CN113215258A - Nucleic acid composition, kit and detection method for detecting methylation of colorectal cancer related genes - Google Patents
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Abstract
The invention discloses a nucleic acid composition, a kit and a detection method for detecting methylation of colorectal cancer related genes; the nucleic acid composition comprises: methylation specific primers and probes of Septin 9 gene target sites, methylation specific primers and probes of MGMT gene target sites, methylation specific primers and probes of VIM gene target sites, methylation specific primers and probes of SDC2 gene target sites, and specific primers and probes of ACTB reference genes; the kit prepared from the nucleic acid composition disclosed by the invention has the advantages of simplicity in operation, high accuracy and high sensitivity in detecting the methylation result of the colorectal cancer related gene and the like.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a nucleic acid composition, a kit and a detection method for detecting colorectal cancer related gene methylation.
Background
Colorectal cancer (CRC) includes colon and rectal cancers, a common lower gastrointestinal malignancy in humans. According to the latest cancer statistics published by the world health organization international agency for research on cancer (IARC) worldwide in 2020, it has been shown that colorectal cancer is third in global morbidity (193 ten thousand) and second in mortality (94 ten thousand). The prevention and treatment of colorectal cancer mainly aims at early screening and early diagnosis of colorectal cancer, realizes early detection and early treatment in the disease onset period, stops the progress of the disease course, prevents the spread or slows down the development of the disease. According to the consensus opinion of 2016 colorectal cancer prevention, the five-year survival rate of stage IV colorectal cancer is 12%, stage II-III is 70%, and stage I of carcinoma in situ is 90%, so that if early diagnosis and early treatment can be realized, the survival time of colorectal cancer patients can be effectively prolonged.
The current means for screening and early diagnosing CRC mainly include electronic enteroscope (imaging technology) and Fecal Occult Blood Test (FOBT). Enteroscopy is the gold standard for colorectal cancer diagnosis, but requires a 1-3 day bowel preparation period for the patient; intestinal pain and discomfort can occur during the detection process: and has some contraindications, such as hypertension, coronary heart disease, etc. These cause limitations of their applicable population and increase the difficulty of their popularization in china. FOBT is also a commonly applied technology for CRC screening, and whether the gastrointestinal tract bleeds or not is judged by detecting the content of hemoglobin in excrement, for example, FOBT can be positive when peptic ulcer, drug-induced gastric mucosal injury, ancylostomiasis, intestinal tuberculosis, Crohn, ulcerative colitis, colon polyp, gastric cancer and colorectal cancer. The positive rate is reported to be 40-70% in peptic ulcer; the positive rate of the digestive tract cancer is 95 percent. It can be seen that FOBT is not sensitive to CRC screening.
With the rapid development of technology, epigenetic changes of specific genes are found as potential candidate biomarkers for early detection of cancer, such as DNA methylation. Aberrant methylation of CpG islands in the promoter region of genes is associated with epigenetic transcriptional silencing of tumor suppressor genes, which is crucial in the early development of CRC. Combined with genome-wide analysis, epigenetic changes are more frequent than those of CRC, and the methylation status of gene promoters has therefore become an important indicator of CRC molecular diagnosis.
The importance of DNA methylation as a novel molecular marker in tumor diagnosis is increasingly emphasized, and the advantages of DNA methylation are mainly as follows: firstly, in the process of tumor formation, the occurrence frequency of promoter hypermethylation is very high, even higher than that of gene mutation, and important genes related to tumor formation are not lacked; secondly, methylation is an important event in the early stages of tumorigenesis; and thirdly, DNA methylation exists stably, and can be detected through a PCR amplification effect. Therefore, methylation detection has potential application value in early diagnosis of colorectal cancer.
The Septin 9 gene is positioned at the position of 17q25.3 of a chromosome, and the Septin 9 gene has 18 transcripts and relates to various cell physiological processes such as actin dynamics, angiogenesis, cell migration, cell proliferation, cell shape, cell division and the like. Vimentin is a protein coding gene, which codes type III intermediate filament protein, is an important structure of eukaryotic cells, and participates in adhesion, migration, growth, apoptosis, immune response, etc. of cells. Expressed primarily in embryonic tissue and adult cells derived from mesenchymal tissue.
The protein encoded by the MGMT (O-6-Methylguanine-DNA Methyltransferase) gene is a DNA repair protein involved in cellular defense against mutagenesis and alkylating agent toxicity. The protein catalyzes the methyl transfer of DNA from O (6) -alkylguanine and other methylated portions of DNA to its own molecule, thereby repairing toxic damage. Its associated pathways include DNA damage reversal and chromatin regulation/acetylation.
The SDC2 (syncan 2) gene encodes a protein that is transmembrane (type I) heparan sulfate proteoglycan, a member of the syncan proteoglycan family. The syndecan-2 protein functions as a whole membrane protein and is an indispensable part of membrane proteins. And participate in cell proliferation, cell migration and cell-matrix interactions through receptors for their extracellular matrix proteins.
