuracy of the sequencing detection of the free nucleic acid can be improved, so that it is possible to further effectively determine that the human body has an abnormal state.
  • step 400 before performing sequencing detection on the nucleic acid, step 400 may be further included, as shown in FIG. 3, that is, using a probe to capture a nucleic acid containing a specific sequence, and then sequencing the nucleic acid containing the specific sequence. Check Measurement.
  • the nucleic acid subjected to nucleic acid sequence analysis can be screened in advance by the probe, and the efficiency of the method for determining the abnormal state of the human body can be further improved.
  • the inventors of the present invention surprisingly found that by this step, the efficiency of detecting free nucleic acid integrated with the human genome can be improved.
  • the type of probe used can be varied depending on the purpose of the assay, i.e., the type of probe used can be selected based on the particular sequence desired.
  • the specific sequence may be an exogenous nucleic acid sequence, a human genome partial sequence containing a mutation site, or a human genome partial sequence containing a modification site such as methylation.
  • the probe is specific for at least one selected from the group consisting of: HBV, HPV, EBV, H.
  • pylori which may be provided in the form of a set of nucleic acid probes comprising a plurality of probes, And it has the following characteristics: (1) one or more biotin-labeled dNTPs per probe; and/or (2) biotin-labeled dNTPs have abundance in the nucleic acid probe set of 1: 6- The entire nucleic acid sequence of the 1:2; and/or (3) nucleic acid probe set covers 70%-100% of the genomic sequence corresponding to at least one virus selected from the group consisting of HBV, HPV, EBV and H. pylori.
  • the nucleic acid probe set of the present invention has 1 to 20,000 nucleic acid probes; preferably, the nucleic acid probe set has 1000 to 5000 nucleic acid probes; more preferably, the nucleic acid probe set has 2,500 Nucleic acid probes.
  • the abundance of the biotinylated dNTP in the nucleic acid probe set is 1:4.
  • the nucleic acid probes are concentrated with partial overlap between the probes.
  • the length of the probe in the set of nucleic acid probes is from 100 to 500 bp; preferably, the length of the probe is from 200 to 300 bp; Preferably, the probe is 250 bp long.
  • the probe is obtained by PCR amplification using a viral genome as a template.
  • the amplification template is a hepatitis B virus (HBV) genome, a hepatitis C virus (HCV) genome, and an AIDS virus.
  • nucleic acid containing a specific sequence such as a consensus sequence in the human genome
  • a specific probe prior to sequencing the nucleic acid, thereby improving a method for determining an abnormal state of a human being. accuracy.
  • a nucleic acid containing a specific sequence may be removed by a probe prior to sequencing detection of the nucleic acid, and then the remaining nucleic acid after the removal may be subjected to sequencing detection.
  • probes for removing a nucleic acid containing a specific sequence are the same as those for capturing a nucleic acid containing a specific sequence, and their types are not particularly limited, and may be a nucleic acid, a protein shield, and any small molecule as long as it is capable of specificity. It can be combined with a specific sequence. Further, in order to remove a nucleic acid containing a specific sequence, the probe to be used can bind to a consensus sequence in the human genome, or can be an antibody or protein capable of binding to a methylation site in the human genome. The type of probe to be used, depending on the specific situation, and whether nucleic acid capture is required for the sample, or whether specific removal is required.
  • the present invention provides a system for determining an abnormal state of a human body, which can effectively implement the above-described method of determining an abnormal state of a human body according to an embodiment of the present invention.
  • the system includes: a nucleic acid sequence information receiver 500, and a nucleic acid sequence information analyzer 600.
  • the nucleic acid sequence information receiver 500 receives the nucleic acid sequence information of the human body sample
  • the nucleic acid sequence information analyzer 600 is connected to the nucleic acid sequence information receiver 500, and determines whether the human body has an abnormal state based on the nucleic acid sequence information of the human body sample.
