WO2022100766A1 - 人微卫星不稳定位点的检测方法及其应用 - Google Patents

人微卫星不稳定位点的检测方法及其应用 Download PDF

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WO2022100766A1
WO2022100766A1 PCT/CN2022/072414 CN2022072414W WO2022100766A1 WO 2022100766 A1 WO2022100766 A1 WO 2022100766A1 CN 2022072414 W CN2022072414 W CN 2022072414W WO 2022100766 A1 WO2022100766 A1 WO 2022100766A1
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msi
seq
primer pair
site
probe
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江萤
黄昕
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江萤
黄昕
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Priority to US18/253,116 priority Critical patent/US20230416839A1/en
Priority to EP22723000.0A priority patent/EP4245858A1/en
Publication of WO2022100766A1 publication Critical patent/WO2022100766A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to the fields of medicine and biotechnology, in particular, to a method for detecting unstable sites of human microsatellites and applications thereof.
  • DNA mismatch repair (MMR) gene mutation or promoter methylation is one of the important causes of cancer.
  • the protein encoded by the MMR gene monitors base mismatches during DNA replication to avoid errors.
  • Mismatch repair proteins include two families, MutS and MutL.
  • the former includes MSH2/MSH3 and MSH6, and the latter includes MLH1, MLH3, PMS1 and PMS2.
  • MSH2 and MLH1 respectively form complexes with their homologous mismatch repair proteins to function.
  • MSH2 forms complexes MutS ⁇ and MutS ⁇ with MSH6 and MSH3, respectively
  • MLH1 forms complexes MutL ⁇ , MutL ⁇ or MutL ⁇ with PMS2, PMS1 or MLH3, respectively.
  • MutS ⁇ or MutS ⁇ mismatch repair base The steps of MutS ⁇ or MutS ⁇ mismatch repair base are as follows: after identifying the mismatched base, it binds to the DNA base, then binds to MutL, activates ATPase, hydrolyzes the mismatched base, and activates endonuclease I at the same time to excise and repair mismatched bases.
  • Microsatellite is a short tandem repeat sequence, the length of each unit is between 1 and 6 bp. It is widely present in prokaryotic and eukaryotic genomes and has high genetic stability. However, when the mismatch repair gene function of cells is abnormal, the number of repeat nucleotides of microsatellites in daughter cells can increase or decrease, resulting in changes in the length of microsatellites. This phenomenon is called microsatellite instability (MSI, MSI). ).
  • MSI DNA mismatch repair function
  • Many important growth regulation-related genes such as type II TGF- ⁇ , IGF2R, PTEN, BAX, contain microsatellites in their coding regions or promoter regions.
  • MSI caused by abnormal mismatch repair can cause missense mutations or frameshift mutations in these genes during the replication process, which makes the accumulation of such replication errors and becomes an important factor leading to tumorigenesis. Therefore, MSI can be used as a molecular marker of tumors.
  • high-frequency MSI has been found in various tumors, such as colorectal cancer, gastric cancer, small bowel cancer, endometrial cancer, etc.
  • the purpose of the present invention is to provide a method for detecting tumor MSI with high sensitivity, high specificity, eliminating the possibility of sample contamination and strong anti-interference ability.
  • the invention also provides a detection method and application of human microsatellite instability (MSI) site.
  • MSI human microsatellite instability
  • a human microsatellite instability (Microsatellite Instability, MSI) site detection reagent for preparing a diagnostic reagent or kit for use in Diagnosing and/or prognosing MSI-related diseases;
  • MSI site is selected from one or more sites of the following group A:
  • the detection reagent is selected from the group consisting of primers, probes, guide RNAs (guild RNAs for CRISPR), chips, or combinations thereof.
  • the MSI site is selected from the following group:
  • the MSI site includes at least 1, at least 2, at least 3, at least 4, or 5 selected from Z1-Z7.
  • the MSI sites include Z2, Z3, Z4, Z5 and Z7.
  • the MSI site includes at least one, at least two, or at least three selected from Z1 to Z10.
  • the MSI site further includes additional MSI sites other than Z1-Z10.
  • the MSI site includes (a) one or more (eg 2, 3, 4, or 5) sites selected from Z2, Z3, Z4, Z5 and Z7; and ( b) Additional MSI sites in addition to Z2, Z3, Z4, Z5 and Z7.
  • the MSI sites include (a) Z2, Z3, Z4, Z5 and Z7; and (b) additional MSI sites other than Z2, Z3, Z4, Z5 and Z7.
  • the additional MSI site is selected from the group consisting of BAT-26, BAT-25, MONO-27, NR-21, NR-24, D5S346, D2S123, D17S250 or a combination thereof.
  • the MSI-related disease is tumor or cancer.
  • the tumor or cancer is selected from the group consisting of colorectal cancer, endometrial cancer, uterine sarcoma, gastric cancer, small bowel cancer, cervical cancer, liver cancer, esophageal cancer, pancreatic cancer, ovarian cancer, gallbladder cancer Cancer, testicular cancer, prostate cancer, fallopian tube cancer, vulvar cancer, adrenocortical cancer, primary abdominal tumor, bile duct cancer, breast cancer, neuroendocrine tumor, thymic cancer, thyroid cancer, small cell lung cancer, tumors of unknown primary origin, etc. .
  • the diagnostic reagent or kit is used for detection selected from the group consisting of serum detection, plasma detection, cell detection, and tissue sample detection.
  • the detection is PCR detection or sequencing detection; preferably, digital PCR (digital PCR, ddPCR) detection.
  • the detection includes single detection or multiple detection (such as n-multiple detection, wherein n is any positive integer from 2 to 20, preferably n is 3-15, more preferably 5- 10).
  • the multiplex detection includes: performing multiplex amplification in one reaction system, and then performing detection.
  • the "subsequent detection” includes fluorescence detection, capillary electrophoresis, sequencing, or a combination thereof.
  • a reagent for detecting a human microsatellite instability (Microsatellite Instability, MSI) site the site is selected from position 1 to position 10 in the hg38 chromosome (SEQ ID NO.: 1 ⁇ 10); wherein, the reagent is selected from the following group:
  • a second primer pair for detecting chr11 site 2 (SEQ ID NO.: 2) MSI, wherein the second primer pair includes the primers shown in SEQ ID NO.: 19 and 20;
  • a fifth primer pair for detecting chr20 site 5 (SEQ ID NO.: 5) MSI, wherein the fifth primer pair includes the primers shown in SEQ ID NO.: 29 and 30;
  • an eighth primer pair for detecting chr6 site 8 (SEQ ID NO.: 8) MSI, wherein the eighth primer pair includes the primers shown in SEQ ID NO.: 17 and 18;
  • sequence where the site 1 is located is located at chr3:30650236-30650508.
  • sequence where the site 2 is located is located at chr11:106739898-106740117.
  • sequence where the site 3 is located is located at chr16:18841298-18841518.
  • sequence where the site 4 is located is located in chr17:19411505-19411722.
  • sequence where the position 5 is located is located in chr20:62921533-62921750.
  • the sequence where the position 6 is located is located at chr2:47408320-47408461.
  • sequence where the position 7 is located is located at chr2:147906719-147906938.
  • the sequence where the position 8 is located is located at chr6:142407071-142407290.
  • sequence where the position 9 is located is located at chr14:93268657-93268877.
  • sequence where the site 10 is located is located in chr20:47779916-47780134.
  • the sites for detecting human microsatellite instability are preferably site 2, site 3, site 4, site 5 and site 7.
  • the reagent also includes:
  • the first probe used in combination with the first primer pair wherein the first probe is selected from the following group: the probe shown in SEQ ID NO.:31, the probe shown in SEQ ID NO.:32 probes or combinations thereof; and/or
  • (b1) a second probe used in conjunction with a second primer pair, wherein the second probe is selected from the group consisting of: the probe shown in SEQ ID NO.:39, the probe shown in SEQ ID NO.:40 probes, or combinations thereof; and/or
  • (c1) the third probe used in conjunction with the third primer pair, wherein the third probe is selected from the following group: the probe shown in SEQ ID NO.:43, the probe shown in SEQ ID NO.:44 probes, or combinations thereof; and/or
  • (d1) the fourth probe used in conjunction with the fourth primer pair, wherein the fourth probe is selected from the following group: the probe shown in SEQ ID NO.:45, the probe shown in SEQ ID NO.:46 probes, or combinations thereof; and/or
  • the fifth probe used in conjunction with the fifth primer pair wherein the fifth probe is selected from the following group: the probe shown in SEQ ID NO.:49, the probe shown in SEQ ID NO.:50 probes, or combinations thereof; and/or
  • (f1) the sixth probe used in conjunction with the sixth primer pair, wherein the sixth probe is selected from the following group: the probe shown in SEQ ID NO.:33, the probe shown in SEQ ID NO.:34 probes, or combinations thereof; and/or
  • (g1) the seventh probe used in conjunction with the seventh primer pair, wherein the seventh probe is selected from the following group: the probe shown in SEQ ID NO.:35, the probe shown in SEQ ID NO.:36 probes, or combinations thereof; and/or
  • the ninth probe used in conjunction with the ninth primer pair wherein the ninth probe is selected from the following group: the probe shown in SEQ ID NO.:41, the probe shown in SEQ ID NO.:42 probes, or combinations thereof; and/or
  • (j1) the tenth probe used in conjunction with the tenth primer pair, wherein the tenth probe is selected from the following group: the probe shown in SEQ ID NO.:47, the probe shown in SEQ ID NO.:48 probe, or a combination thereof.
  • the first probe to the tenth probe are single-stranded nucleic acid probes.
  • L1 is a fluorophore and L3 is a quencher; or L3 is a fluorophore and L1 is a quencher;
  • L2 is the specific complementary nucleic acid sequence of nucleotides
  • "-" is a chemical bond, a linking group, or a linker composed of 1-3 nucleotides.
  • the L2-specific nucleic acid sequence specifically targets position 1 to position 10 in hg38.
  • the L2 contains locked nucleotide modifications.
  • sequence of described L2 is selected from the following group:
  • the fluorescent groups are independently located at the 5' end, the 3' end and the middle of the nucleic acid probe.
  • the fluorescent group and the quenching group are each independently located at the 5' end, the 3' end, and/or the middle.
  • the fluorescent group includes a fluorescent group cross-linked with the DNA probe.
  • the fluorescent group is selected from the group consisting of FAM, VIC, HEX, FITC, BODIPY-FL, G-Dye100, FluorX, Cy3, Cy5, Texas Red, or a combination thereof.
  • the quenching group is selected from the following group: DABCYL, TAMRA, BHQ 1, BHQ 2, BHQ3, MGB, BBQ-650, TQ1-TQ6, QSY 7carboxylic acid, TQ7, eclipse, or its combination.
  • kits comprising the reagent for detecting MSI sites according to the second aspect of the present invention.
  • the MSI site is selected from one or more sites in group A (ie, one or more of Z1-Z23), preferably the MSI site is selected from Z1- One or more of the Z10.
  • the kit is used to detect MSI sites in human DNA samples from tumor cells, tumor tissues or suspected tumor tissues.
  • the product is for detection of circulating cell-free DNA (cell-free DNA, cfDNA) samples.
  • the cfDNA is from the blood, plasma, or serum of the subject.
  • the subject is a tumor patient.
  • the tumor is selected from the group consisting of colorectal cancer, endometrial cancer, uterine sarcoma, gastric cancer, small bowel cancer, cervical cancer, liver cancer, esophageal cancer, pancreatic cancer, ovarian cancer, gallbladder cancer, Testicular cancer, prostate cancer, fallopian tube cancer, vulvar cancer, adrenocortical cancer, primary abdominal tumor, bile duct cancer, breast cancer, neuroendocrine tumor, thymic cancer, thyroid cancer, small cell lung cancer, tumor of unknown primary, etc., or its combination.
  • a combination of detection reagents includes n detection reagents for detecting human microsatellite instability (Microsatellite Instability, MSI) sites, wherein the The MSI sites are selected from the sites of Group A, and n is a positive integer ⁇ 2.
  • MSI human microsatellite instability
  • n is 2-25, more preferably n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, or 23.
  • the MSI site is selected from one or more sites in group A (ie, one or more of Z1-Z23), preferably the MSI site is selected from Z1- One or more of the Z10.
  • the detection reagent is selected from the group consisting of primers, probes, chips, or combinations thereof.
  • the combination of detection reagents is a combination of detection reagents for detecting MSI sites of Z2, Z3, Z4, Z5 and Z7.