At present, the nucleic acid composition and the kit for early detection of colorectal cancer methylation still have the defects of complex operation, low accuracy and low sensitivity, and the invention solves the problems, finally indirectly provides effective information for early diagnosis of human colorectal cancer, and provides a simple, convenient and feasible method for early discovery, early treatment and treatment effect monitoring of colorectal cancer.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a nucleic acid composition, a kit and a detection method for detecting methylation of genes related to colorectal cancer.
In order to achieve the above object, the present invention adopts the following technical solutions:
a nucleic acid composition for detecting methylation of a colorectal cancer-associated gene, comprising: methylation specific primers and probes of Septin 9 gene target sites, methylation specific primers and probes of MGMT gene target sites, methylation specific primers and probes of VIM gene target sites, methylation specific primers and probes of SDC2 gene target sites, and specific primers and probes of ACTB reference genes;
methylation specific primers and probes of Septin 9 gene target sites comprise:
a forward primer: 5 '-CGCGATTCGTGTTTATTAG-3' SEQ ID 01,
reverse primer: 5 '-CACCTTCGAAATCGAAA-3' SEQ ID 02,
detecting a probe: 5 'tag-CGCGTTACGCGAAATCCGACATAAT-3' tag SEQ ID 03;
methylation specific primers and probes for the MGMT gene target site include:
a forward primer: 5 '-GATATGTTGGATAGTTCGC-3' SEQ ID 04,
reverse primer: 5 '-GCACTCTCCGAAACGAAACG-3' SEQ ID 05,
detecting a probe: 5 'tag-CGTTTTGCGTTGACGTTCGTAGGTTTTCGCGGT-3' tag SEQ ID 06;
methylation specific primers and probes for VIM gene target sites include:
a forward primer: 5 '-GGTCGAGTTTTATGGAGTTACGT-3' SEQ ID 07,
reverse primer: 5 '-CCCGAAAACAAACTAAAAACTA-3' SEQ ID 08,
detecting a probe: 5 'tag-CGTATTTAAGTTGGGTAGCGC-3' tag SEQ ID 09;
methylation specific primers and probes for target sites of the SDC2 gene comprise:
a forward primer: 5 '-AAATTAATAATGGAGGGCGTCGC-3' SEQ ID 10,
reverse primer: 5 '-CGAAAACCGAACTGAAACTCGA-3' SEQ ID 11,
detecting a probe: 5 'tag-TTTCGGGCGTAGTTGCGGCGGCGGGA-3' tag SEQ ID 12;
ACTB reference gene specific primers and probes include:
a forward primer: 5 '-GTGATGAGGAGGTTTGTAAGTT-3' SEQ ID 13,
reverse primer: 5 '-GCACTCTCCGAAAACGAACG-3' SEQ ID 14,
detecting a probe: 5 'tag-ACCACACCCAACACACAATACAAACACA-3' tag SEQ ID 15.
A kit for detecting methylation of a colorectal cancer-associated gene, comprising: PCR reaction solution, negative quality control product, positive quality control product and no-template control; the PCR reaction solution comprises: methylation specific primers and probes of Septin 9 gene target sites, methylation specific primers and probes of MGMT gene target sites, methylation specific primers and probes of VIM gene target sites, methylation specific primers and probes of SDC2 gene target sites, specific primers and probes of ACTB reference genes, 2X PCR buffer solution, dNTP, nuclease-free water, genome DNA samples and Taq enzyme;
methylation specific primers and probes of Septin 9 gene target sites comprise:
a forward primer: 5 '-CGCGATTCGTGTTTATTAG-3' SEQ ID 01,
reverse primer: 5 '-CACCTTCGAAATCGAAA-3' SEQ ID 02,
detecting a probe: 5 'tag-CGCGTTACGCGAAATCCGACATAAT-3' tag SEQ ID 03;
methylation specific primers and probes for the MGMT gene target site include:
a forward primer: 5 '-GATATGTTGGATAGTTCGC-3' SEQ ID 04,
reverse primer: 5 '-GCACTCTCCGAAACGAAACG-3' SEQ ID 05,
detecting a probe: 5 'tag-CGTTTTGCGTTGACGTTCGTAGGTTTTCGCGGT-3' tag SEQ ID 06;
methylation specific primers and probes for VIM gene target sites include:
a forward primer: 5 '-GGTCGAGTTTTATGGAGTTACGT-3' SEQ ID 07,
reverse primer: 5 '-CCCGAAAACAAACTAAAAACTA-3' SEQ ID 08,
detecting a probe: 5 'tag-CGTATTTAAGTTGGGTAGCGC-3' tag SEQ ID 09;
methylation specific primers and probes for target sites of the SDC2 gene comprise:
a forward primer: 5 '-AAATTAATAATGGAGGGCGTCGC-3' SEQ ID 10,
reverse primer: 5 '-CGAAAACCGAACTGAAACTCGA-3' SEQ ID 11,
detecting a probe: 5 'tag-TTTCGGGCGTAGTTGCGGCGGCGGGA-3' tag SEQ ID 12;
ACTB reference gene specific primers and probes include:
a forward primer: 5 '-GTGATGAGGAGGTTTGTAAGTT-3' SEQ ID 13,
reverse primer: 5 '-GCACTCTCCGAAAACGAACG-3' SEQ ID 14,
detecting a probe: 5 'tag-ACCACACCCAACACACAATACAAACACA-3' tag SEQ ID 15.