  • the method of analyzing the nucleic acid sequence information according to the embodiment of the present invention is not particularly limited, and according to a specific example, the nucleic acid sequence information can be obtained from the genomic sequence of the normal state of the human body, the genomic sequence of the pathogen, and the normal population.
  • the genomic sequences are aligned to determine if the human body has an abnormal state.
  • the genomic sequence of the human body, the genomic sequence of the pathogen, and the location of the genomic sequence of the normal population are not particularly limited and can be stored in a remote database.
  • At least one selected from the group consisting of: a genomic sequence of a human normal state, a genomic sequence of a pathogen, and a genomic sequence of a normal population may be pre-stored in the nucleic acid sequence information analyzer 500.
  • the pathogen is at least one selected from the group consisting of HBV, HPV, EBV, and H. pylori.
  • nucleic acid sequence information By comparing the nucleic acid sequence information with the genomic sequence of the normal state of the human body (that is, the genomic information of the same body in different states), it is possible to determine the state change of the human body over a period of time. Further, by comparing the nucleic acid sequence information of the human sample with the genomic sequence information of the normal population, an abnormal state compared with a normal person can be known.
  • the source of the nucleic acid sequence information to be analyzed is not particularly limited.
  • the system for determining an abnormal state of a human body of the present invention may further include a nucleic acid sequence detecting device 700.
  • the nucleic acid sequence detecting device 700 is connected to the nucleic acid sequence information receiver 500 for performing nucleic acid sequence detection on the human body sample to obtain nucleic acid sequence information and transporting it to the nucleic acid sequence information receiver 500, thereby performing analysis and determining whether the human body has an abnormal state.
  • the nucleic acid can be directly subjected to sequence detection and transported to the nucleic acid sequence receiver 500, thereby performing nucleic acid sequence analysis to determine whether the human body has an abnormal state, thereby improving the efficiency of determining that the human body has an abnormal state.
  • the nucleic acid sequence detecting device utilizes a second generation sequencing technique or a third generation sequencing technique.
  • the nucleic acid sequence of the human sample can be efficiently detected, and high-throughput deep sequencing can be realized.
  • the inventors of the present invention have found that an efficient and high-precision shield based on the second-generation sequencing technology and the third-generation sequencing technology can realize efficient and high-precision detection of nucleic acid sequence information of human samples, and can be very sensitive to human samples. Trace amounts of nucleic acid are detected.
  • the system for determining an abnormal state of a human body of the present invention may further include a free nucleic acid capture device 800 connected to the nucleic acid sequence detecting device 700 and free
  • the nucleic acid capture device 800 is provided with probes adapted to capture a nucleic acid containing a specific sequence, and deliver the nucleic acid containing the specific sequence to the nucleic acid sequence detecting device 700 for nucleic acid sequence detection.
  • a nucleic acid containing a specific sequence is captured using a probe, and then the nucleic acid containing the specific sequence is subjected to sequencing detection.
  • nucleic acid subjected to nucleic acid sequence analysis can be screened in advance by the probe, whereby the efficiency of the method for determining the abnormal state of the human body can be further improved. Further, the inventors of the present invention surprisingly found that by this step, the efficiency of detecting free nucleic acid integrated with the human genome can be improved.
  • the type of probe used can be selected based on the purpose of the assay, i.e., the type of probe used can be selected based on the particular sequence desired.
  • the specific sequence may be an exogenous nucleic acid sequence, a human genome partial sequence containing a mutation site, or a human genome partial sequence containing a modification site such as methylation.
  • the probe is for at least one selected from the group consisting of Specific: HBV, HPV, EBV, H.
  • pylori which may be provided in the form of a set of nucleic acid probes comprising a plurality of probes, and which have the following characteristics: (1) 1 or more on each probe Biotin-labeled dNTP; and/or (2) biotin-labeled dNTPs have abundance in the nucleic acid probe set of 1: 6-1: 2; and/or (3) full nucleic acid sequence coverage of the nucleic acid probe set 70%-100% of the genomic sequence corresponding to at least one virus selected from the group consisting of HBV, HPV, EBV, and H. pylori.