  • a method for detecting MSI in a sample to be tested comprising the steps of:
  • PCR reaction system contains a sample to be tested as a template and a primer pair for amplification, and the primer pair is selected from the following group:
  • a second primer pair for detecting chr11 site 2 (SEQ ID NO.: 2) MSI, wherein the second primer pair includes the primers shown in SEQ ID NO.: 19 and 20;
  • a fifth primer pair for detecting chr20 site 5 (SEQ ID NO.: 5) MSI, wherein the fifth primer pair includes the primers shown in SEQ ID NO.: 29 and 30;
  • an eighth primer pair for detecting chr6 site 8 (SEQ ID NO.: 8) MSI, wherein the eighth primer pair includes the primers shown in SEQ ID NO.: 17 and 18;
  • step (S2) PCR reaction is performed on the PCR reaction system of step (S1), thereby obtaining an amplification product;
  • MSI-H If the number of MSI sites in the sample P MSI ⁇ 2, it is determined to be highly unstable (MSI-H);
  • MSI-L low-level instability
  • MSI-L low-grade instability
  • the analysis is qualitative analysis, quantitative analysis or semi-quantitative analysis.
  • the reaction system is a sequencing reaction system, including first-generation, second-generation, and third-generation sequencing reaction systems.
  • the PCR reaction system is a digital PCR reaction system.
  • the digital PCR is ddPCR.
  • the method is an in vitro method.
  • Figures 1A-1H show the results of PCR electrophoresis (M: DNA Marker) of 40 sites in the DNA of 293T and HCT116 cells with primers.
  • Figures 2A-2E show the two-dimensional digital PCR map of the 5 selected sites; wherein, Figure A is a two-dimensional digital PCR map of SEQ ID NO.: 1; Figure B is a digital PCR two-dimensional map of SEQ ID NO.: 2 Dimensional diagram; Figure C is the two-dimensional digital PCR diagram of SEQ ID NO.: 3; Figure D is the two-dimensional digital PCR diagram of SEQ ID NO.: 4; Figure E is the two-dimensional digital PCR diagram of SEQ ID NO.: 5 . Among them, gray is no amplified fragment, green is positive signal, and orange is negative signal.
  • Figures 3A-3E show two-dimensional images of digital PCR for MSI detection in HCT-116 cell line; wherein, image A is site 1 (SEQ ID NO.: 1); image B is site 2 (SEQ ID NO.: 2 ); C picture is site 3 (SEQ ID NO.: 3); D picture is site 4 (SEQ ID NO.: 4); E picture is site 5 (SEQ ID NO.: 5).
  • Figures 4A-4E show two-dimensional digital PCR diagrams of RKO cell line MSI detection; wherein, picture A is site 1 (SEQ ID NO.: 1); picture B is site 2 (SEQ ID NO.: 2); Picture C is site 3 (SEQ ID NO.:3); picture D is site 4 (SEQ ID NO.:4); picture E is site 5 (SEQ ID NO.:5).
  • Figures 5A-5E show two-dimensional digital PCR diagrams for MSI detection in SW48 cell line; wherein, the A picture is site 1 (SEQ ID NO.: 1); the B picture is site 2 (SEQ ID NO.: 2); Picture C is site 3 (SEQ ID NO.:3); picture D is site 4 (SEQ ID NO.:4); picture E is site 5 (SEQ ID NO.:5).
  • Figure 6A shows the results of MSI detection of case SY020 by capillary electrophoresis by Human Microsatellite Instability Detection Kit 2B3D (BAT-25, BAT-26, D5S346, D17S250, D2S123).
  • Figure 6B shows the results of MSI detection of novel microsatellite loci (Z2, Z3, Z4, Z5 and Z7) of case SY020 by capillary electrophoresis.
  • Figure 7A shows the results of MSI detection of case SY028 by capillary electrophoresis by Human Microsatellite Instability Detection Kit 2B3D (BAT-25, BAT-26, D5S346, D17S250, D2S123).
  • Figure 7B shows the results of MSI detection of novel microsatellite loci (Z2, Z3, Z4, Z5 and Z7) of case SY028 by capillary electrophoresis.
  • Figure 8A shows the results of MSI detection of novel microsatellite loci (Z2, Z3, Z4, Z5 and Z7) of case SY020 by digital PCR method.
  • Figure 8B shows the results of MSI detection of novel microsatellite loci (Z2, Z3, Z4, Z5 and Z7) of case SY028 by digital PCR method.
  • Figures 9A-9B show two-dimensional digital PCR diagrams of the MSI detection results of case SY029; wherein, picture A is the detection result of case SY029 blood sample site 5; B picture is the detection result of case SY029 tissue sample site 5.
  • the present inventors identified multiple novel MSI loci for the first time. Studies have shown that these new MSI loci are highly correlated with MSI-related diseases (such as colon cancer and other tumors), so they can be used for auxiliary diagnosis and/or prognosis of MSI-related diseases.
  • the inventors have also optimized the primer and/or probe sequences based on these new MSI sites, combined with detection methods such as digital PCR, to effectively improve the detection effect of MSI and use plasma/serum samples for detection, thus breaking through the current situation. There are some problems in gene mutation detection technology, such as pathological tissue as starting material, low accuracy and low sensitivity. On this basis, the present inventors have completed the present invention.
  • the present invention selects 40 microsatellite sites consisting of single nucleotide repeats (polyA or polyT) in the human genome, designs amplification primer pairs respectively, and performs PCR in MSI-unstable colorectal cancer cell lines Amplification. From the microsatellite loci screened by the amplification results, the optimal 10 microsatellite loci were further selected, and probes that could simultaneously detect stable and unstable microsatellite loci were designed. The sequence is optimized, and the optimized primers and probes are used for digital PCR, and the optimal conditions for low-frequency detection are optimized. The method was validated in tumor tissue, blood and other samples of patients with primary colorectal cancer.
  • polyA or polyT single nucleotide repeats
  • the MSI detection method of the present invention has high specificity and high sensitivity, is quick and convenient to operate, and simple to interpret, and can not only accurately detect tissue and body fluid samples, but also process difficult samples such as plasma/serum, and be used for MSI liquid biopsy of different samples .
  • the term “about” may refer to a value or composition within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
  • the expression “about 100” includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the terms "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “consisting essentially of,” or “consisting of.”
  • blood can be plasma, serum, but does not include blood cells.
  • isolated refers to the separation of a substance from its original environment (in the case of a natural substance, the original environment is the natural environment).
  • the original environment is the natural environment.
  • polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances present in the natural state. .
  • Sequence identity is obtained by comparing two aligned sequence, and determine the number of positions where identical residues occur. Typically, this is expressed as a percentage.
  • the measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.
  • Microsatellite is a short tandem repeat sequence, the length of each unit is between 1 and 6 bp. It is widely present in prokaryotic and eukaryotic genomes and has high genetic stability. However, when the mismatch repair gene function of cells is abnormal, the number of repeat nucleotides of microsatellites in daughter cells can increase or decrease, resulting in changes in the length of microsatellites. This phenomenon is called microsatellite instability (MSI, MSI). ).
  • MSI-L colorectal cancer Because there is no significant difference between MSI-L colorectal cancer and MSS tumor in clinical manifestations and pathological changes, MSI-L colorectal cancer is usually classified as MSS tumor type. Clinical studies have found that MSI-H colorectal cancer has a lower risk of metastasis, suggesting a better prognosis than MSS colorectal cancer; MSI-H colorectal cancer is not sensitive to chemotherapy drugs such as 5-fluorouracil and cisplatin, but is less sensitive to irinotecan. and other chemotherapeutic drugs, while MSI colorectal cancer is more sensitive to radiotherapy.
  • chemotherapy drugs such as 5-fluorouracil and cisplatin
  • the detection result has changes in two or more sites, it is determined to be MSI-H, indicating that the patient has a good prognosis, and 5-fluorouracil and platinum should not be used mainly If the test result is MSI-L (only one site changes) or MSS (no site changes), it indicates that the patient's prognosis may be poor, and it is not 5-fluorouracil- and platinum-based regimens were rejected.
  • the "new effective site” in the present invention refers to the selected 40 single nucleotide repeat (polyA or polyT) sites in the human genome. After screening with designed primers PCR, compared with human normal cells, Locuses that can be detected at low frequencies in MSI-unstable colorectal cancer cell lines.
  • the "new effective site" in the MSI site is selected from Group A in Table 4 of Example 1:
  • Digital PCR (digital PCR) technology based on the single-molecule PCR method for counting nucleic acid quantification, is an absolute quantitative method. It mainly adopts the microfluidic or microdroplet method in the current hot research field of analytical chemistry to disperse a large amount of diluted nucleic acid solution into the microreactors or microdroplets of the chip, and the number of nucleic acid templates in each reactor is less than or equal to 1 indivual.
  • the fluorescence signal of each droplet is analyzed after the amplification, the reactor with the nucleic acid molecule template will give a fluorescence signal, and the reactor without the template will have no fluorescence signal. Based on the relative proportions and the volume of the reactor, the nucleic acid concentration of the original solution can be calculated.
  • digital PCR can accurately quantitatively analyze and detect target nucleic acid molecules with high sensitivity.
  • the method of analyzing the results of conventional qPCR is an analog method, wherein the digital PCR method, the results of which are analyzed by digital methods (since the resulting signal has a value of "0" or "1"), has the ability to analyze large volume samples.
  • Digital PCR is a technique that allows absolute quantification of DNA samples using single-molecule counting without a standard curve, and allows for more precise absolute quantification of individual droplets per well by PCR (see Gudrun Pohl and le-Ming Shih). , Principle and applications of digital PCR (Principle and applications of digital PCR), Expert Rev. Mol. Diagn. 4(1), 41-47(2004)).
  • Digital PCR has the advantages of high sensitivity, accurate quantification without standard curve, and simple operation.
  • each droplet containing the sample gene template, amplification primers and fluorescent probes prepared for dilution to an average copy number of 0.5-1 is dispensed into a single well and microemulsion PCR is performed.
  • Wells showing a fluorescent signal are then counted as a value of "1" because samples with a gene copy number of 1 were assigned to the wells and showed a fluorescent signal after amplification, while wells that did not show a signal were counted as "0" because Samples with a gene copy number of 0 were dispensed into the wells and showed no fluorescent signal due to no amplification. In this way, absolute quantification can be achieved.
  • the nucleic acid sequences of the primers include unmodified or modified primer sequences.
  • the inventors can significantly improve the specificity of the probe by modifying the probe with locked nucleotides, thereby improving the sensitivity and specificity of the detection result.
  • the modification is selected from: Phosphorylation, Biotin, Digoxigenin, internal amino modification, 5' amino modification, 3' amino modification, sulfhydryl group (Thiol), Spacer, Phosphorthioate, DeoxyUridine (dU), DeoxyInosine (dI), or a combination thereof.
  • Phosphorylation modification 5' phosphorylation can be used for linkers, cloning and gene construction, and ligase-catalyzed ligation reactions. 3' phosphorylation is resistant to 3' exonuclease digestion in related experiments, and is also used to prevent DNA polymerase-catalyzed DNA chain extension reactions.
  • Biotin modification The primers are labeled with biotin, which can be used for non-radioactive immunoassays to detect proteins, intracellular chemical staining, cell isolation, nucleic acid isolation, hybridization to detect specific DNA/RNA sequences, ion channel conformational changes, etc.
  • Digaoxin is linked to the C5 position of uracil via an 11-atom spacer arm, and the hybridized Digaoxin probe can be detected by anti-Digaoxin antibody.
  • Digao-labeled probes can be used in various hybridization reactions, such as DNA-DNA hybridization (Southern blotting), DNA-RNA hybridization (Northern blotting), dot blotting (Dot blotting), clone hybridization, in situ hybridization and ELISA (ELISA).
  • C6-dT aminolinker is mainly used to add to thymine residues for internal modification.
  • the modified amino group is 10 atoms away from the main chain, which can be used for further labeling and enzymatic ligation (such as alkaline phosphatase), and dT-Dabcyl, dT-Biotin, and dT-Digoxingenin modifications mediated by internal amino modifications are currently available.
  • 5' Amino Modification It can be used to prepare functionalized oligonucleotides, which are widely used in DNA microarrays and multiple labeling diagnostic systems. At present, there are two kinds of 5'C6 amino modification and 5'C12 amino modification. The former can be used to connect some compounds that will not affect their function even if they are close to the oligonucleotide, and the latter is used for the connection of affinity purification groups and some fluorescence labeling, especially when fluorescence may be quenched by the label being too close to the DNA strand.
  • 3' Amino Modifications are currently available. It can be used to design new diagnostic probes and antisense nucleotides, for example, the 5' end can be labeled with highly sensitive 32P or fluorescein while the 3' can be modified with amino groups for additional ligation. In addition, 3' modification can inhibit 3' exonuclease digestion, which can be used in antisense experiments.
  • 5'-Sulfhydryl groups are similar in many ways to amino modifications.
  • Thiol groups can be used to attach various modifications such as fluorescent labels and biotin.
  • thiol-linked fluorescent probes can be prepared in the presence of iodoacetic acid and maleimide derivatives.