In the kit for detecting methylation of genes related to colorectal cancer, the final concentration of the components of the PCR reaction solution is as follows: 1 XPCR buffer solution, 0.4-0.6 MuM Septin 9 forward primer, 0.4-0.6 MuM Septin 9 reverse primer, 0.2-0.3 MuM Septin 9 detection probe, 0.4-0.6 MuM MGMT forward primer, 0.4-0.6 MuM MGMT reverse primer, 0.2-0.3 MuM MGMT detection probe, 0.4-0.6 MuM VIM forward primer, 0.4-0.6 MuM VIM reverse primer, 0.2-0.3 MuM VIM detection probe, 0.4-0.6 MuM SDC2 forward primer, 0.4-0.6 MuM SDC2 reverse primer, 0.2-0.3 MuM SDC2 detection probe, 0.1-0.3 MuM ACTB forward primer, 0.1-0.3 MuM ACTB reverse primer, 0.05 MuM ACTB 0.05-0.05 MuM ACTB enzyme, 0.05-0.05 mM DNA, and 0.05-0.05 mM DNA-free genome DNA.
In the kit for detecting methylation of colorectal cancer related genes, the positive quality control product is genomic DNA of Hct116 cell line treated by SssI methylase.
In the kit for detecting methylation of colorectal cancer related genes, the negative quality control product is genomic DNA of Hct116 cell line with genes DNMT1 and DNMT3b knocked out.
An assay for detecting methylation of a colorectal cancer-associated gene comprising the steps of:
the method comprises the following steps: extracting DNA of a sample to be detected, and taking the DNA after conversion treatment as a genome DNA sample of a PCR template;
step two: performing PCR amplification on a genomic DNA sample by using a kit for detecting methylation of genes related to colorectal cancer;
the kit comprises: PCR reaction solution, negative quality control product, positive quality control product and no-template control; the PCR reaction solution comprises: methylation specific primers and probes of Septin 9 gene target sites, methylation specific primers and probes of MGMT gene target sites, methylation specific primers and probes of VIM gene target sites, methylation specific primers and probes of SDC2 gene target sites, specific primers and probes of ACTB reference genes, 2X PCR buffer solution, dNTP, nuclease-free water, genome DNA samples and Taq enzyme;
methylation specific primers and probes of Septin 9 gene target sites comprise:
a forward primer: 5 '-CGCGATTCGTGTTTATTAG-3' SEQ ID 01,
reverse primer: 5 '-CACCTTCGAAATCGAAA-3' SEQ ID 02,
detecting a probe: 5 'tag-CGCGTTACGCGAAATCCGACATAAT-3' tag SEQ ID 03;
methylation specific primers and probes for the MGMT gene target site include:
a forward primer: 5 '-GATATGTTGGATAGTTCGC-3' SEQ ID 04,
reverse primer: 5 '-GCACTCTCCGAAACGAAACG-3' SEQ ID 05,
detecting a probe: 5 'tag-CGTTTTGCGTTGACGTTCGTAGGTTTTCGCGGT-3' tag SEQ ID 06;
methylation specific primers and probes for VIM gene target sites include:
a forward primer: 5 '-GGTCGAGTTTTATGGAGTTACGT-3' SEQ ID 07,
reverse primer: 5 '-CCCGAAAACAAACTAAAAACTA-3' SEQ ID 08,
detecting a probe: 5 'tag-CGTATTTAAGTTGGGTAGCGC-3' tag SEQ ID 09;
methylation specific primers and probes for target sites of the SDC2 gene comprise:
a forward primer: 5 '-AAATTAATAATGGAGGGCGTCGC-3' SEQ ID 10,
reverse primer: 5 '-CGAAAACCGAACTGAAACTCGA-3' SEQ ID 11,
detecting a probe: 5 'tag-TTTCGGGCGTAGTTGCGGCGGCGGGA-3' tag SEQ ID 12;
ACTB reference gene specific primers and probes include:
a forward primer: 5 '-GTGATGAGGAGGTTTGTAAGTT-3' SEQ ID 13,
reverse primer: 5 '-GCACTCTCCGAAAACGAACG-3' SEQ ID 14,
detecting a probe: 5 'tag-ACCACACCCAACACACAATACAAACACA-3' tag SEQ ID 15;
step three: and determining whether the sample to be detected is methylated or not according to the fluorescence Ct values of the amplification results of Septin 9, MGMT, VIM, SDC2 and ACTB genes.
In the method for detecting methylation of genes related to colorectal cancer, in the step one, the reagent used in the conversion treatment is bisulfite or bisulfite.
In the aforementioned detection method for detecting methylation of genes related to colorectal cancer, in step two, the reaction procedure of PCR amplification is: denaturation at 95 deg.C for 10 min; cycles 45cycles, 95 ℃ for 15sec, 60 ℃ for 30sec (fluorescence acquisition).