  • the nucleic acid probe set of the present invention has 1 to 20,000 nucleic acid probes; preferably, the nucleic acid probe set has 1000 to 5000 nucleic acid probes; more preferably, the nucleic acid probe set has 2,500 Nucleic acid probes.
  • the abundance of the biotinylated dNTP in the concentration of the nucleic acid probe is 1:4.
  • the nucleic acid probes are concentrated with partial overlap between the probes.
  • the length of the probe in the set of nucleic acid probes is from 100 to 500 bp; preferably, the length of the probe is from 200 to 300 bp; Preferably, the probe is 250 bp long.
  • the probe is obtained by PCR amplification using a viral genome as a template.
  • the amplification template is a hepatitis B virus (HBV) genome, a hepatitis C virus (HCV) genome, and an AIDS virus.
  • the amplification template is the B-type HBV genome and/or the C-type HBV genome.
  • the full-length sequence of HPV can be used as a probe, thereby enabling efficient and accurate determination of whether or not HPV in a patient has been integrated with the genome in the patient, thereby judging the degree of cervical lesion in the individual.
  • a specific sequence in the X gene and/or the C gene of HBV can be used as a probe, whereby it is possible to efficiently and accurately determine whether or not the HBV in the patient has been associated with the genome in the patient. Integration occurs to determine whether an individual has liver cancer.
  • a specific sequence of a full-length gene region of HBV can be used as a probe, thereby enabling efficient and accurate determination of whether or not HBV in a patient has been integrated with a genome in a patient, thereby judging The degree of individual hepatitis lesions.
  • a probe capable of removing a nucleic acid containing a specific sequence can be used, so that certain nucleic acids containing a specific sequence can be removed by using a specific probe before sequencing the nucleic acid.
  • a specific probe for example, consensus sequences in the human genome, thereby improving the accuracy of methods for determining abnormal states in humans.
  • a nucleic acid containing a specific sequence may be removed using a probe before sequencing detection of the nucleic acid, and then the remaining nucleic acid after the removal may be subjected to sequencing detection.
  • probes for removing a nucleic acid containing a specific sequence are the same as those for capturing a nucleic acid containing a specific sequence, and their types are not particularly limited, and may be a nucleic acid, a protein shield, and any small molecule as long as it is capable of specificity. It can be combined with a specific sequence.
  • the probe to be used in order to remove a nucleic acid containing a specific sequence, can bind to a consensus sequence in the human genome, or can be an antibody or protein capable of binding to a methylation site in the human genome. The type of probe to be used, depending on the specific situation, and whether nucleic acid capture is required for the sample, or whether specific removal is required.
  • the source of the sample is liver cancer tissue of the same patient, and the liver cancer tissue of this patient has whole genome sequencing information.
  • the fragment added to the adaptor is subjected to PCR to obtain a sample library, and the constructed library carries an Index tag sequence, wherein the Index sequence is as follows:
  • TCTTCCGATCT-3' (SEQ ID NO: 3);
  • TCTTCCGATCT-3' (SEQ ID NO: 4).
  • the designed primers are:
  • PI TTTTTCACCTCTGCCTAATCA ( SEQ ID NO: 5 );
  • P2 AAAAAGTTGCATGGTGCTGG (SEQ ID NO: 6)
  • the PCR reaction was carried out on an AB-9700 PCR machine.
  • the reaction procedure is shown in Table 2.
  • the PCR product was detected by 1% agarose gel electrophoresis, purified with 1.2-1.5 volumes of AMPURE BEADS, and dissolved in 80 ⁇ l of water. It was then purified using 25 OMinElute PCR Purification Kit and dissolved in 60 ⁇ M water. Among them, the results of 1% agarose gel electrophoresis of the PCR product are shown in Fig. 7. The results showed that a fragment of HBV of about 3.2 ⁇ was amplified and purified.