  • the 5' thiol modification mainly uses 5' thiol to modify the monomer (5'-Thiol-Modifier C6-CE Phosphoramidite or Thiol-Modifier C6 S-S CE Phosphoramidite).
  • Spacer can provide the necessary space for oligonucleotide labeling to reduce the interaction between the labeling group and oligonucleotide, mainly used in the study of DNA hairpin structure and double-stranded structure.
  • the C3 spacer is primarily used to mimic the three-carbon spacer between the 3' and 5' hydroxyl groups of ribose, or to "replace" an unknown base in a sequence.
  • 3'-Spacer C3 is used to introduce a 3' spacer arm to prevent 3' exonuclease and 3' polymerase from functioning.
  • Spacer 18 is often used to introduce a strongly hydrophilic group.
  • Thio-modified oligonucleotides are mainly used in antisense experiments to prevent degradation by nucleases. You can choose all-thio, but as the number of thio bases increases, the Tm value of the oligonucleotide will decrease. To reduce this effect, 2-5 bases at both ends of the primer can be thio-modified , usually 3 bases at 5' and 3' can be selected for sulfur modification.
  • Deoxyuracil can be inserted into oligonucleotides to increase the melting point temperature of the duplex to increase the stability of the duplex.
  • the replacement of each deoxythymine by deoxyuracil can increase the double-stranded melting point temperature of 1.7°C.
  • Deoxyhypoxanthine modification Deoxyhypoxanthine is a naturally occurring base. Although it is not a universal base in the true sense, it is relatively more stable than other base mismatches when combined with other bases.
  • the binding ability of deoxyhypoxanthine to other bases is dI:dC>dI:dA>dI:dG>dI:dT. Under the catalysis of DNA polymerase, deoxyhypoxanthine is firstly combined with dC.
  • Circulating free DNA in plasma, also known as “liquid biopsy”, avoids the need for biopsy of tumor tissue and is a very useful diagnostic application in clinical practice.
  • liquid biopsies offers the possibility of repeated blood sampling, allowing the tracking of cfDNA changes during tumorigenesis or during cancer treatment to monitor disease changes (Cell-free nucleic acids as biomarkers in cancer patients).
  • cfDNA detection to accurately and specifically detect gene mutations currently has huge technical challenges.
  • the content of cfDNA in blood varies from person to person, and most cases are very low, and the quality and content of tumor-derived cell-free nucleic acid (Circulating tumor DNA, referred to as "ctDNA”) are even more uneven.
  • ctDNA tumor-derived cell-free nucleic acid
  • Douillard et al reported that the detection of EGFR mutations using plasma was only 65% consistent with the results of tumor tissue detection.
  • the absolute quantification method of the present invention can determine whether the microsatellite sites have changed (Table 1) through the two-dimensional fluorescence map, and determine whether MSI-H is MSI-H according to the number of changes (Table 2).
  • Table 1 The detection and determination criteria of the MSI of the present invention are shown in the following table:
  • the designed primers are aimed at the sequences of the microsatellite loci detected by MSI, and can specifically amplify stable microsatellites and unstable microsatellites; the designed probes, one probe (P1) covers stable type microsatellite site, another probe (P2) covers the sequence near the microsatellite site, the 5' end of the P1 probe is modified with fluorophores such as FAM, Cy5, Texas Red or ROX, and the 5' end of the P2 probe has Group modification such as HEX or Vic.
  • the applicable sample types of the present invention can be used in addition to fresh tissue samples and paraffin sections commonly used in general methods, peripheral blood samples can also be used; and , due to the uniqueness of its digital PCR platform, the reaction system can be divided into about 20,000 or more small systems, and the interfering substances can be divided into the same number, which can greatly reduce the influence of the interfering substances on the reaction. Detect samples with more complex backgrounds. This is something no other platform can do.
  • the positive interpretation method is simple: the present invention obtains the final test result after the PCR reaction is completed, and has incomparable timeliness compared with other current MSI detection methods.
  • NGS Next Generation Sequencing
  • the sequencing depth of NGS greatly exceeds that of tumor tissue-derived DNA. , which directly leads to an increase in the cost of sequencing.
  • complex preprocessing and bioinformatics analysis are required to obtain correct sequencing information, which makes it take a long time to obtain the detection report.
  • ddPCR-based MSI detection does not have such problems. Generally, the detection can be completed within two hours, and the results can be obtained at the same time as the detection is completed. There is no need for complex result analysis, which greatly shortens the waiting time of tumor patients, and can be used as soon as possible. Medication.
  • the chromosomal positions of the sequences are all based on hg38, and the nucleic acid sequence information of the primers, probes and DNA sites used are shown in Table 3:
  • the probes are all fluorescent probes with a fluorophore at one end and a quenching group at the other end.
  • + means expansion and difference between 2 cell lines
  • - means no difference or no expansion.
  • polyA or polyT 40 single nucleotide repeat (polyA or polyT) sites were selected from the human genome, and several pairs of primers were designed (primers were synthesized from IDT Corporation, USA) for screening.
  • primers were synthesized from IDT Corporation, USA.
  • the specific human genome sites and primer sequences are shown in Table 3.
  • Screening PCR system Takara Taq HS Perfect Mix 10ul, primer 1 1ul, primer 2 1ul, human renal epithelial cell 293T or human colon cancer cell HCT116 cell genomic DNA template 5ul, the system is filled with water to 20ul. Screening PCR program: 32 cycles (94°C for 5s, 56°C for 1s, 68°C for 20s).
  • probes were designed respectively (the probes were synthesized from IDT Corporation, USA).
  • the designed probe sequences are shown in Table 3. To optimize the probe, the steps are as follows:
  • Templates were added to the sample preparation area in the following order: blank control, negative control (293T cell DNA), and positive control (HCT116 cell DNA).
  • Droplet generation is performed according to the requirements of the droplet generator.
  • PCR was performed according to the following procedure: 95°C for 10 min, 40 cycles (94°C for 30s, 56°C for 30s, 72°C for 30s), 98°C for 10min, and the ramp rate was 2°C/s.
  • Example 2 From Example 2, 5 sites (Z2, Z3, Z4, Z5 and Z7) were selected, and the DNA of colorectal cancer cell lines HCT-116, RKO, and SW48 were detected by the method in Example 2, and HCT-116 was determined. , RKO and SW48 are MSI-H.
  • the digital PCR two-dimensional map is shown in Figure 3-5, and the detection results are shown in Table 5.
  • Figures 3A-E show the detection results of HCT-116 cells.
  • Figure 3A shows only negative signal points (orange points), which are determined to be MSS;
  • Figures 3B, 3C, and 3E only have positive signal points (green points), which are determined to be MSI;
  • Figure 3D both The negative signal point is displayed, and the positive signal point is also displayed, which is also judged as MSI. Therefore, for HCT-116 cells, 4 out of 5 loci were judged as MSI, so the whole was judged as MSI-H.
  • Rectal cancer cell line HCT-116 RKO SW48 Site 2 (SEQ ID NO.: 2) MSS MSI MSI Site 3 (SEQ ID NO.:3) MSI MSI MSS Site 4 (SEQ ID NO.: 4) MSI MSI MSI Site 5 (SEQ ID NO.: 5) MSI MSI MSI Site 7 (SEQ ID NO.:7) MSI MSI MSI result judgment MSI-H MSI-H MSI-H
  • Genomic nucleic acid was extracted from its FFPE specimens. The extracted nucleic acid was quantified by Qubit, and stored in a -20°C refrigerator in the sample preparation area after quantification.
  • one end of the primer was labeled with a fluorescent group to amplify the genomic nucleic acids of the tumor specimens and adjacent tissues of SY020 and SY028, respectively, and detected by capillary electrophoresis.
  • Example 2 From Example 2, 5 sites (Z2, Z3, Z4, Z5 and Z7) were selected, and 263 cases of colorectal cancer tumor tissues were detected by digital PCR method according to the method in Example 2.
  • PCR reaction system was formed into droplets according to the same method as in Example 2.
  • PCR was performed according to the optimized PCR program: 95°C for 10 min, 40 cycles (94°C for 30s, 56°C for 15s, 72°C for 15s), 98°C for 10min, and the ramp rate was 2°C/s. Turn on the instrument, set up as required, and start reading the plate.
  • the first centrifugation 4 °C, 1900 ⁇ g, 10min.
  • the second high-speed centrifugation 4°C, 16000 ⁇ g, 10min.
  • About 4.5 ml of plasma were isolated.
  • Extraction of plasma free nucleic acid Use Kangwei Century Free Nucleic Acid Extraction Kit, operate according to the instructions, and elute free nucleic acid with 50ul nuclease-free water.
  • the clinical significance of distinguishing MSI colorectal cancer includes the following three aspects:
  • stage II colorectal cancer Clinical studies have found that in stage II colorectal cancer, the proportion of MSI-H colorectal cancer (22%) is higher than that in stage III colorectal cancer (12%), but only 3.5% in stage IV tumors. %. This suggests that MSI-H colorectal cancer has a lower risk of metastasis.
  • MSI status can be used as an independent predictor of prognosis: MSI-H tumors have a better prognosis than MSS tumors.
  • Risk factors for recurrence of stage II colorectal cancer include poorly differentiated tumor histological type, vascular/nerve invasion, less than 12 lymph nodes detected, obstruction or perforation, and positive surgical margins. However, if the tumor is of MSI-H type, poorly differentiated cancer is not a high-risk factor.
  • MSI-H colorectal cancer is not sensitive to chemotherapy drugs such as 5-fluorouracil and cisplatin, but is sensitive to chemotherapy drugs such as irinotecan, and MSI colorectal cancer is more sensitive to radiotherapy.
  • NCN National Comprehensive Cancer Network
  • mismatch repair proteins by immunohistochemistry: The functional loss of one or more mismatch repair proteins can cause MSI-H. Therefore, detection of mismatch repair protein deficiency can indirectly reflect the MSI status of tumors.
  • detection of mismatch repair protein deficiency can indirectly reflect the MSI status of tumors.
  • tissue fixation, staining conditions, and antibody clone numbers false positives or false positives may occur. Negative result. This method cannot be used for the detection of blood samples.
  • MSI detection based on conventional PCR The principle of PCR detection of MSI is to amplify specific microsatellite sequences by PCR method, and to judge whether there is MSI phenomenon by comparing the difference in the length of microsatellite sequences between tumor tissue and normal tissue. Due to the limited sensitivity of this method, it is not suitable for the detection of blood samples, and generally requires the acquisition of normal tissues and tumor tissues of patients. Generally, this method is carried out by two detection means:
  • Capillary electrophoresis So far, the most commonly used method is to use capillary electrophoresis to analyze the length distribution of the amplified product of the marker for diagnosis.
  • the most well-known method of analysis is "multiplex fluorescent PCR amplification and capillary electrophoresis", a method of analysis by extracting DNA from normal and tumor tissues, amplifying the DNA by fluorescent PCR targeting satellite markers, and then analyzing the DNA using capillary electrophoresis. Fluorescence method to analyze MSI.
  • this method requires an expensive capillary electrophoresis device to operate in two steps. There is a possibility of laboratory contamination during the process of adding samples for capillary electrophoresis after PCR, and it takes a long time. The result requires an experienced and well-trained laboratory. personnel to analyze.
  • Isotope-labeled PCR method Using the sensitivity of isotopes, PCR primers can be labeled with isotopes, and the amplified PCR products can be electrophoresed and exposed to light to detect the length of the PCR products. This method requires isotope operation, which is unfavorable to human health, takes a long time, and requires special operation space and treatment of experimental wastes, which pollutes the environment, and is now rare.
  • MSI detection based on next-generation sequencing The principle of NGS detection of MSI is to directly read out specific microsatellite sequences to compare the difference in the length of microsatellite sequences between tumor tissue and normal tissue to determine whether there is MSI phenomenon.
  • NGS next-generation sequencing
  • MSI detection technologies are cumbersome, time-consuming, highly subjective in positive interpretation, and have high requirements for the type and quality of samples, and most of them need to provide tissue samples.

