WO2021004215A1 - Réactif de détection combiné multigénique - Google Patents

Réactif de détection combiné multigénique Download PDF

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WO2021004215A1
WO2021004215A1 PCT/CN2020/094963 CN2020094963W WO2021004215A1 WO 2021004215 A1 WO2021004215 A1 WO 2021004215A1 CN 2020094963 W CN2020094963 W CN 2020094963W WO 2021004215 A1 WO2021004215 A1 WO 2021004215A1
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seq
sequence
methylation
detection
gene
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PCT/CN2020/094963
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Chinese (zh)
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吴孝林
刘相林
罗茵
邹鸿志
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广州市康立明生物科技有限责任公司
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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
    • C12Q2600/154—Methylation markers

Definitions

  • This application belongs to the field of biomedicine, and particularly relates to primers, capture reagents, nucleic acid probes, methylation detection reagents, kits and applications thereof.
  • Colorectal cancer also known as colorectal cancer
  • colorectal cancer is a common malignant tumor of the digestive tract.
  • the incidence rate is increasing year by year in my country.
  • some coastal areas of my country, such as Shanghai and Guangzhou the incidence of colorectal cancer has jumped to the second place, second only to lung cancer.
  • the formation of bowel cancer is the result of accumulation of genetic defects and epigenetic defects.
  • Early onset of colorectal cancer is hidden, often without obvious symptoms, and symptoms such as blood in the stool, abdominal pain, and diarrhea may appear in the late stage. When the symptoms appear, it is often in the late stage, which brings great pain and expensive treatment to the patient. Therefore, early detection, early diagnosis and early treatment are important measures to reduce the incidence and mortality of colorectal cancer.
  • the current screening methods for colorectal cancer mainly include occult blood test and colonoscopy.
  • the occult blood test is vulnerable to food or the detection rate of adenomas is not high.
  • colonoscopy is the gold standard for the diagnosis of bowel cancer, it is not highly adherent to the population when used as a screening method. Therefore, there is an urgent need for a colorectal cancer screening method with high accuracy and high compliance.
  • the existing methods for detecting single methylation markers of colorectal cancer have the following two limitations: First, the existing research and product detection methods are to detect a marker in a PCR reaction well, and the detection method cannot Realize multiple detection of methylation markers in a PCR reaction well. Second, when the existing methylation markers for colorectal cancer detection, such as SDC2, ITAG4, Septin 9, etc., detect colorectal cancer by detecting the methylation level of a single gene, the sensitivity of the detection is limited by genetic factors.
  • the detection sensitivity of methylated SDC2 gene is 84.2%, and the specificity is 97.9%; the detection sensitivity of methylated ITGA4 is 83.8%, and the specificity is 95.2%; the detection sensitivity of methylated Septin9 is 79.3%. 94.3%.
  • the specificity of these single markers for colorectal cancer detection can reach more than 90%, but when the specificity reaches more than 90%, the sensitivity cannot reach more than 90%.
  • this application provides a primer and its application in preparing reagents or kits for detecting colorectal tumors.
  • this application provides a capture sequence and its application in preparing reagents or kits for detecting colorectal tumors.
  • the present application provides a probe and its application in preparing reagents or kits for detecting colorectal tumors.
  • this application provides a multi-gene methylation combined detection reagent, the genes are SDC2, COL4A1/COL4A2 and ITGA4.
  • this application provides a colorectal tumor detection reagent and kit with strong specificity and high sensitivity.
  • the present application provides a primer, which includes a primer such as SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and their complementary sequences have at least 85% or at least 90% or at least 91% or at least At least any one of 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or 98% or at least 99% or 100% identical sequence.
  • a primer such as SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID
  • the primers include SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25 have at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least Any one of a sequence that is 95% or at least 96% or at least 97% or 98% or at least 99%, or 100% identical.
  • the primers include SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 18 and SEQ ID NO: 19 and SEQ ID NO: 24 and SEQ ID NO: 25 have at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least At least one primer pair in a sequence that is 95% or at least 96% or at least 97% or 98% or at least 99%, or 100% identical.
  • the primers include SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 7 and SEQ ID NO: 8, and SEQ ID NO: 11 and SEQ ID NO: 12. .
  • the primers include the primer pairs shown in SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 18 and SEQ ID NO: 19, and SEQ ID NO: 24 and SEQ ID NO: 25 .