  • HBV genome sequence Hepatitis B virus serotype adr, complete genome .
  • the purified PCR products were all transferred to Covaris interrupted tubules and supplemented with TE buffer to a total volume of 80 ⁇ (Nanodrop was detected as a total of 5 g), and Covaris S2 instrument (Gene Co., Ltd.) was interrupted by Thus, a fragmented product was obtained in which the breaking conditions are shown in Table 3.
  • the size of the fragmented product was detected by 2% agarose gel electrophoresis. The results are shown in Fig. 8. The results showed that the main band of the fragmented product was 250-300 bp, indicating that the obtained fragmented product could be used as a probe for hybridization.
  • the fragment product was purified using a MinElute PCR Purification Kit, dissolved in 40 ⁇ M buffer, and the probe DNA concentration was measured with a Nanodrop instrument so that the probe concentration was about 120 ng/ ⁇ .
  • the resulting probe can be stored at -20 C or -80 °C.
  • the probe must be denatured at 95 °C for 10 minutes before use and then rapidly placed on water to form a single strand.
  • the amount of the library is 1 g
  • the amount of the probe is 600 ng (Quantification of Nanodrop)
  • the blocking molecule is added.
  • the ratio of the amount of the blocking molecule to the amount of the library is l nmol: lg
  • the tag blocking molecule is 1 nmol: 1 ⁇ g.
  • the closed molecular sequence of the linker is:
  • the tag blocking molecular sequence is:
  • CTTCCGATCT-3' (SEQ ID NO: 9); CTTCCGATCT-3' (SEQ ID NO: 10).
  • a 200 ⁇ PCR vial was placed on the PCR machine and hybridized for 24 h at 47 °C.
  • the hybridization mixture was aspirated and added to the magnetic beads prepared in the previous step; the mixture was pipetted 10 times with a pipette; the tube was placed on a PCR machine and incubated at 47 ° C for 45 min (the vortex was shaken every 15 min 3 s to prevent precipitation of magnetic beads); after incubation for 45 min, the mixture was transferred from a 0.2 mL vial into a 1.5 mL EP tube.
  • PFX polymerase purchased from Invirtogen
  • PFX reaction buffer (10 X ) PFX reaction buffer
  • dNTP (10 mM) dNTP
  • CAAGCAGAAGACGGCATACGA SEQ ID NO: 12
  • the purified PCR product was determined by 2100 Bioanalyzer (Agilent) and the size of the insert was shown in Figure 9 and Figure 10.
  • the purified product was 271 bp and 876 bp, respectively.
  • QPCR was accurately quantified and sequenced. In this example, the sequencing of the upper machine was carried out in accordance with the specifications of c-Bot and HISEQ2000Hiseq 2000 published by Illumina/Solexa. 7.
  • the results in Table 7 are the results obtained by using the data on the machine.
  • the samples L-170, L-800, Genome are all from the same liver cancer.
  • L-170 is an insert of 170 bp
  • L-800 is an 800 bp library
  • genome is whole genome sequencing.
  • the accuracy of the self-made probe for capturing the gene fragment, as well as the effect of fragment length, can be derived from Table 7.
  • Stable and reliable sites are fully available by the method of the present invention, and the amount of data required is only about 1% of the genome-wide sequencing data.
  • Example 2 Whole genome analysis of peripheral blood samples from patients with cervical cancer
  • a patient who has cervical cancer is subjected to venous blood sampling to obtain a peripheral blood sample of a patient with cervical cancer, and a plasma sample is obtained by centrifugation.
  • DNA was extracted from plasma samples according to the Tiangen Micro Kit (DP316) genome-wide protocol and quantified using Qubit (Invitrogen, the Quant-iTTM dsDNAHS Assay Kit). The total amount of DNA extracted was 5 to 50 ng.
  • the extracted DNA was constructed according to the standard library-building procedures provided by the manufacturer (see the Illumina standard library specification provided at http://www.illumina.com/).