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Abstract

提供一种人微卫星不稳定（MSI）位点的检测方法及其应用，提供了用于检测MSI的引物、探针、检测体系。该方法和试剂盒可用于检测肿瘤患者是否存在MSI-H，并根据检测结果来指导用药或指导风险评估，具有高灵敏度、高特异性、抗干扰能力强等优点。

Description

人微卫星不稳定位点的检测方法及其应用 技术领域
本发明涉及医学和生物技术领域，具体地，涉及人微卫星不稳定位点的检测方法及其应用。
背景技术
DNA的错配修复(mismatch repair,MMR)基因突变或启动子甲基化是引发癌症的重要原因之一。MMR基因编码的蛋白质在DNA复制过程中对碱基错配进行监察，以避免错误的发生。错配修复蛋白包括MutS和MutL两大家族，前者包括MSH2/MSH3和MSH6等，后者包括MLH1、MLH3、PMS1和PMS2。MSH2和MLH1分别与其同源错配修复蛋白组成复合体以发挥作用。例如，MSH2分别与MSH6、MSH3组成复合体MutSα、MutSβ；MLH1则分别与PMS2、PMS1或MLH3形成复合体MutLα、MutLβ或MutLγ。MutSα或MutSβ错配修复碱基的步骤如下：识别错配碱基后，与DNA碱基结合，再与MutL结合，激活ATP酶，水解错配的碱基，同时激活核酸内切酶I，切除并修复错配的碱基。
微卫星(microsatellite)是一种短串联重复序列，每单元长度在1～6bp之间。其广泛存在于原核及真核生物基因组中，具有较高的遗传稳定性。但在细胞的错配修复基因功能发生异常时，子代细胞微卫星的重复核苷酸数量可增多或减少，导致微卫星的长度发生变化，这种现象称微卫星不稳定(microsatellite instability，MSI)。
错配修复基因发生突变或启动子甲基化可引起DNA错配修复功能的降低，导致MSI。很多重要的生长调节相关基因，如Ⅱ型TGF-β、IGF2R、PTEN、BAX，其编码区或启动子区含有微卫星。错配修复异常导致的MSI可引起这些基因在复制过程中发生错义突变或移码突变，使得这种复制错误不断累积，成为导致肿瘤发生的重要因素。因此MSI可作为肿瘤的分子标志物。目前，已在多种肿瘤中发现了高频率的MSI，如结直肠癌、胃癌、小肠癌、子宫内膜癌等。
此外，目前虽然有一些MSI检测的方法，但现有MSI检测技术操作繁琐，样品之间容易相互污染，耗时长，阳性判读主观性强，且对于样本的类型与质 量要求高，大多需要同时提供肿瘤与癌旁组织样本，且肿瘤组织样本中的肿瘤细胞比例不低于20％。
综上所述，本领域迫切需要开发一种高灵敏度、高特异性、消除样品污染可能性、抗干扰能力强的检测肿瘤MSI的方法。
发明内容
本发明的目的就是提供一种高灵敏度、高特异性、消除样品污染可能性、抗干扰能力强的检测肿瘤MSI的方法。
本发明还提供了一种人微卫星不稳定(MSI)位点的检测方法及其应用。
在本发明的第一方面，提供了一种人微卫星不稳定(Microsatellite Instability，MSI)位点检测试剂的用途，用于制备一种诊断试剂或试剂盒，所述诊断试剂或试剂盒用于诊断MSI相关疾病和/或用于对MSI相关疾病进行预后；
其中，所述的MSI位点选自下组A的一个或多个位点：
(Z1)chr3:30650236-30650508；
(Z2)chr11:106739898-106740117；
(Z3)chr16:18841298-18841518；
(Z4)chr17:19411505-19411722；
(Z5)chr20:62921533-62921750；
(Z6)chr2:47408320-47408461；
(Z7)chr2:147906719-147906938；
(Z8)chr6:142407071-142407290；
(Z9)chr14:93268657-93268877；
(Z10)chr20:47779916-47780134；
(Z11)chr7:143306180-143306440；
(Z12)chr1:201819424-201819543；
(Z13)chr1:201771449-201771558；
(Z14)chr2:61827581-61827677；
(Z15)chr2:147857500-147857584；
(Z16)chr4:82821524-82821646；
(Z17)chr5:172998578-172998712；
(Z18)chr6:142337918-142338138；
(Z19)chr14:93244766-93244900；
(Z20)chr15:45593287-45593508；
(Z21)chr15:33349068-33349160；
(Z22)chr15:33764931-33765150；
(Z23)chr16:18854033-18854164。
在另一优选例中，所述的检测试剂选自下组：引物、探针、向导RNA(guild RNA，用于CRISPR)、芯片、或其组合。
在另一优选例中，所述的MSI位点选自下组：
(Z1)chr3:30650236-30650508；
(Z2)chr11:106739898-106740117；
(Z3)chr16:18841298-18841518；
(Z4)chr17:19411505-19411722；
(Z5)chr20:62921533-62921750；
(Z6)chr2:47408320-47408461；
(Z7)chr2:147906719-147906938；
(Z8)chr6:142407071-142407290；
(Z9)chr14:93268657-93268877；
(Z10)chr20:47779916-47780134；
(Y1)Z1～Z10的任意组合。
在另一优选例中，所述的MSI位点包括选自Z1～Z7中至少1个，至少2个，至少3个，至少4个，或5个。
在另一优选例中，所述的MSI位点包括Z2、Z3、Z4、Z5和Z7。
在另一优选例中，所述的MSI位点包括选自Z1～Z10中至少1个，至少2个，至少3个。
在另一优选例中，所述的MSI位点还包括除Z1～Z10的额外的MSI位点。
在另一优选例中，所述的MSI位点包括(a)选自Z2、Z3、Z4、Z5和Z7中的一个 或多个(如2、3、4、或5)位点；和(b)除Z2、Z3、Z4、Z5和Z7的额外的MSI位点。
在另一优选例中，所述的MSI位点包括(a)Z2、Z3、Z4、Z5和Z7；和(b)除Z2、Z3、Z4、Z5和Z7的额外的MSI位点。
在另一优选例中，所述的额外的MSI位点选自下组：BAT-26、BAT-25、MONO-27、NR-21、NR-24、D5S346、D2S123、D17S250或其组合。
在另一优选例中，所述的MSI相关疾病为肿瘤或癌症。
在另一优选例中，所述的肿瘤或癌症选自下组：结直肠癌、子宫内膜癌、子宫肉瘤、胃癌、小肠癌、宫颈癌、肝癌、食管癌、胰腺癌、卵巢癌、胆囊癌、睾丸癌、前列腺癌、输卵管癌、外阴癌、肾上腺皮质癌、原发性腹腔肿瘤、胆管癌、乳腺癌、神经内分泌肿瘤、胸腺癌、甲状腺癌、小细胞肺癌、原发未知的肿瘤等。
在另一优选例中，所述诊断试剂或试剂盒用于选自下组的检测：血清检测、血浆检测、细胞检测、组织样品检测。
在另一优选例中，所述的检测为PCR检测或测序检测；较佳地，为数字PCR(digital PCR，ddPCR)检测。
在另一优选例中，所述的检测包括单一检测或多重检测(如n重检测，其中，n为2-20的任一正整数，较佳地n为3-15，更佳地5-10)。
在另一优选例中，所述的多重检测包括：在一个反应体系中进行多重扩增，并随后进行检测。
较佳地，所述的“随后进行检测”包括荧光检测、毛细管电泳、测序、或其组合。
在本发明的第二方面，提供了一种用于检测人微卫星不稳定(Microsatellite Instability，MSI)位点的试剂，所述位点选自hg38染色体中的位点1～位点10(SEQ ID NO.:1～10)；其中，所述试剂选自下组：
(a)用于检测chr3位点1(SEQ ID NO.:1)MSI的第一引物对，其中第一引物对包括SEQ ID NO.:11和12所示的引物；
(b)用于检测chr11位点2(SEQ ID NO.:2)MSI的第二引物对，其中第二引物对包括SEQ ID NO.:19和20所示的引物；
(c)用于检测chr16位点3(SEQ ID NO.:3)MSI的第三引物对，其中第三引物对 包括SEQ ID NO.:23和24所示的引物；
(d)用于检测chr17位点4(SEQ ID NO.:4)MSI的第四引物对，其中第四引物对包括SEQ ID NO.:25和26所示的引物；
(e)用于检测chr20位点5(SEQ ID NO.:5)MSI的第五引物对，其中第五引物对包括SEQ ID NO.:29和30所示的引物；
(f)用于检测chr2位点6(SEQ ID NO.:6)MSI的第六引物对，其中第六引物对包括SEQ ID NO.:13和14所示的引物；
(g)用于检测chr2位点7(SEQ ID NO.:7)MSI的第七引物对，其中第七引物对包括SEQ ID NO.:15和16所示的引物；
(h)用于检测chr6位点8(SEQ ID NO.:8)MSI的第八引物对，其中第八引物对包括SEQ ID NO.:17和18所示的引物；
(i)用于检测chr14位点9(SEQ ID NO.:9)MSI的第九引物对，其中第九引物对包括SEQ ID NO.:21和22所示的引物；
(j)用于检测chr20位点10(SEQ ID NO.:10)MSI的第十引物对，其中第十引物对包括SEQ ID NO.:27和28所示的引物；
上述(a)至(j)任一项的组合。
在另一优选例中，所述的位点1所在序列位于chr3:30650236-30650508。
在另一优选例中，所述的位点2所在序列位于chr11:106739898-106740117。
在另一优选例中，所述的位点3所在序列位于chr16:18841298-18841518。
在另一优选例中，所述的位点4所在序列位于chr17:19411505-19411722。
在另一优选例中，所述的位点5所在序列位于chr20:62921533-62921750。
在另一优选例中，所述的位点6所在序列位于chr2:47408320-47408461。
在另一优选例中，所述的位点7所在序列位于chr2:147906719-147906938。
在另一优选例中，所述的位点8所在序列位于chr6:142407071-142407290。
在另一优选例中，所述的位点9所在序列位于chr14:93268657-93268877。
在另一优选例中，所述的位点10所在序列位于chr20:47779916-47780134。
在另一优选例中，所述检测人微卫星不稳定(Microsatellite Instability，MSI)的位点优选为位点2、位点3、位点4、位点5和位点7。
在另一优选例中，所述试剂还包括：
(a1)与第一引物对配合使用的第一探针，其中所述的第一探针选自下组：SEQ ID NO.:31所示的探针、SEQ ID NO.:32所示的探针或其组合；和/或
(b1)与第二引物对配合使用的第二探针，其中所述的第二探针选自下组：SEQ ID NO.:39所示的探针、SEQ ID NO.:40所示的探针、或其组合；和/或
(c1)与第三引物对配合使用的第三探针，其中所述的第三探针选自下组：SEQ ID NO.:43所示的探针、SEQ ID NO.:44所示的探针、或其组合；和/或
(d1)与第四引物对配合使用的第四探针，其中所述的第四探针选自下组：SEQ ID NO.:45所示的探针、SEQ ID NO.:46所示的探针、或其组合；和/或
(e1)与第五引物对配合使用的第五探针，其中所述的第五探针选自下组：SEQ ID NO.:49所示的探针、SEQ ID NO.:50所示的探针、或其组合；和/或
(f1)与第六引物对配合使用的第六探针，其中所述的第六探针选自下组：SEQ ID NO.:33所示的探针、SEQ ID NO.:34所示的探针、或其组合；和/或
(g1)与第七引物对配合使用的第七探针，其中所述的第七探针选自下组：SEQ ID NO.:35所示的探针、SEQ ID NO.:36所示的探针、或其组合；和/或
(h1)与第八引物对配合使用的第八探针，其中所述的第八探针选自下组：SEQ ID NO.:37所示的探针、SEQ ID NO.:38所示的探针、或其组合；和/或
(i1)与第九引物对配合使用的第九探针，其中所述的第九探针选自下组：SEQ ID NO.:41所示的探针、SEQ ID NO.:42所示的探针、或其组合；和/或
(j1)与第十引物对配合使用的第十探针，其中所述的第十探针选自下组：SEQ ID NO.:47所示的探针、SEQ ID NO.:48所示的探针、或其组合。
在另一优选例中，所述的第一探针到第十探针为单链的核酸探针。
在另一优选例中，所述第一探针到第十探针的结构(5'-3')如式I所示：
L1-L2-L3    I
其中，
L1为荧光基团而L3为淬灭基团；或者L3为荧光基团而L1为淬灭基团；
L2为核苷酸的特异性互补核酸序列；
“-”为化学键、连接基团、或1-3个核苷酸构成的接头。
在另一优选例中，所述的L2特异性核酸序列特异性靶向hg38中的位点1到位点10。
在另一优选例中，所述的L2含有锁核苷酸修饰。
在另一优选例中，所述的L2的序列选自下组：
Figure PCTCN2022072414-appb-000001
在另一优选例中，所述的荧光基团各自独立地位于所述核酸探针的5'端、3'端和中部。