  • this application also provides a capture sequence
  • the capture sequence includes the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, And their complementary sequence has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or At least any one of the 100% identical sequences.
  • the capture sequence includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, which has at least 85% or at least 90% or at least 91% or At least one of at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or 100% identical sequence.
  • the capture sequence includes the sequence shown in SEQ ID NO: 1, SEQ ID NO: 6 and SEQ ID NO: 10.
  • the capture sequence includes the sequence shown in SEQ ID NO: 2, SEQ ID NO: 6 and SEQ ID NO: 10.
  • the present application also provides a nucleic acid probe, the nucleic acid probe comprising the sequence shown in SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, and their complements
  • the sequence has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or 98% or at least 99%, or 100% identity At least any one of the sequence.
  • the nucleic acid probe includes at least one of SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13.
  • the nucleic acid probe comprises the sequence shown in SEQ ID NO: 5, SEQ ID NO: 9 and SEQ ID NO: 13.
  • the application also provides the application of the above-mentioned primers or capture sequences or probes in preparing reagents or kits for detecting colorectal tumors.
  • the application also provides applications of the above-mentioned primers or capture sequences or probes or reagents or kits.
  • the application also provides the application of the above-mentioned primers or capture sequences or probes or reagents or kits in the detection of colorectal tumors.
  • this application also provides a multi-gene methylation combined detection reagent, including a combination of SDC2, COL4A1/COL4A2 and ITGA4 gene combination detection reagent.
  • the obtained capture sequences, primers and/or probes for each gene in the combination of SDC2, COL4A1/COL4A2 and ITGA4 genes are included.
  • capture sequences, primers, and/or probes obtained for the CpG islands of each gene in the SDC2, COL4A1/COL4A2, and ITGA4 gene combination are included.
  • the methylation detection reagents provided in the present application detect the morphology of each gene in the combination of SDC2, COL4A1/COL4A2, and ITGA4 genes, intergenic regions or promoter regions, and regions near the promoter regions. Basic level.
  • the methylation detection reagent provided in the present application includes a capture sequence obtained from the promoter region of each gene in the SDC2, COL4A1/COL4A2, and ITGA4 gene combination or the CpG island in the vicinity of the promoter region , Primers and/or probes.
  • SDC2, COL4A1/COL4A2, and ITGA4 can each be detected for methylation levels. In other embodiments, SDC2, COL4A1/COL4A2, and ITGA4 can be simultaneously detected for methylation levels, such as using multiple real-time fluorescent quantitative PCR to detect the methylation levels of multiple genes. In some aspects, it is more convenient for SDC2, COL4A1/COL4A2 and ITGA4 to be detected simultaneously by multiplex PCR.
  • COL4A1/COL4A2 in this application means “COL4A1 or COL4A2".
  • the human genes COL4A1 and COL4A2 are closely linked at the far end of the long arm of chromosome 13. These two genes are in a "head-to-head” positional relationship, and they are transcribed in opposite directions. The 5'ends of the COL4A1 and COL4A2 genes are close to each other, with an interval of 127bp.
  • This sequence is a bidirectional promoter region shared by the two genes [1]. Therefore, the sequence designed according to the bidirectional promoter shared by the two can target both COL4A1 and COL4A2. In this application, whether COL4A1 or COL4A2 is used to label sequence information, there is no difference between the two.
  • Detection in this application is the same as “diagnosis”. In addition to the early diagnosis of colorectal tumors, it also includes the diagnosis of the middle and late stages of colorectal tumors, and also includes colorectal tumor screening, risk assessment, prognosis, disease identification, and disease stages. Selection of diagnostic and therapeutic targets.
  • colorectal tumor marker combination SDC2, COL4A1/COL4A2 and ITGA4 makes the early diagnosis of colorectal tumors possible.
  • a gene methylated in a cancer cell is methylated in a clinically or morphologically normal cell, this indicates that the normal cell is developing into cancer.
  • colorectal cancer can be diagnosed at an early stage by the methylation of the colorectal tumor-specific SDC2, COL4A1/COL4A2, and ITGA4 gene combination in normal appearance cells.
  • early diagnosis refers to the possibility of detecting cancer before metastasis, preferably before the morphological changes of tissues or cells can be observed.
  • the reagents/kits of this application are also expected to be used for colorectal tumor screening, risk assessment, prognostic diagnosis, disease identification, diagnosis of disease stages, and selection of therapeutic targets.