  • a linker for sequencing is added to both ends of the DNA molecule, and a different tag sequence is added, and then hybridized with a complementary linker on the surface of the sequencing chip to cluster the nucleic acid molecules, and then pass through 100 rounds on the Illumina HiSeq 2000.
  • the deep sequencing cycle yielded a DNA fragment sequence of length lOObp.
  • the DNA samples obtained from the peripheral blood of the tumor patient were subjected to the sequencing operation in batches according to the manufacturer's instructions (see Illumina official publication specification).
  • the sequence information measured in the DNA sequencing part of the step is graphically transformed to obtain sequencing sequence information, and the sequencing shield is removed.
  • the ELA D alignment results for the human genome reference sequence of NCBI version 36 can finally be obtained after a low amount of sequence.
  • the obtained data were compared using the SOAP software package.
  • the sequences of both ends were aligned to the human genome, and one of the strands was aligned to the sequence of the human genome, and the other sequence was The one-end sequence is aligned into the HPV genome sequence to obtain information on HPV recombination in the human genome, including recombination locations in the human genome as well as HPV types.
  • HPV16 type According to the data analysis process in the data analysis section, through the use of high-throughput sequencing platform for deep sequencing and data analysis of cervical cancer samples, 45 HP V integrated genes supported by more than 10 sequencing sequences were detected, and integrated HPV regions were generated. For the E1 area, the integrated HPV type is HPV16 type.
  • Peripheral blood DNA extraction and library preparation methods are the same as in Example 2 except that the sample source is a plurality of liver cancer patients.
  • a nucleic acid probe chip (Mmblegen) will be used to capture a nucleic acid fragment containing a foreign sequence region.
  • the experimental process is as follows:
  • the nucleic acid probe is a HBV genome having a nucleic acid sequence of SEQ ID NO: 7 (Hepatitis B virus serotype adr, complete genome) as a template, with a probe length of 60 bp, and a probe at intervals of 5 bp. Needle, designed to get.
  • PE Block 1.0
  • PE Block 2.0 CTTCCGATCT-3' (SEQ ID NO: 14).
  • Chip washing and sample elution e. Chip washing and sample elution:
  • PCR-amplified the captured DNA library which is divided into 6 tubes and 50 reactions for PCR.
  • the composition of the PCR reaction is as follows:
  • PE Post Primer 1.0 AATGATACGGCGACCACCGAGATC (SEQ ID NO: 15);
  • reaction conditions of PCR are as follows: ( a ) . 98 °C 30 s
  • peripheral blood DNA samples obtained from the tumor patient were subjected to a sequencing operation in batches according to the manufacturer's instructions (see the Illumina/Solexa officially published cBot).
  • a DNA fragment sequence of 100 bp in length was obtained by 100 cycles of sequencing.
  • the sequence information measured in the high-throughput sequencing part of the step is graphically transformed to obtain the sequencing sequence information, and removed.
  • the results of the ELA D alignment of the human genome reference sequence for NCBI version 36 were finally obtained after sequencing the low-profile sequence.
  • the obtained data was analyzed by SOAP software package.
  • the sequences of both ends were aligned to the human genome, and one of the strands was aligned to the sequence of the human genome, and the other was The end sequences are aligned into the HBV genomic sequence to obtain HBV recombination information in the human genome, including recombination positions in the human genome as well as HBV types.
  • the HBV virus integration information in the liver cancer samples was measured, that is, a 733 bp HBV sequence was integrated into the chromosome 1 region.
  • Example 4 Capture and analysis of peripheral blood and tissues of liver cancer samples through target regions
  • a liver cancer patient is subjected to venous blood sampling, a peripheral blood sample of the patient is obtained, and a plasma sample is obtained by centrifugation.
  • DNA was extracted from plasma samples according to the Tiangen Micro Kit (DP316) microgenome protocol Do not quantify with Qubit (Invitrogen, the Quant-iTTM dsDNAHS Assay Kit). The total amount of DNA extracted from each sample is 5 to 50 ng.