The invention has the advantages that:
the invention finds that the nucleic acid composition of Septin 9, MGMT, VIM, SDC2 specific forward primer, specific reverse primer and specific probe has synergistic effect on the sensitivity and specificity of detecting colorectal cancer related gene methylation;
the gene ACTB is used as an internal reference gene to control the quality of a sample, the situation that housekeeping genes are possibly methylated is considered, the position without CpG sites is selected when an internal reference primer and a probe are designed, and the sequence after bisulfite treatment is designed, so that the quality control of the housekeeping genes on the sample is ensured;
the kit has the characteristics of high speed, high flux, sensitivity and good specificity by designing the primers and the probes with high specificity, configuring the kit with convenient use and reliable detection results and combining a scientific and reasonable PCR reaction system, and when the kit is used for screening the colorectal cancer, the positive detection rate (sensitivity) is 100 percent and the negative detection rate (specificity) is 80 percent, so that the rapid and accurate measurement of the methylation degree of the genes related to the colorectal cancer is realized, the colorectal cancer can be indirectly and effectively diagnosed and treated in time, the medical cost is reduced, the social resources are saved, and the life quality is improved.
Drawings
FIG. 1 is a graph showing amplification curves of methylation positive samples of Septin 9 gene according to the present invention;
FIG. 2 is a graph showing the amplification of a methylation positive sample of the MGMT gene of the present invention;
FIG. 3 is a graph showing the amplification of a methylation-positive sample of VIM gene according to the present invention;
FIG. 4 is a graph of amplification of a methylation positive sample of the SDC2 gene according to the present invention;
FIG. 5 is a graph of the amplification curve of a negative sample according to the present invention;
FIG. 6 is a schematic view showing a curve of the present invention in which neither a target gene nor an internal reference gene is amplified.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
An assay for detecting methylation of a colorectal cancer-associated gene comprising the steps of:
the method comprises the following steps: extracting DNA of a sample to be detected, and taking the DNA after conversion treatment as a genome DNA sample of a PCR template;
the reagent adopted in the conversion treatment is bisulfite or bisulfite, and the sequence after the bisulfite treatment is adopted for design, so that the quality control of housekeeping genes on the sample can be ensured; the examples herein are not exhaustive, and any examples are applicable to the present invention as long as the sequences can be pre-processed.
Step two: performing PCR amplification on a genomic DNA sample by using a kit for detecting methylation of genes related to colorectal cancer;
preferably, the reaction procedure for PCR amplification is: denaturation at 95 deg.C for 10 min; cycles 45cycles, 95 ℃ 15sec, 60 ℃ 30sec (fluorescence acquisition), noted are: the amplification procedure of PCR is not limited, and any procedure capable of amplifying a genomic DNA sample is applicable to the present invention;
the kit comprises: PCR reaction solution, negative quality control product, positive quality control product and no-template control; the positive quality control product is the genome DNA of the Hct116 cell line treated by SssI methylase, and can methylate C in all CpG sequences of the genome at the position of C5; the negative quality control product is the genome DNA of the Hct116 cell line with genes DNMT1 and DNMT3b knocked out; the no template control means that no human genomic DNA was contained.
The PCR reaction solution comprises: methylation specific primers and probes of Septin 9 gene target sites, methylation specific primers and probes of MGMT gene target sites, methylation specific primers and probes of VIM gene target sites, methylation specific primers and probes of SDC2 gene target sites, specific primers and probes of ACTB reference genes, 2X PCR buffer solution, dNTP, nuclease-free water, genome DNA samples and Taq enzyme;
as an example, the final concentrations of the components of the PCR reaction solution were: 1 XPCR buffer solution, 0.4-0.6 MuM Septin 9 forward primer, 0.4-0.6 MuM Septin 9 reverse primer, 0.2-0.3 MuM Septin 9 detection probe, 0.4-0.6 MuM MGMT forward primer, 0.4-0.6 MuM MGMT reverse primer, 0.2-0.3 MuM MGMT detection probe, 0.4-0.6 MuM VIM forward primer, 0.4-0.6 MuM VIM reverse primer, 0.2-0.3 MuM VIM detection probe, 0.4-0.6 MuM SDC2 forward primer, 0.4-0.6 MuM SDC2 reverse primer, 0.2-SDC 0.3 MuM 2 detection probe, 0.1-0.3 MuM ACTB forward primer, 0.1-0.3 MuM ACTB reverse primer, 0.05 MuM ACTB 0.05-0.05 MuM ACTB enzyme, 0.05-0.05 mM DNA sample, and no Taq DNA/5 MuM DNA; it should be noted that: the reagent formulation of the reaction solution is not limited, and is provided as a preferred embodiment, and other reaction solution formulations capable of being matched with the amplification of the sample DNA are also applicable to the present invention.