  • diagnosis can be made by measuring the degree of methylation of the combination of SDC2, COL4A1/COL4A2 and ITGA4 genes obtained from a sample through the progress of colorectal tumors in different stages or periods.
  • SDC2, COL4A1/COL4A2, and ITGA4 gene combination methylation degree of nucleic acids isolated from samples of each stage of colorectal cancer with one or one isolated from samples from intestinal tissues without abnormal cell proliferation
  • the methylation degree of SDC2, COL4A1/COL4A2 and ITGA4 gene combination of multiple nucleic acids can detect the specific stage of colorectal tumor in the sample.
  • CpG islands refer to regions rich in CpG dinucleotides, usually located in the promoter and its vicinity.
  • the detection sites of methylation in this application include not only CpG islands, but also other regions such as CpG sites that are heterozygously methylated in the genome or intergenic regions, or isolated CpG sites.
  • the methylation joint detection reagent of the combination of SDC2, COL4A1/COL4A2 and ITGA4 genes may be a methylation detection reagent in the prior art.
  • MSP methylation-specific PCR
  • qMSP methylation-specific quantitative PCR
  • DNA binding protein PCR quantitative PCR and DNA chips
  • methylation-sensitive restriction endonucleases bisulfite sequencing or pyrosequencing, etc.
  • other methylation detection methods can be introduced through patent US62007687. Each detection method has its corresponding reagents, which can be used in this application to detect the methylation of SDC2, COL4A1/COL4A2 and ITGA4 gene combinations.
  • the primers and/or probes detect the methylation of each gene in the SDC2, COL4A1/COL4A2, and ITGA4 gene combination by quantitative Methylation-Specific PCR (qMSP).
  • qMSP quantitative Methylation-Specific PCR
  • the SDC2 gene capture sequence in the methylation combined detection reagent provided in the present application includes any one of the following nucleotide sequences:
  • the nucleotide sequence shown in SEQ ID NO: 1 or 2 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% Or a sequence of at least 97% or 98% or at least 99%, or 100% identity;
  • the capture sequence for the methylation detection of the COL4A1/COL4A2 gene includes any one of the following nucleotide sequences:
  • the nucleotide sequence shown in SEQ ID NO: 6 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence;
  • the capture sequence for the methylation detection of the ITGA4 gene includes any one of the nucleotide sequences shown below:
  • the nucleotide sequence shown in SEQ ID NO: 10 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence;
  • the upstream primer in the primers for methylation detection of the SDC2 gene provided in this application includes any one of the nucleotide sequences shown below:
  • the nucleotide sequence shown in SEQ ID NO: 3 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence;
  • the downstream primer in the primer for methylation detection of the SDC2 gene contains any one of the following nucleotide sequences:
  • nucleotide sequence shown in IX, SEQ ID NO: 4 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence;
  • the upstream primer in the primer for methylation detection of the COL4A1/COL4A2 gene contains any one of the following nucleotide sequences:
  • nucleotide sequence shown in any one of XI, SEQ ID NO: 7 or SEQ ID NO: 18 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% Or at least 96% or at least 97% or 98% or at least 99%, or 100% identical sequence;
  • the downstream primer in the primer for methylation detection of the COL4A1/COL4A2 gene contains any one of the following nucleotide sequences:
  • nucleotide sequence shown in any of XIII, SEQ ID NO: 8 or SEQ ID NO: 19 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% Or at least 96% or at least 97% or 98% or at least 99%, or 100% identical sequence;
  • the primer pair for methylation detection of the COL4A1/COL4A2 gene is shown in SEQ ID NO: 7 and SEQ ID NO: 8;
  • the primer pair for methylation detection of the COL4A1/COL4A2 gene is shown in SEQ ID NO: 18 and SEQ ID NO: 19;
  • the upstream primer in the primer for methylation detection of ITGA4 gene contains any one of the following nucleotide sequences:
  • nucleotide sequence shown in any one of XV, SEQ ID NO: 11 or SEQ ID NO: 24 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% Or at least 96% or at least 97% or 98% or at least 99%, or 100% identical sequence;
  • the downstream primer in the primer for methylation detection of ITGA4 gene contains any one of the following nucleotide sequences:
  • nucleotide sequence shown in any one of XVII, SEQ ID NO: 12 or SEQ ID NO: 25 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% Or at least 96% or at least 97% or 98% or at least 99%, or 100% identical sequence;
  • the primer pair for methylation detection of the ITGA4 gene is shown in SEQ ID NO: 11 and SEQ ID NO: 12;
  • the primer pair for methylation detection of the ITGA4 gene is shown in SEQ ID NO: 24 and SEQ ID NO: 25;
  • the methylation detection probe of the SDC2 gene in the methylation detection reagent provided in the present application includes any one of the following nucleotide sequences:
  • nucleotide sequence shown in XIX SEQ ID NO: 5 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence;
  • the COL4A1/COL4A2 gene methylation detection probe contains any one of the following nucleotide sequences:
  • SEQ ID NO: 9 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence;
  • the ITGA4 gene methylation detection probe contains any one of the following nucleotide sequences:
  • SEQ ID NO: 13 has at least 85% or at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% Or 98% or at least 99%, or 100% identical sequence;
  • the nucleic acid probe further includes one or more of a label such as a radioisotope, a fluorescent group, a bioluminescent compound, a chemiluminescent compound, a metal chelate and an enzyme.