在另一优选例中，所述的荧光基团和淬灭基团各自独立地位于所述的5'端、3'端、和/或中部。
在另一优选例中，所述荧光基团包括与DNA探针交联的荧光基团。
在另一优选例中，所述荧光基团选自下组：FAM、VIC、HEX、FITC、BODIPY-FL、G-Dye100、FluorX、Cy3、Cy5、Texas Red，或其组合。
在另一优选例中，所述淬灭基团选自下组：DABCYL、TAMRA、BHQ 1、BHQ 2、BHQ3、MGB、BBQ-650、TQ1-TQ6、QSY 7carboxylic acid、TQ7、eclipse，或其组合。
在本发明的第三方面，提供了一种试剂盒，所述试剂盒含有在本发明的第二方面所述的用于检测MSI位点的试剂。
在另一优选例中，所述的MSI位点选自组A的一个或多个位点(即Z1～Z23中的一个或多个)，优选地所述的MSI位点为选自Z1～Z10中的一个或多个。
在另一优选例中，所述试剂盒用于检测来自肿瘤细胞、肿瘤组织或疑似肿瘤组织中人DNA样品中的MSI位点。
在另一优选例中，所述的产品是针对循环游离DNA(cell-free DNA，cfDNA)样本进行检测。
在另一优选例中，所述的cfDNA来自对象的血液、血浆、或血清。
在另一优选例中，所述的对象为肿瘤患者。
在另一优选例中，所述的肿瘤选自下组：结直肠癌、子宫内膜癌、子宫肉瘤、胃癌、小肠癌、宫颈癌、肝癌、食管癌、胰腺癌、卵巢癌、胆囊癌、睾丸癌、前列腺癌、输卵管癌、外阴癌、肾上腺皮质癌、原发性腹腔肿瘤、胆管癌、乳腺癌、神经内分泌肿瘤、胸腺癌、甲状腺癌、小细胞肺癌、原发未知的肿瘤等，或其组合。
在本发明的第四方面，提供了一种检测试剂的组合，所述的检测试剂组合包括用于检测人微卫星不稳定(Microsatellite Instability，MSI)位点的n种检测试剂，其中，所述的MSI位点选自组A的位点，并且n为≥2的正整数。
较佳地，n为2-25，更佳地n为2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、或23。
在另一优选例中，所述的MSI位点选自组A的一个或多个位点(即Z1～Z23中的一个或多个)，优选地所述的MSI位点为选自Z1～Z10中的一个或多个。
在另一优选例中，所述的检测试剂选自下组：引物、探针、芯片、或其组合。
在另一优选例中，所述的检测试剂的组合为检测Z2、Z3、Z4、Z5和Z7的MSI位点的检测试剂所构成的组合。
在本发明的第五方面，提供了一种检测待测样本中MSI的方法，包括步骤：
(S1)提供一PCR反应体系，所述PCR反应体系中含有作为模板的待测样本、以及用于扩增的引物对，所述的引物对选自下组：
(a)用于检测chr3位点1(SEQ ID NO.:1)MSI的第一引物对，其中第一引物对包括SEQ ID NO.:11和12所示的引物；
(b)用于检测chr11位点2(SEQ ID NO.:2)MSI的第二引物对，其中第二引物对包括SEQ ID NO.:19和20所示的引物；
(c)用于检测chr16位点3(SEQ ID NO.:3)MSI的第三引物对，其中第三引物对包括SEQ ID NO.:23和24所示的引物；
(d)用于检测chr17位点4(SEQ ID NO.:4)MSI的第四引物对，其中第四引物对包括SEQ ID NO.:25和26所示的引物；
(e)用于检测chr20位点5(SEQ ID NO.:5)MSI的第五引物对，其中第五引物对包括SEQ ID NO.:29和30所示的引物；
(f)用于检测chr2位点6(SEQ ID NO.:6)MSI的第六引物对，其中第六引物对包括SEQ ID NO.:13和14所示的引物；
(g)用于检测chr2位点7(SEQ ID NO.:7)MSI的第七引物对，其中第七引物对包括SEQ ID NO.:15和16所示的引物；
(h)用于检测chr6位点8(SEQ ID NO.:8)MSI的第八引物对，其中第八引物对包括SEQ ID NO.:17和18所示的引物；
(i)用于检测chr14位点9(SEQ ID NO.:9)MSI的第九引物对，其中第九引物对包括SEQ ID NO.:21和22所示的引物；
(j)用于检测chr20位点10(SEQ ID NO.:10)MSI的第十引物对，其中第十引物对包括SEQ ID NO.:27和28所示的引物；
上述(a)至(j)任一项的组合。
(S2)对步骤(S1)的所述PCR反应体系进行PCR反应，从而获得扩增产物；
(S3)对步骤(S2)中产生的扩增产物进行分析，从而获得所述待测样本MSI情况；其中，以检测的位点数P 总＝5为例，
若样本中的MSI位点数P MSI≥2，则判定为高度不稳定(MSI-H)；
若样本中的MSI位点数P MSI＝1，则判定为低度不稳定(MSI-L)；
若样本中的MSI位点数P MSI＝0，则判定为稳定(MSS)。
在另一优选例中，若检测的位点数P 总≥5时，样本中的MSI位点数百分比为P(P MSI/P 总)，
若P≥40％时，则判定为高度不稳定(MSI-H)；
若10≤P＜40％时，则判定为低度不稳定(MSI-L)；
若P＝0时，则判定为稳定(MSS)。
在另一优选例中，所述的分析为定性分析、定量分析或半定量分析。
在另一优选例中，所述的反应体系为测序反应体系，包括一代、二代、三代测序反应体系。
在另一优选例中，所述的PCR反应体系为数字PCR反应体系。
在另一优选例中，所述的数字PCR为ddPCR。
在另一优选例中，所述的方法为体外方法。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1A-1H显示了293T和HCT116细胞DNA中40个位点对引物PCR电泳结果(M：DNA Marker)。
图2A-2E显示了筛选的5个位点的数字PCR二维图；其中，A图为SEQ ID NO.:1的数字PCR二维图；B图为SEQ ID NO.:2的数字PCR二维图；C图为SEQ ID NO.:3的数字PCR二维图；D图为SEQ ID NO.:4的数字PCR二维图；E图为SEQ ID NO.:5的数字PCR二维图。其中，灰色为无扩增片段，绿色为阳性信号，橙色为阴性信号。
图3A-3E显示了HCT-116细胞系MSI检测的数字PCR二维图；其中，A图为位点1(SEQ ID NO.:1)；B图为位点2(SEQ ID NO.:2)；C图为位点3(SEQ ID NO.:3)；D图为位点4(SEQ ID NO.:4)；E图为位点5(SEQ ID NO.:5)。
图4A-4E显示了RKO细胞系MSI检测的数字PCR二维图；其中，A图为位点1(SEQ ID NO.:1)；B图为位点2(SEQ ID NO.:2)；C图为位点3(SEQ ID NO.:3)；D图为位点4(SEQ ID NO.:4)；E图为位点5(SEQ ID NO.:5)。
图5A-5E显示了SW48细胞系MSI检测的数字PCR二维图；其中，A图为位点1(SEQ ID NO.:1)；B图为位点2(SEQ ID NO.:2)；C图为位点3(SEQ ID NO.:3)；D图为位点4(SEQ ID NO.:4)；E图为位点5(SEQ ID NO.:5)。
图6A显示了通过毛细管电泳法对病例SY020通过人微卫星不稳定性检测试剂盒2B3D(BAT-25、BAT-26、D5S346、D17S250、D2S123)的MSI检测结果。
图6B显示了通过毛细管电泳法对病例SY020的新微卫星位点(Z2、Z3、Z4、Z5和Z7)的MSI检测结果。
图7A显示了通过毛细管电泳法对病例SY028通过人微卫星不稳定性检测试剂盒2B3D(BAT-25、BAT-26、D5S346、D17S250、D2S123)的MSI检测结果。
图7B显示了通过毛细管电泳法对病例SY028的新微卫星位点(Z2、Z3、Z4、Z5和Z7)的MSI检测结果。
图8A显示了通过数字PCR法对病例SY020的新微卫星位点(Z2、Z3、Z4、Z5和Z7)的MSI检测结果。
图8B显示了通过数字PCR法对病例SY028的新微卫星位点(Z2、Z3、Z4、Z5和Z7)的MSI检测结果。
图9A-9B显示了病例SY029的MSI检测结果的数字PCR二维图；其中，A图为病例SY029血液样本位点5的检测结果；B图为病例SY029组织样本位点5的检测结果。
具体实施方式
本发明人经过广泛而深入的研究，通过大量筛选，首次鉴别出多个新颖的MSI位点。研究表明，这些新的MSI位点与MSI相关疾病(如结肠癌等肿瘤)存在高相关性，因此可用于MSI相关疾病的辅助诊断和/或预后。此外，本发明人还对基于这些新MSI位点的引物和/或探针序列进行优化，再结合数字PCR等检测方法，有效提高MSI检测效果并可用血浆/血清样本进行检测，从而突破了现有基因突变检测技术中病理组织作为起始材料、准确度低、灵敏度低等问题。在此基础上，本发明人完成了本发明。
具体地，本发明选取了人基因组中40个由单核苷酸重复(polyA或polyT)组 成的微卫星位点，分别设计扩增引物对，在MSI不稳定的结直肠癌细胞株中进行PCR扩增。从扩增结果筛选的微卫星位点中，进一步选取了最优的10个微卫星位点，设计了能同时检测到稳定型及不稳定型微卫星位点的探针，对引物和探针序列进行优化，采用优化后的引物和探针进行数字PCR，并优化出低频检出的最佳条件。应用该方法在原发结直肠癌病人肿瘤组织、血液等样本中进行了验证。本发明的MSI检测方法具有高特异性和高灵敏度，操作快捷方便，判读简单，不仅可以准确检测组织、体液样本，还可以处理血浆/血清等难度高的样本，用于不同样本的MSI液体活检。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
如本文所用，术语“血液”可以为血浆、血清，但不包括血细胞。
如本文所用，术语“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分离纯化的，但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。
序列同一性通过沿着预定的比较窗(其可以是参考核苷酸序列或蛋白的长度的50％、60％、70％、80％、90％、95％或100％)比较两个对齐的序列，并且确定出现相同的残基的位置的数目来确定。通常地，这表示为百分比。核苷酸序列的序列同一性的测量是本领域技术人员熟知的方法。
微卫星位点
微卫星(microsatellite)是一种短串联重复序列，每单元长度在1～6bp之间。其广泛存在于原核及真核生物基因组中，具有较高的遗传稳定性。但在细胞的错配修复基因功能发生异常时，子代细胞微卫星的重复核苷酸数量可增多或减少，导致微卫星的长度发生变化，这种现象称微卫星不稳定(microsatellite instability，MSI)。
人类基因组中有许多微卫星位点。1998年，MSI国际研究合作组织推荐对BAT26、BAT25、D5S346、D2S123和D17S250五个位点进行检测。以结直肠癌为例，约15％的结直肠癌经MSI途径发生，其中3％左右为家族遗传性(Lynch综合征)，12％为散发性。根据微卫星位点不稳定的数量，将结直肠癌分为高频MSI(MSI-H，具有两个或两个以上位点改变)、低频MSI(MSI-L，仅有一个位点发生改变)以及微卫星稳定型(MSS，没有位点发生改变)。由于在临床表现和病理改变上，MSI-L结直肠癌与MSS肿瘤无明显差别，因此MSI-L结直肠癌通常被归入MSS肿瘤型。临床研究发现，MSI-H结直肠癌发生转移的风险较低，提示预后好于MSS结直肠癌；MSI-H结直肠癌对5-氟尿嘧啶和顺铂等化疗药物不敏感，而对伊立替康等化疗药物敏感，同时MSI结直肠癌对放疗比较敏感。
使用本发明方法检测结直肠癌患者MSI情况，若检测结果具有两个或两个以上位点发生改变，则判定为MSI-H，提示患者预后良好，且不宜采用5-氟尿嘧啶和铂类为主的治疗方案，可以考虑伊立替康等化疗药物；若检测结果为MSI-L(仅有一个位点发生改变)或MSS(没有位点发生改变)，则提示患者预后情况可能不佳，也不排斥5-氟尿嘧啶和铂类为主的治疗方案。
如本文所用，本发明中“新的有效位点”，指人基因组中挑选的40个单核苷酸重复(polyA或polyT)位点中，经设计引物PCR筛选，与人正常细胞相比，在MSI不稳定的结直肠癌细胞株中能被低频检出的位点。在另一优选例中，所述MSI位点中“新的有效位点”为选自实施例1表4中的组A：
(Z1)chr3:30650236-30650508
(Z2)chr11:106739898-106740117
(Z3)chr16:18841298-18841518
(Z4)chr17:19411505-19411722
(Z5)chr20:62921533-62921750
(Z6)chr2:47408320-47408461
(Z7)chr2:147906719-147906938
(Z8)chr6:142407071-142407290
(Z9)chr14:93268657-93268877
(Z10)chr20:47779916-47780134
(Z11)chr7:143306180-143306440
(Z12)chr1:201819424-201819543
(Z13)chr1:201771449-201771558
(Z14)chr2:61827581-61827677
(Z15)chr2:147857500-147857584
(Z16)chr4:82821524-82821646
(Z17)chr5:172998578-172998712
(Z18)chr6:142337918-142338138
(Z19)chr14:93244766-93244900