Methylation specific primers and probes of Septin 9 gene target sites comprise:
a forward primer: 5 '-CGCGATTCGTGTTTATTAG-3' SEQ ID 01,
reverse primer: 5 '-CACCTTCGAAATCGAAA-3' SEQ ID 02,
detecting a probe: 5 'tag-CGCGTTACGCGAAATCCGACATAAT-3' tag SEQ ID 03, preferably, the 5 'tag is FAM and the 3' tag is BHQ 1;
methylation specific primers and probes for the MGMT gene target site include:
a forward primer: 5 '-GATATGTTGGATAGTTCGC-3' SEQ ID 04,
reverse primer: 5 '-GCACTCTCCGAAACGAAACG-3' SEQ ID 05,
detecting a probe: 5 'tag-CGTTTTGCGTTGACGTTCGTAGGTTTTCGCGGT-3' tag SEQ ID 06, preferably, the 5 'tag is VIC and the 3' tag is BHQ 1;
methylation specific primers and probes for VIM gene target sites include:
a forward primer: 5 '-GGTCGAGTTTTATGGAGTTACGT-3' SEQ ID 07,
reverse primer: 5 '-CCCGAAAACAAACTAAAAACTA-3' SEQ ID 08,
detecting a probe: 5 'tag-CGTATTTAAGTTGGGTAGCGC-3' tag SEQ ID 09, as a preferred, 5 'tag is ROX and 3' tag is BHQ 2;
methylation specific primers and probes for target sites of the SDC2 gene comprise:
a forward primer: 5 '-AAATTAATAATGGAGGGCGTCGC-3' SEQ ID 10,
reverse primer: 5 '-CGAAAACCGAACTGAAACTCGA-3' SEQ ID 11,
detecting a probe: 5 'tag-TTTCGGGCGTAGTTGCGGCGGCGGGA-3' tag SEQ ID 12, preferably, the 5 'tag is TAMRA and the 3' tag is BHQ 2;
ACTB reference gene specific primers and probes include:
a forward primer: 5 '-GTGATGAGGAGGTTTGTAAGTT-3' SEQ ID 13,
reverse primer: 5 '-GCACTCTCCGAAAACGAACG-3' SEQ ID 14,
detecting a probe: 5 'tag-ACCACACCCAACACACAATACAAACACA-3' tag SEQ ID 15, preferably, the 5 'tag is CY5 and the 3' tag is BHQ 2;
remarking: the purity of the primers should be PAGE or HPLC grade.
BHQ1 and BHQ2 are preferable as the quenching marker group, and a fluorescent marker is also preferable, and any product that can be used for labeling can be applied to the present invention.
Step three: and determining whether the sample to be detected is methylated or not according to the fluorescence Ct values of the amplification results of Septin 9, MGMT, VIM, SDC2 and ACTB genes.
Here, it is to be emphasized that: the detection method of the present invention is not limited, and any detection method using the nucleic acid composition of the present invention is not specifically exemplified herein, as long as it is within the scope of the present invention.
The technical effects of the invention are verified through experiments as follows:
first, a kit for an experiment was prepared using the following procedure.
Firstly, materials: a kit for detecting methylation of a colorectal cancer-associated gene, comprising: methylation specific primers of Septin 9 gene target sites; septin 9 gene specific hydrolysis probe; methylation specific primers for the MGMT gene target site; a hydrolysis probe specific for the MGMT gene; methylation specific primers of VIM gene target sites; VIM gene specific hydrolysis probes; methylation specific primers for target sites of the SDC2 gene; a hydrolysis probe specific for the SDC2 gene; primers for the ACTB reference gene; hydrolysis probes for ACTB reference genes;
methylation specific primers and probes of Septin 9 gene target sites comprise:
a forward primer: 5 '-CGCGATTCGTTGTTTATTAG-3' (SEQ ID NO.1)
Reverse primer: 5 '-CACCTTCGAAATCCGAAA-3' (SEQ ID NO.2)
Detecting a probe: 5 'FAM-CGCGTTAACCGCGAAATCCGACATAAT-3' BHQ1(SEQ ID NO.3)
Methylation specific primers and probes for the MGMT gene target site include:
a forward primer: 5 '-GATATGTTGGGATAGTTCGC-3' (SEQ ID NO.4)
Reverse primer: 5 '-GCACTCTTCCGAAAACGAAACG-3' (SEQ ID NO.5)
Detecting a probe: 5 'VIC-CGTTTTGCGTTTCGACGTTCGTAGGTTTTCGCGGT-3' BHQ1(SEQ ID NO.6)
Methylation specific primers and probes for VIM gene target sites include:
a forward primer: 5 '-GGTCGAGTTTTAGTCGGAGTTACGT-3' (SEQ ID NO.7)
Reverse primer: 5 '-CCCGAAAACGAAACGTAAAAACTA-3' (SEQ ID NO.8)
Detecting a probe: 5 'ROX-CGTATTTATAGTTTGGGTAGCGC-3' BHQ2(SEQ ID NO.9)
Methylation specific primers and probes for target sites of the SDC2 gene comprise:
a forward primer: 5 '-AAATTAATAAGTGAGAGGGCGTCGC-3' (SEQ ID NO.10)
Reverse primer: 5 '-CGAAAACTCGAACTCGAAACTCGA-3' (SEQ ID NO.11)
Detecting a probe: 5 'TAMRA-TTTCGGGGCGTAGTTGCGGGCGGCGGGA-3' BHQ2(SEQ ID NO.12)
ACTB reference gene order-specific primers and probes include:
a forward primer: 5 '-GTGATGGAGGAGGTTTAGTAAGTT-3' (SEQ ID NO.13)