  • liver cancer patients and cancer tissue samples were subjected to whole genome extraction of tissue samples, and 3 micrograms were routinely constructed.
  • the main insert of the library insert was 170 bp.
  • the extracted DNA was constructed according to the standard library-building procedures provided by the manufacturer (see the Illumina standard library specification provided at http://www.illumina.com/).
  • a linker for sequencing is added to both ends of the DNA molecule, and a different tag sequence is added, and then hybridized with a complementary linker on the surface of the sequencing chip to cluster the nucleic acid molecules, and then PE101 is sequenced on an Illumina HiSeq 2000.
  • a DNA fragment sequence of length lOObp was obtained.
  • a nucleic acid fragment containing a foreign sequence region will be captured using a HBV (Nimblegen) nucleic acid probe chip.
  • the HBV nucleic acid probe is designed from the eight types of genomes of HBV A, B, C, D, E, F, G, H, and the genome sequence of each is specifically visible in the HB V genome sequence in the well-known database. Specifically, a 60-90 bp-length probe with Bio-tin labeling was synthesized by sliding 10 bp on the genome each time according to the length of the HB V genome. The specific probe is commissioned by the corresponding company.
  • Chip washing and sample elution Elution and recovery according to the standard EZ elution procedure.
  • Flowcell-primer-F 1 AATGATACGGCGACCACCGAGATC ( SEQ ID NO: 17 );
  • Flowcell-primer-Rl CAAGCAGAAGACGGCATACGA (SEQ ID NO: 18).
  • each sample was purified using 1.5 volumes of Amprue Beads.
  • the recovered PCR product was dissolved in 30 ⁇ M ultrapure water and the concentration of Nanodrop 100 was measured at 10
  • peripheral blood DNA samples obtained from the tumor patient were subjected to a sequencing operation in batches according to the manufacturer's instructions (see the Illumina/Solexa officially published cBot).
  • the lower data is removed from the duplicate and the joint contaminated by the joint, and the basic information of the data (the length of the library; the length of the reads; the number of reads; the number of bases; the repetition rate) are counted; and the 50 bp of the two reads of the PE are respectively intercepted.
  • Bases forming a pair of 50bp new reads, ie PE50 readsrcis
  • the new PE50 reads using the soap comparison software (-r 1 -v 2 ) with the human reference sequence (hgl9) and HBV various reference sequences, respectively
  • the alignment selects a pair of reads that are read to the human reference sequence and another to the HBV reference sequence; such reads are likely to cross the HBV insertion site; assemble this part of the reads , using BWA to find the insertion hotspots in the human genome.
  • the plasma DNA of patient A, the amount of 5G (base) data after hybridization is as follows:
  • cervical exfoliated cells were sampled from HPV patients, and the samples were according to Tiangen Micro Kit.
  • the extracted DNA was constructed into a 170 bp DNA library according to the manufacturer's standard library protocol (see http: Il www.illumina.com/ for the Illumina standard library specification).
  • a linker for sequencing is added to both ends of the DNA molecule, and a different tag sequence is added, and then hybridized with a complementary linker on the surface of the sequencing chip to cluster the nucleic acid molecules, and then PE101 is sequenced on an Illumina HiSeq 2000. A DNA fragment sequence of 100 bp in length was obtained.
  • a nucleic acid fragment containing a foreign sequence region will be captured using a (Mygenostics) HPV nucleic acid probe chip.
  • the experimental process is as follows:
  • HPV nucleic acid probes are designed from the genomes of HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, 66, 68, 69, 82
  • the genomic sequences are specifically visible in the HPV genome in a well-known database. Specifically, according to the length of the HPV genome, each time a 10 bp sliding movement on the genome was performed, a probe of 60-90 bp in length and carrying a Bio-tin label was synthesized. The specific probe is commissioned by the corresponding company.