(Z20)chr15:45593287-45593508
(Z21)chr15:33349068-33349160
(Z22)chr15:33764931-33765150
(Z23)chr16:18854033-18854164
数字PCR(digital PCR)技术
数字PCR(digital PCR)技术，基于单分子PCR方法来进行计数的核酸定量，是一种绝对定量的方法。主要采用当前分析化学热门研究领域的微流控或微滴化方法，将大量稀释后的核酸溶液分散至芯片的微反应器或微滴中，每个反应器的核酸模板数少于或者等于1个。这样经过PCR循环之后，扩增结束后对各个微滴的荧光信号进行分析，有核酸分子模板的反应器就会给出荧光信号，没有模板的反应器就没有荧光信号。根据相对比例和反应器的体积，就可以推算出原始溶液的核酸浓度。
数字PCR与常规的qPCR相比，其能够精确地定量分析和高灵敏度地检测靶 核酸分子。分析常规qPCR的结果的方法是模拟方法，其中，所述数字PCR方法，其结果是通过数字方法(因为得到的信号具有“0”或“1”的数值)分析的，具有可分析大体积样本、可同时检测不同样本以及同时进行不同测试的优点。数字PCR技术是一种可使用不具有标准曲线的单分子计数法来绝对定量DNA样本的技术，并且可以通过PCR对每孔单个液滴进行更精确的绝对定量(参见Gudrun Pohl和le-Ming Shih，数字PCR的原理和应用(Principle and applications of digital PCR)，Expert Rev.Mol.Diagn.4(1),41-47(2004))。数字PCR具有灵敏度高、无需标准曲线即可准确定量、操作简单等优点。
在数字PCR中，含有经制备从而可用于稀释至平均拷贝数目为0.5-1的样本基因模板、扩增引物和荧光探针的各液滴分配到单个孔中，并进行微乳液PCR。然后，显示荧光信号的孔计数为数值“1”，因为具有基因拷贝数目为1的样本分配到所述孔中并且扩增后显示荧光信号，而没有显示信号的孔计数为“0”，因为具有基因拷贝数目为0的样本分配到所述孔中，由于无扩增，不显示荧光信号。采用这种方式，可以实现绝对定量。
引物和探针的修饰
在本发明中，所述引物的核酸序列包括未修饰的或经修饰的的引物序列。
优选地，本发明人通过对探针用锁核苷酸进行修饰，可以显著提高探针的特异性，从而提高检测结果的灵敏度和特异性。
在本发明的一个优选例中，所述修饰的方式选自：磷酸化(Phosphorylation)、生物素(Biotin)、地高新(Digoxigenin)、内部氨基修饰、5'氨基修饰、3'氨基修饰、巯基(Thiol)、间臂(Spacer)、硫代(Phosphorthioate)、脱氧脲嘧啶(DeoxyUridine，dU)、脱氧次黄嘌呤(deoxyInosine，dI)、或其组合。
磷酸化修饰：5'磷酸化可用于接头、克隆和基因构建以及连接酶催化的连接反应。3'磷酸化可抗3'外切酶消化的相关实验中，也用于阻止DNA聚合酶催化的DNA链延伸反应。
生物素修饰：引物生物素标记，可用于非放射性免疫分析来检测蛋白质、胞内化学染色、细胞分离、核酸分离、杂交检测特异性的DNA/RNA序列、离子通道构象变化等。
地高新修饰：地高新经由一个11个原子的间臂连接到脲嘧啶的C5位置， 杂交的地高新探针可以由抗地高新抗体来检测。地高新标记的探针可用于各种杂交反应，如DNA-DNA杂交(Southern blotting)、DNA-RNA杂交(Northern blotting)、斑点杂交(Dot blotting)、克隆杂交、原位杂交以及酶联免疫分析(ELISA)。
内部氨基修饰：主要用C6-dT aminolinker来加到胸腺嘧啶残基上来进行内部修饰。修饰后氨基与主链相距10个原子距离，可用于进一步的标记和酶连接(如碱性磷酸酶)，目前提供内部氨基修饰介导的dT-Dabcyl、dT-Biotin和dT-Digoxingenin修饰。
5'氨基修饰：可用于制备功能化的寡核苷酸，广泛应用在DNA芯片(DNA Microarray)和多重标记诊断系统。目前提供5'C6氨基修饰和5'C12氨基修饰两种，前者可用于连接一些即便靠近寡核苷酸也不会影响其功能的化合物，后者用于亲和纯化基团的连接和一些荧光标记，尤其是当荧光可能会因标记太靠近DNA链而被淬灭时。
3'氨基修饰：目前提供3'C6氨基修饰。它可用于设计新的诊断探针和反义核苷酸，例如5'端可用高度敏感的32P或荧光素标记的同时3'可用氨基修饰以进行其他的连接。此外，3'修饰可以抑制3'外切酶酶解，从而可用于反义实验。
巯基修饰：5'-巯基在很多方面与氨基修饰类似。巯基可用于加附各种修饰如荧光标记物和生物素。例如可以在碘乙酸和马来酰亚胺衍生物存在下来制作巯基连接的荧光探针。5'的巯基修饰主要用5'巯基修饰单体(5'-Thiol-Modifier C6-CE Phosphoramidite或Thiol-Modifier C6 S-S CE Phosphoramidite)。用5'-Thiol-Modifier C6-CE单体修饰后必须进行硝酸银氧化以去除保护基(trityl)，而Thiol-Modifier C6 S-S CE单体修饰后须用DTT将二硫键还原成巯基。
间臂修饰：Spacer可为寡核苷酸标记提供必要的间隔以减少标记基团与寡核苷酸间的相互作用，主要应用于DNA发夹结构和双链结构研究。C3 spacer主要用于模仿核糖的3'和5'羟基间的三碳间隔，或"替代"一个序列中未知的碱基。3'-Spacer C3用于引进一个3'间臂从而阻止3'端外切酶和3'端聚合酶发挥作用。Spacer 18常用于引进一个强亲水基团。
硫代修饰：硫代修饰的寡核苷酸主要用于反义实验中防止被核酸酶降解。您可以选择全硫代，但随着硫代碱基的增加，寡核苷酸的Tm值会降低，为了降低这种这种影响，可以对引物两端2-5个碱基进行硫代修饰，通常可以选择 5'和3'各3个碱基进行硫代修饰。
脱氧脲嘧啶修饰：脱氧脲嘧啶可以插进寡核苷酸来增加双链的熔点温度从而增长双链的稳定性。每个脱氧胸腺嘧啶被脱氧脲嘧啶替代可以增长双链熔点温度1.7℃。
脱氧次黄嘌呤修饰：脱氧次黄嘌呤是一个自然存在的碱基，虽然不是真正意义上的通用碱基，但当与其它碱基结合时，会比其它碱基错配相对更稳定。脱氧次黄嘌呤与其它碱基的结合能力为dI:dC>dI:dA>dI:dG>dI:dT.在DNA聚合酶的催化下，脱氧次黄嘌呤首选与dC结合。
cfDNA
血浆中的游离核酸(Circulating free DNA,简称“cfDNA”),又称“液体活检”，避免了需要肿瘤组织的活检，在临床上是一种非常有用的诊断应用。使用液体活检提供了重复采血的可能性，从而允许在肿瘤发生过程中或者癌症治疗期间追踪cfDNA的变化，进而监控病情变化(Cell-free nucleic acids as biomarkers in cancer patients)。但是应用cfDNA检测准确并特异地检测基因突变目前存在巨大的技术挑战。首先，血液中的cfDNA含量因人而异，而且大部分情况都非常低,而其中肿瘤来源的游离核酸(Circulating tumor DNA,简称“ctDNA”)质量更是参差不齐、含量高低不一。不仅如此，cfDNA检测方法特异性有待提高。Douillard et al报道，使用血浆检测EGFR突变与肿瘤组织检测结果的符合度仅为65％。
检测和判定标准
本发明的绝对定量的方法可通过荧光的二维图，判定微卫星位点是否发生改变(表1)，并根据发生改变的个数来判定是否MSI-H(表2)。本发明的MSI的检测和判定标准如下表所示：
表1.检测结果表
Figure PCTCN2022072414-appb-000002
Figure PCTCN2022072414-appb-000003
表2.MSI判定(以5个位点为例)
1 ≥2个位点阳性 MSI-H ≥40％
2 1个位点阳性 MSI-L 20-40％
3 0个位点阳性 MSS 0
在本发明中，例如当采用≥5个位点时，阳性位点的百分比P为0时，可判定为MSS；百分比P为10-40％时，可判定为MSI-L；百分比P为≥40％时，可判定为MSI-H。
本发明的主要优点包括
1、灵敏度高：由于本方法采用数字PCR平台，能将反应体系分成20000或更多个微小反应，理论上能够检测到单个拷贝的变化，具有其他技术无法匹敌的灵敏度优势。现有MSI检测基于的普通PCR方法一般需要肿瘤组织样品，由于数字PCR优异的灵敏度，使在cfDNA中检测微量肿瘤DNA带有的MSI成为可能。
2、特异性强：设计的引物分别针对MSI检测的微卫星位点所在的序列，能特异性扩增稳定型微卫星和不稳定微卫星；设计的探针，一条探针(P1)覆盖稳定型微卫星位点，另一条探针(P2)覆盖微卫星位点附近的序列，P1探针5'端有FAM、Cy5、Texas Red或ROX等荧光基团修饰，P2探针5'端有HEX或Vic等基团修饰。
3、对样本的类型与质量要求宽松，抗干扰力强：由于其灵敏度高的特性，本发明适用的样本类型除了一般方法常用的新鲜组织样本、石蜡切片，还可以使用的外周血样本；并且，由于其数字PCR平台的独特性，即能将反应体系分成约20000或更多个小体系，同时也能将干扰物质分成同样份数，能够大大减少干扰物质对反应的影响，当然也就可以检测更复杂背景的样本。这是其他平台无法做到的。
4、阳性判读方法简单：本发明在PCR反应结束即得到最终检验结果，与目前其它MSI检测方法相比具有不可比拟的时效性。
5、成本低廉：虽然目前肿瘤液体活检中二代测序(Next generation sequencing,NGS)也能进行MSI的检测，但是由于血液中ctDNA浓度整体偏低， NGS测序深度大大超过肿瘤组织来源DNA的测序深度，直接导致测序成本增加。而且由于在深度测序时测序本身的错误开始显现，需要进行复杂的前处理和生物信息学分析来得到正确的测序信息，使取得检测报告所需要的时间较长。而基于ddPCR的MSI检测则不存在此类问题，一般可以在两个小时内即完成检测，并且在检测完成的同时得到结果，无需复杂的结果分析，大大缩短了肿瘤患者的等待时间，可以尽快用药。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
引物、探针和位点的序列
实施例中，序列的染色体位置皆以hg38为准，使用的引物、探针及DNA位点的核酸序列信息如表3所示：
表3.引物、探针及DNA位点的核酸序列
Figure PCTCN2022072414-appb-000004
Figure PCTCN2022072414-appb-000005
Figure PCTCN2022072414-appb-000006
注：探针均为荧光探针，一端带有荧光基团，另一端带有淬灭基团。
实施例1微卫星位点的筛选
表4.筛选的微卫星位点
序号 位置 长度 泳道 结论 备注
#1-1 chr3:30650236-30650508 131 图1A-1,2 + 位点1
#1-2 chr2:47408320-47408461 108 图1A-3,4 + 位点6
#1-3 chr7:143306180-143306440 120 图1A-5,6 +  
#1-4 chr1:201677226-201677336 111 图1A-7,8 -  
#1-5 chr1:201819424-201819543 120 图1A-9,10 +  
#1-6 chr1:201771449-201771558 110 图1A-11,12 +  
#1-7 chr2:147906719-147906938 129 图1B-13,14 + 位点7
#1-8 chr2:107849601-107849715 115 图1B-15,16 -  
#1-9 chr2:61827581-61827677 97 图1B-17,18 +  
#1-10 chr2:147857500-147857584 85 图1B-19,20 +  
#1-11 chr4:82849514-82849731 97 图1B-21,22 -  
#1-12 chr4:82820034-82820251 132 图1B-23,24 -  
#1-13 chr4:82821524-82821646 123 图1C-25,26 +  
#1-14 chr5:173027224-173027443 126 图1C-27,28 -  
#1-15 chr5:172998578-172998712 135 图1C-29,30 +  
#1-16 chr6:142337918-142338138 120 图1C-31,32 +  
#1-17 chr6:142394554-142394674 121 图1C-33,34 -  
#1-18 chr6:142407071-142407290 125 图1D-35,36 + 位点8
#1-19 chr7:1741030-1741248 132 图1D-37,38 -  
#1-20 chr7:1722166-1722384 135 图1D-39,40 -  
#2-1 chr11:106698028-106698162 135 图1E-1,2 -  
#2-2 chr11:106739898-106740117 115 图1E-3,4 + 位点2
#2-3 chr11:106777416-106777549 134 图1E-5,6 -  
#2-4 chr14:93244766-93244900 135 图1E-7,8 +  
#2-5 chr14:93268657-93268877 102 图1E-9,10 + 位点9
#2-6 chr14:93289781-93289901 121 图1E-11,12 -  