Reverse primer: 5 '-GCACTCTTCCGAAAACGAAACG-3' (SEQ ID NO.14)
Detecting a probe: 5 'CY 5-ACCACCACCCAACACACAATAACAAACACA-3' BHQ2(SEQ ID NO.15)
Selecting a positive quality control product and a negative quality control product:
positive quality control product and negative quality control product. The positive quality control product is the genome DNA of the Hct116 cell line treated by SssI methylase, and can methylate C in all CpG sequences in the genome at the position of C5; the negative quality control product is the genome DNA of the Hct116 cell line with genes DNMT1 and DNMT3b knocked out;
thirdly, PCR reaction solution composition:
the kit comprises a PCR reaction solution containing the specific primer and the probe, wherein the PCR reaction solution comprises:
Septin 9 reverse primer: 5' -CACCTTCGAAATCCGAAA-3
MGMT forward primer: 5' -GATATGTTGGGATAGTTCGC-3
MGMT reverse primer: 5' -GCACTCTTCCGAAAACGAAACG-3
MGMT detection probe: 5 'VIC-CGTTTTGCGTTTCGACGTTCGTAGGTTTTCGCGGT-3' BHQ1
VIM forward primer: 5' -GGTCGAGTTTTAGTCGGAGTTACGT-3
VIM reverse primer: 5' -CCCGAAAACGAAACGTAAAAACTA-3
VIM detection probe: 5 'ROX-CGTATTTATAGTTTGGGTAGCGC-3' BHQ2
SDC2 forward primer: 5' -AAATTAATAAGTGAGAGGGCGTCGC-3
SDC2 reverse primer: 5' -CGAAAACTCGAACTCGAAACTCGA-3
SDC2 detection probe: 5 'TAMRA-TTTCGGGGCGTAGTTGCGGGCGGCGGGA-3' BHQ2
ACTB forward primer: 5' -GTGATGGAGGAGGTTTAGTAAGTT-3
ACTB reverse primer: 5' -GCACTCTTCCGAAAACGAAACG-3
ACTB detection probe: 5 'CY 5-ACCACCACCCAACACACAATAACAAACACA-3' BHQ2
2X PCR buffer solution, Taq enzyme, dNTP and nuclease-free water. Were purchased from biotechnology, inc.
The final concentration of the PCR reaction solution components was:
1 XPCR buffer, 0.6. mu.M (. mu.mol/L) Septin 9 forward primer, 0.6. mu.M (. mu.mol/L) Septin 9 reverse primer, 0.3. mu.M (. mu.mol/L) Septin 9 detection probe, 0.6. mu.M (. mu.mol/L) MGMT forward primer, 0.6. mu.M (. mu.mol/L) MGMT reverse primer, 0.3. mu.M (. mu.mol/L) MGMT detection probe, 0.6. mu.M (. mu.mol/L) VIM forward primer, 0.6. mu.M (. mu.mol/L) VIM reverse primer, 0.3. mu.M (. mol/L) VIM detection probe, 0.6. mu.M (. mol/L) SDC2 forward primer, 0.6. mu.M (. mu.mol/L) 2 reverse primer, 0.3. mu.M (. mol/L) SDC2 detection probe, 0.2. mu.M SDC forward primer, 0.2. mu.M ACTB (ACTB) reverse primer, 0. mu.M ACTB (ACTB/L) detection probe, 0.25mM (mmol/L) dNTP.
(II) detecting the methylation of the colorectal cancer related gene by using the kit sample prepared by the method:
first, technical principle
A pair of specific primers and probes are designed by combining promoter regions of colorectal cancer related genes and internal reference genes in a human genome. Then, the primers and the probes are used for amplifying the sample DNA converted by the bisulfite, whether the sample to be detected is methylated or not is determined according to the Ct value of the relative fluorescence value of the PCR amplification result of the related genes, and the colorectal cancer risk is indirectly determined according to the methylation.
Second, detection method
The method comprises the following steps: and extracting NDA of a sample to be detected, carrying out bisulfite conversion treatment on the NDA, and carrying out qPCR amplification by using the converted DNA as a PCR template.
Wherein, the reagent used for the Conversion treatment is Bisulfite or Bisulfite, and other auxiliary reagents (corresponding reagents are purchased from epiect Fast bisulfate Conversion Kit of QIAGEN, Germany);
step two: providing the kit for detecting the methylation of the colorectal cancer related gene, and carrying out PCR amplification on the template;
step three: determining whether the sample to be detected is methylated or not according to the relative fluorescence value Ct of the PCR amplification result of the related gene;
the specific detection method comprises the following steps:
one) plasma separation
1. 10ml of peripheral blood was drawn using a free DNA sampling tube and plasma separation was performed the first time after arrival at the laboratory.
2. Centrifuging at 1600rpm for 20min at 4 deg.C, and packaging the plasma in sterile centrifuge tubes.
3. The plasma after the primary separation was centrifuged at 16,000rpm for 10min at 4 ℃ to remove residual cells. After centrifugation, the plasma was dispensed into sterile centrifuge tubes.