  • a label such as a radioisotope, a fluorescent group, a bioluminescent compound, a chemiluminescent compound, a metal chelate and an enzyme.
  • the label of the probe is a fluorescent group
  • these fluorescent groups include but are not limited to one or more of VIC, ROX, FAM, Cy5, HEX, TET, JOE, NED, and Texas Red.
  • the probes of the SDC2, COL4A1/COL4A2, and ITGA4 genes are labeled with the same fluorophore, so that the sum of the methylation levels of these genes can be easily detected through a fluorescence channel, instead of Each gene needs to be detected with a different fluorescence channel, which reduces the complexity of the experiment.
  • the application also provides a kit for detecting tumors, including the methylation detection reagent.
  • the kit provided by the present application includes: a first container, which contains a capture reagent; a second container, which contains a primer pair for amplification; and a third container, which contains a probe.
  • the kit provided in this application also includes the commonly used reagents in the kit, such as the commonly used conversion agent in qMSP, which is used to convert all unmethylated cytosine bases into uracil, The methylated cytosine base remains unchanged.
  • the conversion agent is not particularly limited.
  • the reagents reported in the prior art that can convert cytosine to uracil can be used, such as hydrazine salt, bisulfite and bisulfite (such as sodium metabisulfite, subsulfite).
  • One or more of potassium bisulfate, cesium bisulfite, ammonium bisulfite, etc. Another example is DNA polymerase, dNTPs, Mg 2+ ions and buffers commonly used in the amplification of COL4A1 gene.
  • the kit includes: a first container, which contains a capture reagent; a second container, which contains a primer pair for amplification; and a third container, which contains a probe.
  • the fourth container contains a conversion reagent for converting unmethylated cytosine.
  • the kit further includes instructions.
  • the kit further includes nucleic acid extraction reagents.
  • the kit further includes a sampling device.
  • This application also provides the above-mentioned methylation detection reagents, kits, capture sequences, primers and/or probes in the preparation of reagents or kits for methylation detection, or the preparation of reagents or kits for detecting colorectal tumors. application.
  • the application also provides the application of the above-mentioned methylation detection reagents, kits, capture sequences, primers and/or probes in methylation detection, or applications in the detection of colorectal tumors.
  • This application also provides a tumor detection system, which includes the following components:
  • the methylation detection component contains a methylation detection instrument
  • the methylation detection component further contains one or more of the methylation detection reagents, kits, capture sequences, primers, and probes.
  • the methylation detection instrument includes one or more of a fluorescent quantitative PCR machine, a PCR machine, and a sequencer.
  • the data processing component contains a data processing machine.
  • the data processing machine includes any equipment or instrument or device that can perform data processing that can be used by those skilled in the art.
  • the data processing machine includes one or more of a calculator and a computer.
  • the computer is loaded with any software or program that can be used by those skilled in the art that can perform data processing or statistical analysis.
  • the computer includes a computer loaded with one or more software of SPSS, SAS, and Excel.
  • the result output member includes a result output device.
  • the output device includes any equipment or instrument or device that can display the data processing result as readable content.
  • the result exporter includes one or more of a screen and a paper report.
  • the data processor is configured to a. receive the test data of the test sample and the normal control sample; b. store the test data of the test sample and the normal control sample; c. compare the same type The test data of the test sample and the normal control sample; d. According to the comparison result, respond to the probability or possibility of the subject suffering from the tumor.
  • the result output component is used to output the probability or likelihood that the subject has a tumor.