#2-7 chr15:33832903-33833121 130 图1E-13,14 -  
#2-8 chr15:45593287-45593508 117 图1F-15,16 +  
#2-9 chr15:33349068-33349160 93 图1F-17,18 +  
#2-10 chr15:33764931-33765150 103 图1F-19,20 +  
#2-11 chr16:18841298-18841518 116 图1F-21,22 + 位点3
#2-12 chr16:18854033-18854164 132 图1F-23,24 +  
#2-13 chr16:18888721-18888940 125 图1F-25,26 -  
#2-14 chr17:19411505-19411722 110 图1G-27,28 + 位点4
#2-15 chr20:47779916-47780134 110 图1G-29,30 + 位点10
#2-16 chr20:62921533-62921750 118 图1G-31,32 + 位点5
#2-17 chr20:47752536-47752622 87 图1G-33,34 -  
#2-18 chr22:29285033-29285166 134 图1G-35,36 -  
#3-1 chr17:7423470-7423603 134 图1H-1,2 -  
#3-2 chr12:6442426-6442543 118 图1H-3,4 -  
注：+表示有扩增并且2个细胞系有差异，-表示无差异或无扩增。
其中，表4中所有新的有效位点为下表A：
序号 位置 长度 泳道 结论 备注
Z1 chr3:30650236-30650508 131 图1A-1,2 + 位点1
Z2 chr11:106739898-106740117 115 图1E-3,4 + 位点2
Z3 chr16:18841298-18841518 116 图1F-21,22 + 位点3
Z4 chr17:19411505-19411722 110 图1G-27,28 + 位点4
Z5 chr20:62921533-62921750 118 图1G-31,32 + 位点5
Z6 chr2:47408320-47408461 108 图1A-3,4 + 位点6
Z7 chr2:147906719-147906938 129 图1B-13,14 + 位点7
Z8 chr6:142407071-142407290 125 图1D-35,36 + 位点8
Z9 chr14:93268657-93268877 102 图1E-9,10 + 位点9
Z10 chr20:47779916-47780134 110 图1G-29,30 + 位点10
Z11 chr7:143306180-143306440 120 图1A-5,6 +  
Z12 chr1:201819424-201819543 120 图1A-9,10 +  
Z13 chr1:201771449-201771558 110 图1A-11,12 +  
Z14 chr2:61827581-61827677 97 图1B-17,18 +  
Z15 chr2:147857500-147857584 85 图1B-19,20 +  
Z16 chr4:82821524-82821646 123 图1C-25,26 +  
Z17 chr5:172998578-172998712 135 图1C-29,30 +  
Z18 chr6:142337918-142338138 120 图1C-31,32 +  
Z19 chr14:93244766-93244900 135 图1E-7,8 +  
Z20 chr15:45593287-45593508 117 图1F-15,16 +  
Z21 chr15:33349068-33349160 93 图1F-17,18 +  
Z22 chr15:33764931-33765150 103 图1F-19,20 +  
Z23 chr16:18854033-18854164 132 图1F-23,24 +  
如表4所示，从人基因组中挑选40个单核苷酸重复(polyA或polyT)位点，分别设计若干对引物对(引物合成自美国IDT公司)，进行筛选。具体人基因组 位点和引物序列如表3所示。
筛选PCR体系:Takara Taq HS Perfect Mix 10ul，引物1 1ul，引物2 1ul，人肾上皮细胞293T或人结肠癌细胞HCT116细胞基因组DNA模板5ul，水补齐体系至20ul。筛选PCR程序：32个循环(94℃ 5s，56℃ 1s，68℃ 20s)。
经过PCR筛选，最终确定的位点目的片段扩增效率较高，293T和HCT116扩增产物大小不同，且杂带较少，整体效果最好。其中部分有代表性的电泳图见图1。其中电泳结果有差异或有扩增的MSI新的有效位点见表A中的Z1-Z23。
基于图1的结果，优选10个位点(Z1-Z10)。
实施例2数字PCR法检测MSI的方法筛选
根据实施例1筛选出的10个位点的序列，分别设计探针(探针合成自美国IDT公司)，设计的探针序列如表3所示。对探针进行优化，步骤如下：
1.PCR体系的配置。在试剂准备区按照下表配置PCR体系：
2×ddPCRSupermix(No dUTP) 11ul
引物1(F) 1.1ul
引物2(R) 1.1ul
探针1(P1) 0.55ul
探针2(P2) 0.55ul
模板 5.5ul
加水补齐至 22ul
2.加模板。在样本制备区按照以下顺序加入模板：空白对照、阴性对照(293T细胞DNA)、阳性对照(HCT116细胞DNA)。
3.微滴的生成。按照微滴生成仪要求进行微滴生成。
4.PCR：按照以下程序进行PCR：95℃ 10min，40个循环(94℃ 30s，56℃ 30s，72℃ 30s)，98℃ 10min，升降温速率2℃/s。
5.数据读取。
根据实验结果，挑出5个位点，其阳性信号(仅有HEX信号，绿色)与阴性信号(同时有FAM及HEX信号，橙色)分离较好，其数字PCR二维图见图2A-2E。其中，橙色信号为阴性微滴信号，绿色信号为阳性(MSI)微滴信号。
实施例3数字PCR法检测肿瘤细胞系的MSI
从实施例2中，挑选5个位点(Z2、Z3、Z4、Z5和Z7)，以实施例2中的方法检测结直肠癌细胞系HCT-116、RKO、SW48的DNA，判断HCT-116、RKO和SW48为MSI-H。其数字PCR二维图见图3-5，检测结果如表5所示。
图3A～E为HCT-116细胞检测结果，图3A显示只有阴性信号点(橙色点)，判定为MSS；图3B、3C、3E只有阳性信号点(绿色点)，判定为MSI；图3D既显示阴性信号点，也显示阳性信号点，同样判定为MSI。因此，对于HCT-116细胞，5个位点中有4个判定为MSI，因此整体判定为MSI-H。
RKO与SW48的判定与此相同。
表5.几种直肠癌细胞系中不同位点的MSI检测结果
直肠癌细胞系 HCT-116 RKO SW48
位点2(SEQ ID NO.:2) MSS MSI MSI
位点3(SEQ ID NO.:3) MSI MSI MSS
位点4(SEQ ID NO.:4) MSI MSI MSI
位点5(SEQ ID NO.:5) MSI MSI MSI
位点7(SEQ ID NO.:7) MSI MSI MSI
结果判定 MSI-H MSI-H MSI-H
实施例4毛细管电泳法对比新位点与2B3D位点
病例“SY020”和“SY028”为结直肠癌患者。其FFPE标本抽提基因组核酸。抽提核酸经Qubit定量，定量后储存于样本制备区-20℃冰箱内。
以实施例2中的引物序列，在一端引物标记荧光基团，分别扩增SY020和SY028的肿瘤标本和癌旁组织的基因组核酸，用毛细管电泳法检测。
人微卫星不稳定性检测试剂盒(2B3D)采购自上海桐树生物科技有限公司，按说明书方法分别扩增SY020和SY028的肿瘤标本和癌旁组织的基因组核酸，用毛细管电泳法检测。此试剂盒要求肿瘤标本中肿瘤细胞的比例不低于20％。
结果表明，SY020的2B3D的5个位点中有2个为阳性(BAT-25、BAT-26)，3个位点为阴性(D5S346、D17S250、D2S123)，判定为MSI-H(见图6A)；SY020的新位点(Z2、Z3、Z4、Z5和Z7)的5个位点皆为阳性，判定为MSI-H(见图6B)。
SY028的2B3D的5个位点均为阴性，判读为MSS(见图7A)，新位点(Z2、Z3、Z4、Z5和Z7)的5个位点均为阴性，判读为MSS(见图7B)。
按此方法对比了共263例结直肠癌标本，其结果总结如表6。
表6.新位点与2B3D的比较
Figure PCTCN2022072414-appb-000007
结果说明，新位点的结果与2B3D的结果相当。
实施例5数字PCR法检测MSI
从实施例2中，挑选5个位点(Z2、Z3、Z4、Z5和Z7)，以实施例2中的方法用数字PCR法检测结直肠癌肿瘤组织263例。
按照实施例2相同方法将PCR反应体系生成微滴。按照优化PCR程序进行PCR：95℃ 10min，40个循环(94℃ 30s，56℃ 15s，72℃ 15s)，98℃ 10min，升降温速率2℃/s。打开仪器，按照要求进行设置，开始读板。
结果显示，数字PCR法与毛细管电泳法判定结果的一致性为100％。以SY020(MSI-H)和SY028(MSS)为例，其数字PCR法检测结果见图8A和8B。
实施例6基于血液样本的MSI检测
病例“SY029”的血液样本，收集于Streck游离核酸保存管(Streck Cell-Free DNA Blood Collection Tube)中，共10ml。
血浆的分离：第一次离心，4℃、1900×g、10min。小心吸取血浆上清至干净15ml离心管中，不要碰到白细胞层；第二次高速离心，4℃、16000×g、10min。小心移取上清至50ml离心管，不要碰到沉淀。分离得约4.5ml血浆。
血浆游离核酸的提取：使用康为世纪游离核酸提取试剂盒，按说明书操作，以50ul无核酸酶水洗脱游离核酸。
按实施例4中的方法，检测位点5，同时以病例“SY029”的组织样本DNA为对照，结果表明，在血液中同样可以检出组织样本中存在的MSI。(见附图9A、 9B)。
9A：病例“SY029”血液样本位点5的检测结果，检测结果为MSI，不稳定位点比例46.7％；
9B：病例“SY029”组织样本位点5的检测结果，检测结果为MSI，不稳定位点比例71％；
讨论
区分MSI结直肠癌的临床意义包括以下三个方面：
1、提示预后：临床研究发现，在Ⅱ期结直肠癌中，MSI-H结直肠癌的比例(22％)高于Ⅲ期结直肠癌(12％)，而在Ⅳ期肿瘤中仅占3.5％。这提示MSI-H结直肠癌发生转移的风险较低。特别是在Ⅱ期结直肠癌患者中，MSI状态可作为提示预后的独立预测因子：MSI-H肿瘤预后好于MSS肿瘤。Ⅱ期结直肠癌复发的高危因素包括：肿瘤组织学类型为低分化癌、有脉管/神经侵犯、淋巴结检出数量不足12个、发生梗阻或穿孔以及切缘阳性。但如肿瘤为MSI-H型时，则低分化癌不属高危因素。
2、指导治疗：研究发现，MSI-H结直肠癌对5-氟尿嘧啶和顺铂等化疗药物不敏感，而对伊立替康等化疗药物敏感，同时MSI结直肠癌对放疗比较敏感。美国国立综合癌症网络(NCCN)的结直肠癌临床实践指南明确指出，对MSI-H结直肠癌不宜采用5-氟尿嘧啶和铂类为主的治疗方案。
3、Lynch综合征患者筛查：Lynch综合征患者中，同时性或异时性多发结直肠癌较常见。如果术前能明确Lynch综合征的诊断，外科医师有可能采取更为广泛的切除术式。因此，针对结直肠癌患者进行MSI检测极为必要。
目前，MSI检测的主要方法有：
1、免疫组织化学检测错配修复蛋白：一种或多种错配修复蛋白的功能缺失可引起MSI-H，因此，检测错配修复蛋白缺失情况可间接反应肿瘤的MSI状态。目前，4种主要的错配修复蛋白MLH1、MSH2、MSH6、PMS2均已有稳定的商品化抗体，但受组织固定、染色条件、抗体克隆号等不同因素的影响，可能会出现假阳性或假阴性结果。该方法不能用于血液样本的检测。
2、基于常规PCR的MSI检测：PCR检测MSI的原理是通过PCR方法扩增特 定的微卫星序列，通过比较肿瘤组织与正常组织微卫星序列长度的差异判断是否存在MSI现象。由于该方法灵敏度有限，不适用于血液样本的检测，而且一般需要取得患者的正常组织和肿瘤组织。一般该方法通过两种检测手段进行：
(1)毛细管电泳法：迄今为止，最常用的方法是使用毛细管电泳分析标记物扩增产物的长度分布来诊断。最广为人知的“多重荧光PCR扩增和毛细管电泳”的分析方法是一种通过从正常组织和肿瘤组织中提取DNA、通过靶向卫星标记物的荧光PCR扩增DNA，然后使用毛细管电泳分析DNA的荧光来分析MSI的方法。然而，该方法需要昂贵的毛细管电泳装置分两步操作，在PCR结束后加样进行毛细管电泳的过程中存在实验室污染的可能，并且耗时较长，结果需要经验丰富训练有素的实验室人员进行分析。
(2)同位素标记PCR法：利用同位素的灵敏度，可以用同位素标记PCR引物，将扩增后的PCR产物电泳后进行曝光而检测PCR产物的长度。该方法需要进行同位素操作，对人体健康不利，耗时长，而且需要专门的操作空间和进行实验废物的处理，污染环境，目前已经不多见。
3、基于二代测序(NGS)的MSI检测：NGS检测MSI的原理是通过直接读出特定的微卫星序列来比较肿瘤组织与正常组织微卫星序列长度的差异判断是否存在MSI现象。虽然NGS可以用血液样本进行MSI检测，但是其成本和时效性均不能与数字PCR方法相比。综上所述，现有MSI检测技术操作繁琐，耗时长，阳性判读主观性强，且对于样本的类型与质量要求高，大多需要提供组织样本。