II) extracting plasma DNA:
the DNA extraction kit was purchased from Jivan Biotechnology (Beijing) Ltd:
1. 2ml of plasma samples were taken and the corresponding amounts of reagents were added as shown in Table 1 according to the following system:
TABLE 1
| Volume of plasma | Proteinase K | Buffer GHP | FineMag Particles K |
| 2ml | 200μl | 3.0ml | 30μl |
2. After the sample and the reagent are fully mixed, the mixture is incubated at room temperature for 20min, and the mixture is inverted and mixed for 10sec every 3-5min, so that the magnetic beads and the nucleic acids are fully combined. After incubation, removing liquid drops on the inner wall of the tube wall by brief centrifugation;
3. placing the centrifugal tube in a magnetic force and standing for 2min, carefully removing liquid when the magnetic beads are completely adsorbed, and taking down the centrifugal tube;
4. adding 1ml buffer RBP, shaking and mixing for 1min to make the magnetic beads fully suspended, and centrifuging briefly to remove the dropping liquid on the inner wall;
5. placing the centrifugal tube in a magnetic force and standing for 1min, carefully removing liquid when the magnetic beads are completely adsorbed, and taking down the centrifugal tube;
6. adding 1ml 80% ethanol (ready for use), shaking and mixing for 1min each time to suspend the magnetic beads thoroughly, and centrifuging briefly to remove the inner wall dripping liquid.
7. Placing the centrifugal tube on magnetic force and standing for 1min, carefully removing liquid when the magnetic beads are completely adsorbed, and taking down the centrifugal tube.
8. Repeat step 6-7 once, total 2 ethanol elution.
9. Placing the centrifugal tube on magnetic force, and air drying at room temperature for 5-10 min.
10. And (3) taking down the centrifugal tube from the magnetic force, adding 45 mu l of buffer solution EB, shaking and uniformly mixing to suspend the magnetic beads, incubating for 5min at 56 ℃, slightly shaking the centrifugal tube every 2min during the incubation to fully absorb and remove the nucleic acid, and then centrifuging for a short time to remove the liquid on the tube wall and the tube cover.
11. The centrifuge tube was placed on a magnetic rack and allowed to stand for 2min, and when the magnetic beads were completely adsorbed, the DNA solution was carefully transferred to a 1.5ml collection tube and stored under appropriate conditions.
12. Taking 1 mul of DNA eluent to carry out the concentration determination of the Qubit HS, recording the data result, and storing the rest samples in a refrigerator at the temperature of-20 ℃ if the experiment is not continued.
Third) DNA transformation procedure:
1. the extracted DNA was formulated with the bisulfite conversion system as shown in Table 2 according to the following formulation:
TABLE 2
| Reagent composition | Reaction volume (μ l) |
| Extracted DNA | 40 |
| Sulfite salt | 85 |
| DNA protective solution | 15 |
| Total of | 140 |
2. After the transformation system is configured, placing the obtained product in a PCR instrument for reaction according to the following reaction conditions, wherein the reaction program is shown in Table 3;
TABLE 3
3. DNA purification after bisulfite conversion:
4. the PCR tube was transiently detached and the bisulfite converted product was transferred to a 1.5ml EP tube.
5. Add 310. mu.l of buffer BL reagent to each sample
6. Add 250. mu.l of absolute ethanol to each sample, vortex the solution for 15 seconds, and centrifuge briefly
7. Transferring the mixture to a spin column, and centrifuging at 14000rpm for 1min
8. The filtrate was discarded, 500. mu.l of buffer BW was added, and the mixture was centrifuged at 14000rpm for 1min
9. Discarding the filtrate, adding 500. mu.l buffer BD, incubating for 15min, and centrifuging at 14000rpm for 1min
10. The filtrate was discarded, 500. mu.l of buffer BW was added, and the mixture was centrifuged at 14000rpm for 1min
11. Repeat step 10
12. Discarding the filtrate, adding 250 μ l of anhydrous ethanol, centrifuging at 14000rpm for 1min
13. Discarding the filtrate, leaving at 14000rpm for 1min
14. Drying at room temperature for 3-5min to remove residual ethanol;
15. add 15. mu.l buffer EB and incubate and elute at room temperature for 1min, 14000rpm centrifuge for 1min, collect DNA and store at-20 ℃.