  • Chip washing and sample elution elute according to the standard (MyGenostics) elution process, and the specific steps are as follows:
  • step e repeat "step a to step d" twice;
  • each sample was purified with 1.5 volumes of Amprue Beads and the recovered PCR product was dissolved.
  • DNA samples from cervical exfoliated cells obtained from HPV patients were batched in accordance with the manufacturer's instructions (see Illumina/Solexa officially published cBot).
  • the DNA fragment sequence of 100 bp in length was obtained by 100 cycles of sequencing.
  • the lower data is removed from the duplicate and the joint contaminated by the joint, and the basic information of the data (the length of the library; the length of the reads; the number of reads; the number of bases; the repetition rate) are counted; and the 50 bp of the two reads of the PE are respectively intercepted.
  • HPV16 type infection and the degree of cervical lesions were CIN stage 4 patients, the data volume was 1G, and the results were as follows: Chr2 133034596 16
  • the above method uses the cervical lesions in the fourth stage of CIN patients, and the infected cervical exfoliated cells are tested. The result is that the high-frequency integrated position is accurately found, which proves that the Ming Dynasty is too clear.
  • the system and method for determining abnormal state of a human body can be effectively applied to non-invasive detection of human diseases, and by analyzing nucleic acid sequence information of a human sample, the human body can be accurately determined according to information contained in the nucleic acid sequence. Whether there is an abnormal state.

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Abstract

L'invention concerne un système et une méthode de diagnostic d'un corps humain ayant un état anormal. Le procédé de détermination du corps humain ayant un état anormal comprend : l'apport d'informations de séquence nucléotidique à propos d'un échantillon humain, les informations de séquence nucléotidique à propos de l'échantillon humain étant obtenues sur la base d'un essai d'échantillon humain; et la détermination de savoir si le corps humain présente un état anormal sur la base des informations de séquence nucléotidique à propos de l'échantillon humain.
PCT/CN2012/077175 2011-06-24 2012-06-19 Système et méthode de diagnostic d'un corps humain ayant un état anormal Ceased WO2012175013A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105874068A (zh) * 2013-10-18 2016-08-17 台湾大学 循环癌症生物标志物及其应用
CN108753922A (zh) * 2018-06-04 2018-11-06 广州微芯生物科技有限公司 一种构建转录组测序文库的方法及相应的接头序列和试剂盒
CN109957492A (zh) * 2017-12-26 2019-07-02 安诺优达基因科技(北京)有限公司 一种用于二代测序dna文库构建的自动化液体处理工作站

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11319602B2 (en) 2017-02-07 2022-05-03 Tcm Biotech Internationl Corp. Probe combination for detection of cancer

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101921874A (zh) * 2010-06-30 2010-12-22 深圳华大基因科技有限公司 基于Solexa测序法的检测人类乳头瘤病毒的方法

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CN1932033A (zh) * 2006-09-22 2007-03-21 东南大学 基于微阵列芯片的核酸测序方法
US20080268442A1 (en) * 2007-04-24 2008-10-30 Igd Intel, Llc Method and system for preparing a blood sample for a disease association gene transcript test
EP2334802A4 (fr) * 2008-09-09 2012-01-25 Life Technologies Corp Procédés de génération de bibliothèques spécifiques de gènes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101921874A (zh) * 2010-06-30 2010-12-22 深圳华大基因科技有限公司 基于Solexa测序法的检测人类乳头瘤病毒的方法

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105874068A (zh) * 2013-10-18 2016-08-17 台湾大学 循环癌症生物标志物及其应用
CN105874068B (zh) * 2013-10-18 2020-04-10 台湾大学 游离核酸和生物标志物
CN109957492A (zh) * 2017-12-26 2019-07-02 安诺优达基因科技(北京)有限公司 一种用于二代测序dna文库构建的自动化液体处理工作站
CN108753922A (zh) * 2018-06-04 2018-11-06 广州微芯生物科技有限公司 一种构建转录组测序文库的方法及相应的接头序列和试剂盒

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