  • This application also provides a method for diagnosing colorectal tumors, which includes the following steps:
  • the diagnostic method of the present application can be used before and after the treatment of colorectal tumors or in combination with the treatment of colorectal tumors, after treatment, such as evaluating the success of the treatment or monitoring the remission, recurrence and/or progress (including metastasis) of the colorectal tumor after treatment.
  • Another aspect of the present application provides a method for treating colorectal tumors, the method comprising administering surgery, chemotherapy, radiotherapy, radiotherapy and chemotherapy, immunotherapy, oncolytic virus therapy to patients diagnosed with colorectal tumors by the above diagnosis method , Or any other types of colorectal tumor treatment methods used in the art and combinations of these treatment methods.
  • the data processing component in the tumor detection system, or the judgment standard of the colorectal tumor diagnosis method is: judging the tumor based on the cutoff value of the Ct value of methylation-specific quantitative PCR (qMSP) Specimen and normal specimens.
  • qMSP methylation-specific quantitative PCR
  • the cutoff value of the Ct value is about 36-39.
  • the cutoff value of the Ct value is about 38.
  • the above-mentioned cutoff value of the Ct value is about 36-39 or the sample targeted for 38 is a stool sample.
  • the Ct value of the test sample when the Ct value of the test sample is less than the threshold value of the Ct value, it is determined as a tumor sample, and when the Ct value of the test sample is greater than or equal to the threshold value of the Ct value, it is determined as Normal sample.
  • the tumor described in this application is a colorectal tumor.
  • the tumor described in this application is colorectal cancer or adenoma.
  • the sample or sample type targeted by this application includes tissue, body fluid, or excrement.
  • the tissue described herein comprises intestinal tissue.
  • the body fluid described in this application includes blood, extracellular fluid, tissue fluid, lymph fluid, cerebrospinal fluid, or aqueous humor.
  • the blood described in this application includes, serum, and plasma.
  • the excrement comprises sputum, urine, saliva or feces.
  • the excrement comprises feces.
  • a method for treating colorectal tumors comprising the following steps:
  • test sample derived from the subject with the primer of claim 1 or 2, or the capture sequence of claim 3 or 4, or the nucleic acid probe of claim 5 or 6, or claim 7. -9 contact with the kit of any of the reagents or the kit of claim 10 to detect the methylation level of the gene in the test sample;
  • methylation-specific quantitative PCR (qMSP) is used to detect the methylation level of genes.
  • the colorectal tumor sample and the normal sample are judged based on the cutoff value of the Ct value quantified by methylation fluorescence.
  • the methylation level of the promoter region of each gene in the SDC2, COL4A1/COL4A2 and ITGA4 gene combination can be detected by the primers, capture reagents, and nucleic acid probes. It is good to distinguish colorectal cancer specimens from stool specimens. And the detection sensitivity and specificity for bowel cancer are extremely high.
  • the primers, capture reagents, nucleic acid probes, SDC2, COL4A1/COL4A2 and ITGA4 gene combinations and technical solutions provided in this application can be detected with extremely high sensitivity and specificity
  • the detection sensitivity and specificity of the technical solution of the present application for colorectal cancer are higher than 90%.
  • the specific points are as follows:
  • a technical solution of this application performs joint detection of methylation of SDC2, COL4A1/COL4A2 and ITGA4 gene combination. This method can realize joint detection of multiple genes, which greatly reduces the complexity of the test and improves the detection effectiveness.
  • the methylation detection reagent of the combination of SDC2, COL4A1/COL4A2 and ITGA4 genes can detect 91.36% of colorectal cancers in stool samples with a specificity of 95.28%, which can be easily Stool is used as a test sample to make a reliable diagnosis of colorectal cancer.
  • the stool sample is very easy to obtain, the sampling is non-invasive and simple, and it will not cause any pain and inconvenience to the patient.
  • the above-mentioned another technical solution contains methylation detection reagents of SDC2, COL4A1/COL4A2 and ITGA4 gene combination.
  • the extraction detection method can easily and accurately judge colorectal cancer and normal people.
  • the methylation of this gene combination Chemical testing reagents are expected to be used in stool genetic testing kits and serve the clinical testing of bowel cancer.
  • the reagent/kit in the other technical solution mentioned above is to detect and diagnose cancer by methylation level. More and more studies have confirmed that methylation change is an early event in the process of tumorigenesis, and methylation is detected. Abnormalities are easier to find early lesions.