近年来，许多研究证实了单核苷酸重复序列的微卫星标志物检测比双核苷酸重复的微卫星标志物检测更具有灵敏度和特异性，MSI国际研究合作组织推荐的5个位点在数字PCR上的表现不尽如人意，因此本发明中重新筛选了一组全新的MSI位点，并开发了相应的数字PCR检测方法。本发明的检测MSI的方法具有灵敏度高、无需标准曲线即可准确定量、操作简单等优点。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申 请所附权利要求书所限定的范围。

Claims (17)

  1. 一种人微卫星不稳定(Microsatellite Instability，MSI)位点检测试剂的用途，其特征在于，用于制备一诊断试剂或试剂盒，所述诊断试剂或试剂盒用于诊断MSI相关疾病和/或用于对MSI相关疾病进行预后；
    其中，所述的MSI位点选自下组A的一个或多个位点：
    (Z1)chr3:30650236-30650508；
    (Z2)chr11:106739898-106740117；
    (Z3)chr16:18841298-18841518；
    (Z4)chr17:19411505-19411722；
    (Z5)chr20:62921533-62921750；
    (Z6)chr2:47408320-47408461；
    (Z7)chr2:147906719-147906938；
    (Z8)chr6:142407071-142407290；
    (Z9)chr14:93268657-93268877；
    (Z10)chr20:47779916-47780134；
    (Z11)chr7:143306180-143306440；
    (Z12)chr1:201819424-201819543；
    (Z13)chr1:201771449-201771558；
    (Z14)chr2:61827581-61827677；
    (Z15)chr2:147857500-147857584；
    (Z16)chr4:82821524-82821646；
    (Z17)chr5:172998578-172998712；
    (Z18)chr6:142337918-142338138；
    (Z19)chr14:93244766-93244900；
    (Z20)chr15:45593287-45593508；
    (Z21)chr15:33349068-33349160；
    (Z22)chr15:33764931-33765150；
    (Z23)chr16:18854033-18854164。
  2. 如权利要求1所述的用途，其特征在于，所述的MSI位点选自下组：
    (Z1)chr3:30650236-30650508；
    (Z2)chr11:106739898-106740117；
    (Z3)chr16:18841298-18841518；
    (Z4)chr17:19411505-19411722；
    (Z5)chr20:62921533-62921750；
    (Z6)chr2:47408320-47408461；
    (Z7)chr2:147906719-147906938；
    (Z8)chr6:142407071-142407290；
    (Z9)chr14:93268657-93268877；
    (Z10)chr20:47779916-47780134；
    (Y1)Z1～Z10的任意组合。
  3. 如权利要求1所述的用途，其特征在于，所述的MSI位点包括(a)选自Z2、Z3、Z4、Z5和Z7中的一个或多个(如2、3、4、或5)位点；和(b)除Z2、Z3、Z4、Z5和Z7的额外的MSI位点。
  4. 如权利要求3所述的MSI位点，其特征在于，所述的额外的MSI位点选自下组：BAT-26、BAT-25、MONO-27、NR-21、NR-24、D5S346、D2S123、D17S250或其组合。
  5. 如权利要求1所述的用途，其特征在于，所述的MSI相关疾病为肿瘤或癌症。
  6. 如权利要求5所述的用途，其特征在于，所述的肿瘤或癌症选自下组：结直肠癌、子宫内膜癌、子宫肉瘤、胃癌、小肠癌、宫颈癌、肝癌、食管癌、胰腺癌、卵巢癌、胆囊癌、睾丸癌、前列腺癌、输卵管癌、外阴癌、肾上腺皮质癌、原发性腹腔肿瘤、胆管癌、乳腺癌、神经内分泌肿瘤、胸腺癌、甲状腺癌、小细胞肺癌、原发未知的肿瘤等。
  7. 如权利要求1所述的用途，其特征在于，所述诊断试剂或试剂盒用于选自下组的检测：血清检测、血浆检测、细胞检测、组织样品检测。
  8. 如权利要求1所述的用途，其特征在于，所述的检测为PCR检测或测序检测；较佳地，为数字PCR(digital PCR，ddPCR)检测。
  9. 一种用于检测人微卫星不稳定(Microsatellite Instability，MSI)位点的试剂， 其特征在于，所述位点选自hg38染色体中的位点1～位点10(SEQ ID NO.:1～10)；其中，所述试剂选自下组：
    (a)用于检测chr3位点1(SEQ ID NO.:1)MSI的第一引物对，其中第一引物对包括SEQ ID NO.:11和12所示的引物；
    (b)用于检测chr11位点2(SEQ ID NO.:2)MSI的第二引物对，其中第二引物对包括SEQ ID NO.:19和20所示的引物；
    (c)用于检测chr16位点3(SEQ ID NO.:3)MSI的第三引物对，其中第三引物对包括SEQ ID NO.:23和24所示的引物；
    (d)用于检测chr17位点4(SEQ ID NO.:4)MSI的第四引物对，其中第四引物对包括SEQ ID NO.:25和26所示的引物；
    (e)用于检测chr20位点5(SEQ ID NO.:5)MSI的第五引物对，其中第五引物对包括SEQ ID NO.:29和30所示的引物；
    (f)用于检测chr2位点6(SEQ ID NO.:6)MSI的第六引物对，其中第六引物对包括SEQ ID NO.:13和14所示的引物；
    (g)用于检测chr2位点7(SEQ ID NO.:7)MSI的第七引物对，其中第七引物对包括SEQ ID NO.:15和16所示的引物；
    (h)用于检测chr6位点8(SEQ ID NO.:8)MSI的第八引物对，其中第八引物对包括SEQ ID NO.:17和18所示的引物；
    (i)用于检测chr14位点9(SEQ ID NO.:9)MSI的第九引物对，其中第九引物对包括SEQ ID NO.:21和22所示的引物；
    (j)用于检测chr20位点10(SEQ ID NO.:10)MSI的第十引物对，其中第十引物对包括SEQ ID NO.:27和28所示的引物；
    上述(a)至(j)任一项的组合。
  10. 如权利要求9所述的试剂，其特征在于，所述试剂还包括：
    (a1)与第一引物对配合使用的第一探针，其中所述的第一探针选自下组：SEQ ID NO.:31所示的探针、SEQ ID NO.:32所示的探针或其组合；和/或
    (b1)与第二引物对配合使用的第二探针，其中所述的第二探针选自下组：SEQ ID NO.:39所示的探针、SEQ ID NO.:40所示的探针、或其组合；和/或
    (c1)与第三引物对配合使用的第三探针，其中所述的第三探针选自下组：SEQ  ID NO.:43所示的探针、SEQ ID NO.:44所示的探针、或其组合；和/或
    (d1)与第四引物对配合使用的第四探针，其中所述的第四探针选自下组：SEQ ID NO.:45所示的探针、SEQ ID NO.:46所示的探针、或其组合；和/或
    (e1)与第五引物对配合使用的第五探针，其中所述的第五探针选自下组：SEQ ID NO.:49所示的探针、SEQ ID NO.:50所示的探针、或其组合；和/或
    (f1)与第六引物对配合使用的第六探针，其中所述的第六探针选自下组：SEQ ID NO.:33所示的探针、SEQ ID NO.:34所示的探针、或其组合；和/或
    (g1)与第七引物对配合使用的第七探针，其中所述的第七探针选自下组：SEQ ID NO.:35所示的探针、SEQ ID NO.:36所示的探针、或其组合；和/或
    (h1)与第八引物对配合使用的第八探针，其中所述的第八探针选自下组：SEQ ID NO.:37所示的探针、SEQ ID NO.:38所示的探针、或其组合；和/或
    (i1)与第九引物对配合使用的第九探针，其中所述的第九探针选自下组：SEQ ID NO.:41所示的探针、SEQ ID NO.:42所示的探针、或其组合；和/或
    (j1)与第十引物对配合使用的第十探针，其中所述的第十探针选自下组：SEQ ID NO.:47所示的探针、SEQ ID NO.:48所示的探针、或其组合。
  11. 如权利要求10所述的试剂，其特征在于，所述第一探针到第十探针的结构(5'-3')如式I所示：
    L1-L2-L3  I
    其中，
    L1为荧光基团而L3为淬灭基团；或者L3为荧光基团而L1为淬灭基团；
    L2为核苷酸的特异性互补核酸序列；
    “-”为化学键、连接基团、或1-3个核苷酸构成的接头。
  12. 一种试剂盒，其特征在于，所述试剂盒含有权利要求9所述的用于检测MSI位点的试剂。
  13. 如权利要求12所述的试剂盒，其特征在于，所述的MSI位点选自组A的一个或多个位点(即Z1～Z23中的一个或多个)，优选地所述的MSI位点为选自Z1～Z10中的一个或多个。
  14. 如权利要求12所述的试剂盒，其特征在于，所述试剂盒用于检测来自肿瘤细胞、肿瘤组织或疑似肿瘤组织中人DNA样品中的MSI位点。
  15. 一种检测试剂的组合，其特征在于，所述的检测试剂组合包括用于检测 人微卫星不稳定(Microsatellite Instability，MSI)位点的n种检测试剂，其中，所述的MSI位点选自组A的位点，并且n为≥2的正整数；较佳地，n为2-25，更佳地n为2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、或23。
  16. 一种检测待测样本中MSI的方法，其特征在于，包括步骤：
    (S1)提供一PCR反应体系，所述PCR反应体系中含有作为模板的待测样本、以及用于扩增的引物对，所述的引物对选自下组：
    (a)用于检测chr3位点1(SEQ ID NO.:1)MSI的第一引物对，其中第一引物对包括SEQ ID NO.:11和12所示的引物；
    (b)用于检测chr11位点2(SEQ ID NO.:2)MSI的第二引物对，其中第二引物对包括SEQ ID NO.:19和20所示的引物；
    (c)用于检测chr16位点3(SEQ ID NO.:3)MSI的第三引物对，其中第三引物对包括SEQ ID NO.:23和24所示的引物；
    (d)用于检测chr17位点4(SEQ ID NO.:4)MSI的第四引物对，其中第四引物对包括SEQ ID NO.:25和26所示的引物；
    (e)用于检测chr20位点5(SEQ ID NO.:5)MSI的第五引物对，其中第五引物对包括SEQ ID NO.:29和30所示的引物；
    (f)用于检测chr2位点6(SEQ ID NO.:6)MSI的第六引物对，其中第六引物对包括SEQ ID NO.:13和14所示的引物；
    (g)用于检测chr2位点7(SEQ ID NO.:7)MSI的第七引物对，其中第七引物对包括SEQ ID NO.:15和16所示的引物；
    (h)用于检测chr6位点8(SEQ ID NO.:8)MSI的第八引物对，其中第八引物对包括SEQ ID NO.:17和18所示的引物；
    (i)用于检测chr14位点9(SEQ ID NO.:9)MSI的第九引物对，其中第九引物对包括SEQ ID NO.:21和22所示的引物；
    (j)用于检测chr20位点10(SEQ ID NO.:10)MSI的第十引物对，其中第十引物对包括SEQ ID NO.:27和28所示的引物；
    上述(a)至(j)任一项的组合；
    (S2)对步骤(S1)的所述PCR反应体系进行PCR反应，从而获得扩增产物；
    (S3)对步骤(S2)中产生的扩增产物进行分析，从而获得所述待测样本MSI情况；其中，以检测的位点数P 总＝5为例，
    若样本中的MSI位点数P MSI≥2，则判定为高度不稳定(MSI-H)；
    若样本中的MSI位点数P MSI＝1，则判定为低度不稳定(MSI-L)；
    若样本中的MSI位点数P MSI＝0，则判定为稳定(MSS)。
  17. 如权利要求16所述的方法，其特征在于，若所述检测的位点数P 总≥5时，样本中的MSI位点数百分比为P(P MSI/P 总)，
    若P≥40％时，则判定为高度不稳定(MSI-H)；
    若10≤P＜40％时，则判定为低度不稳定(MSI-L)；
    若P＝0时，则判定为稳定(MSS)。
PCT/CN2022/072414 2020-11-16 2022-01-17 人微卫星不稳定位点的检测方法及其应用 WO2022100766A1 (zh)

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