  • Figure 1 is the amplification curve of the primer pair S0 in Example 1;
  • Figure 2 is the amplification curve of the primer pair S0 in Example 1;
  • Figure 3 is an amplification curve of the primer pair S0 in Example 1;
  • Figure 4 is the amplification curve of the primer pair CO in Example 2.
  • Figure 5 is an amplification curve of the primer pair C1 in Example 2.
  • Figure 6 is the amplification curve of primer pair C2 in Example 2.
  • Figure 7 is the amplification curve of the primer pair I0 in Example 3.
  • Figure 8 is the amplification curve of the primer pair I1 in Example 3.
  • Figure 9 is the amplification curve of the primer pair I0 in Example 3.
  • Figure 10 shows the ROC curve of 935 stool specimens in Example 4 for the combined detection of SDC2, COL4A1/COL4A2 and ITG4 genes for colorectal cancer and adenomas ( ⁇ 1cm);
  • Figure 11 shows the detection results of 23 cases of colorectal cancer patients with preoperative and postoperative stool samples detected by the method of combined detection of SDC2, COL4A1/COL4A2 and ITG4 genes in Example 5;
  • Figure 12 shows the ROC curve of 240 stool specimens in Comparative Example 1, SOX21 gene detection for colorectal cancer and adenoma;
  • Figure 13 shows the ROC curve of the combined detection of colorectal cancer with ITGA4, COL4A1/COL4A2 genes in 109 stool specimens in Comparative Example 2.
  • the "capture sequence”, "primer” or “probe” in the present application refers to an oligonucleotide that contains a region complementary to a sequence of at least 6 consecutive nucleotides of a target nucleic acid molecule (for example, a target gene). In some embodiments, at least a portion of the sequence of the primer or probe is not complementary to the amplified sequence. In some embodiments, the primer or probe contains at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 relative to the target molecule. A region where the sequence of consecutive nucleotides is complementary.
  • the primer or probe When a primer or probe contains a region "complementary to at least x consecutive nucleotides of the target molecule", the primer or probe is at least 95% of at least x consecutive or discontinuous block nucleotides of the target molecule Complementary.
  • the primer or probe is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, 96%, at least 97%, at least 98%, at least 99%, or 100% complementary.
  • normal samples refer to samples of the same type isolated from individuals known to be free of the cancer or tumor.
  • the "subject” is a mammal, such as a human.
  • the samples for methylation detection in this application include but are not limited to DNA, or RNA, or DNA and RNA samples containing mRNA, or DNA-RNA hybrids.
  • the DNA or RNA can be single-stranded or double-stranded.
  • methylation level and “methylation degree” can usually be expressed as the percentage of methylated cytosine, which is the number of methylated cytosine divided by the number of methylated cytosine and the amount of unmethylated cytosine.
  • the sum of the number of methylated cytosines; and the method of dividing the number of methylation target genes by the number of internal reference genes is generally used to express the methylation level; and other methods of expressing the methylation level in the prior art.
  • sample is the same as “specimen”.
  • the composition may include A alone; B alone; C alone; D alone Contains the combination of A and B; contains the combination of A and C; contains the combination of A and D; contains the combination of B and C; contains the combination of B and D; contains the combination of C and D; contains the combination of A, B and C Combination; including A, B and D combination; including A, C and D combination; including B, C and D combination; or A, B, C and D in combination.
  • the above 3 pairs of primers were used to detect DNA from stool samples of 2 patients with colorectal cancer and DNA from 2 normal human stool samples to analyze the specificity of amplification.
  • the above 3 pairs of primers were used to detect the DNA of the stool samples of 3 patients with colorectal cancer and the DNA of 3 normal human stool samples to analyze the specificity of amplification.
  • the above 3 pairs of primers were used to detect the DNA of the stool samples of 3 patients with colorectal cancer and the DNA of 3 normal human stool samples to analyze the specificity of amplification.
  • the qMSP reaction system of this example 30ul (nuclease-free water 2.98ul, 5 ⁇ Colorless GoTaq Flexi Buffer 6ul, MgCl 2 (25mM) 5ul, dNTPs (10mM) 1ul, GoTaq Hot Start polymerase 0.6ul, ACTB-FP (100uM) 0.08ul, ACTB-RP (100uM) 0.08ul, ACTB-Probe (100uM) 0.06ul, SDC2-FP (100uM) 0.12ul, SDC2-RP (100uM) 0.12ul, SDC2-Probe (100uM) 0.04ul, COL4A2- FP (100uM) 0.06ul, COL4A2-RP (100uM) 0.06ul, COL4A2-RP (100uM) 0.06ul, COL4A2-Probe (100uM) 0.04ul, ITGA4-FP (100uM) 0.06ul, ITGA4-RP (100uM) 0.06ul, IT
  • the capture and PCR reaction uses ACTB as the internal reference gene, and finally judges the methylation level in the specimen according to the CT value.
  • the target gene is judged as positive with a CT value ⁇ 38, and it is judged as negative with a CT value> 38.
  • the 5'ends of the COL4A1 and COL4A2 genes are close to each other, they are separated by 127bp, which is a shared bidirectional promoter region.
  • the methylation region detected in this example is the bidirectional promoter region of the COL4A1 and COL4A2 genes. Therefore, in this embodiment, the gene direction of COL4A2 of the two genes is selected to annotate sequence information.
  • methylation sites of SDC2, COL4A1/COL4A2 and ITGA4 genes are mainly located in the promoter region or nearby CpG islands.
  • the same fluorescent group is used to label the PCR probes of SDC2, COL4A2 and ITGA4, so the sum of the methylation levels of these genes can be easily detected through a fluorescent channel, without the need for each gene Using different fluorescence channels for detection reduces the complexity of the experiment.
  • SEQ ID NO. 1 SDC2 capture sequence 1:
  • SEQ ID NO. 2 Capture sequence of SDC2 2:
  • SEQ ID NO.3 SDC2-FP: 5’-GAGGAAGCGAGCGTTTTC-3’
  • SEQ ID NO. 4 SDC2-RP: 5’-AAAATACCGCAACGATTACGA-3’
  • SEQ ID NO.5 SDC2-Probe: 5’-AGTTTCGAGTTCGAGTTTTCGAGTTTG-3’
  • SEQ ID NO.6 Capture sequence of COL4A1/COL4A2:
  • SEQ ID NO.7 COL4A1/COL4A2-FP: 5’-AGAGAGTTTAGTAAGGTCGGGC-3’
  • SEQ ID NO. 8 COL4A1/COL4A2-RP: 5’-GACTTCAAAAACTACTACCCG-3’
  • SEQ ID NO.10 Capture sequence of ITGA4:
  • SEQ ID NO.11 ITGA4-FP: 5’-ACGCGAGTTTTGCGTAGAC-3’
  • SEQ ID NO.12 ITGA4-RP: 5’-GCTAAATAAAATCCCGAACG-3’
  • SEQ ID NO.13 ITGA4-Probe: 5’-ACGGAGTTCGGTTTTGCGTTTTC-3’
  • IBM SPSS statistics 20 software was used to draw the ROC curve of the marker combination for detecting colorectal cancer and adenoma, as shown in Figure 9.
  • the capture sequence, primer probe, and qMSP reaction system and procedures of SOX21 are the same as those in Comparative Example 1; the capture sequence, primer probe, and qMSP reaction system and procedures of SDC2 are the same as those in Example 4.
  • the test results showed that the sensitivity of methylated SDC2 gene detection was 88.6%, the specificity was 93.9%; the area under the curve was 0.939.
  • the detection sensitivity of methylated SOX21 gene is 79.7%, the specificity is 86.6%; the area under the curve is 0.924.
  • the detection sensitivity of methylated SOX21 gene and methylated SDC2 gene combination was 87.34%, and the specificity was 93.9%.
  • the detection sensitivity of the combined detection of methylated SDC2 gene and methylated SOX21 gene is lower than the detection sensitivity of methylated SDC2 gene alone as a colorectal cancer marker, and it is also lower than that of the application of SDC2, COL4A1/COL4A2 and ITG4 gene A Based on the sensitivity of combined detection.

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Abstract

La présente invention concerne un réactif de détection de méthylation, un kit et une application associée. Dans la présente invention, des échantillons de cancer colorectal peuvent être très bien dissociés des échantillons de selles par détection du niveau de méthylation dans une combinaison génique de SDC2, COL4A1/COL4A2 et ITGA4. La présente invention utilise un réactif de détection de méthylation contenant la combinaison génique pour détecter un cancer colorectal.
PCT/CN2020/094963 2019-07-11 2020-06-08 Réactif de détection combiné multigénique WO2021004215A1 (fr)

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