Four) qPCR reaction configuration
1. The qPCR instrument was an ABI 7500 model real-time fluorescent quantitative PCR system (available from life technology, Inc.) of USA, and the reaction system was 20 μ l
2. qPCR reaction system configuration and conditions, as shown in table 4 below:
TABLE 4
| Composition (I) | Reaction volume (μ l) |
| DNA after bisulfite conversion | 2.0 |
| Primer and method for producing the same | 5.6 |
| Probe needle | 1.4 |
| RNAse-free Water | 1.0 |
| 2X PCR Mix | 10.0 |
| Total of | 20 |
3. PCR reaction sample addition layout:
the DNA sample to be tested, the positive quality control product, the negative quality control product and the no-template control are all subjected to 3 parallel detection tests, and the 96-hole sample adding layout of the PCR instrument is shown in the following table 5. In the table, PC represents a Positive Control (Positive Control), NC represents a Negative Control (Negative Control), NTC represents a No-Template Control (No Template Control), and S represents a test sample (sample)
TABLE 5
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | PC | PC | PC | S7 | S7 | S7 | ||||||
| B | NC | NC | NC | S8 | S8 | S8 | ||||||
| C | S1 | S1 | S1 | S9 | S9 | S9 | ||||||
| D | S2 | S2 | S2 | S10 | S10 | S10 | ||||||
| E | S3 | S3 | S3 | S1.. | S1.. | S1.. | ||||||
| F | S4 | S4 | S4 | NTC | NTC | NTC | ||||||
| G | S5 | S5 | S5 | |||||||||
| H | S6 | S6 | S6 |
4. The PCR reaction procedure is as follows in table 6:
TABLE 6
5. Analysis of detection results
1) Running amplification curve analysis without template control (NTC), wherein no amplification curve is required, which indicates that the experiment is pollution-free and can continue to analyze;
2) the internal reference genes (ACTB) of the quality control product should have amplification signals and present an S-shaped amplification curve, and the internal reference Ct values should accord with the results of the following table 7; the target gene of the negative quality control product rises due to no amplification curve, the Ct value of the gene of the positive quality control product accords with the following table, and when the quality control product detection meets the following conditions, the experiment is proved to be effective, and the analysis can be continued;
TABLE 7
3) The internal reference genes of the sample have amplification signals and are in an S-shaped amplification curve, and the PCR detection result of the sample is interpreted according to the following table 8;
TABLE 8
| Results of samples | Target gene | ACTB |
| Positive for methylation | Ct≤40 | Ct≤40 |
| Methylation negative | No amplification, or Ct > 40 | Ct≤40 |
| Failure of the experiment | Any result | Ct＞40 |
Referring to fig. 1-6, fig. 1 is a graph showing amplification curves of Septin 9 gene methylation positive samples, fig. 2 is a graph showing amplification curves of MGMT gene methylation positive samples, fig. 3 is a graph showing amplification curves of VIM gene methylation positive samples, fig. 4 is a graph showing amplification curves of SDC2 gene methylation positive samples, and fig. 5 is a graph showing amplification curves of negative samples, it can be seen that the amplification curves of colorectal cancer-related methylation genes and ACTB internal reference genes in different methylation samples are related, and if there is no amplification curve in both target genes and internal reference genes, fig. 6 shows that the sample is unqualified and needs repeated detection or resampling.
(III) the kit samples are used for carrying out sensitivity and specificity detection on colorectal cancer and normal human plasma:
plasma samples from 20 normal persons and 20 colorectal cancer patients were used as subjects to be monitored. Free DNA was extracted from each sample. The extraction of DNA can be carried out by any standard means known in the art, and specifically, in this case, the sample DNA used is extracted by using a specific extraction protocol according to the detection system of example 2.
The DNA sample is then pretreated to convert the 5' unmethylated cytosine to uracil. In this experimental case, the pretreatment was achieved by bisulfite reagent treatment. The modification of bisulfite DNA was pretreated by conversion using the detection system in example 2.
Then, the pretreated DNA samples of 20 normal persons and 20 cases of colorectal cancer were added to the detection system of case 2 to detect Septin 9, MGMT, SDC2, VIM and reference gene ACTB in a multiplex manner. Real-time PCR was performed on bisulfite converted DNA.
Wherein the PCR conditions used in the experimental cases were performed according to the qPCR conditions in case 2.
Finally, tables 9 and 10 below show the results of 20 normal human and 20 colorectal cancer patient samples tested using the multiplex assay of the present invention. As can be seen from tables 9 and 10, the positive detection rate and the negative detection rate of this protocol were high.
TABLE 9 results of plasma samples of colorectal cancer patients Using the kit of the present invention
| Sample positive determination method | Number of samples to be tested | Number of positive samples | Positive rate |
| At least one target positive | 20 | 20 | 100％ |
TABLE 10 results of the measurement of a plasma sample of a normal human by using the kit of the present invention
| Sample positive determination method | Number of samples to be tested | Number of negative samples | Negative rate |
| At least one target positive | 20 | 16 | 80％ |
According to the detection results in the table, when the kit is used for colorectal cancer screening, the positive detection rate (sensitivity) is 100%, and the negative detection rate (specificity) is 80%.
From the above verification experiments, it can be seen that: the nucleic acid composition of the Septin 9, MGMT, VIM, SDC2 specific forward primer, specific reverse primer and specific probe has a synergistic effect on the sensitivity and specificity of detecting the methylation of colorectal cancer related genes, and the detection of colorectal cancer methylation by combining the Septin 9, MGMT, VIM, SDC2 specific forward primer, specific reverse primer and specific probe is an innovative discovery, and is not a result obtained by mechanization experiments.
The invention does not aim at diagnosing diseases, and the obtained detection can not directly judge whether the diseases exist, indirectly provides effective information for early diagnosis of the human colorectal cancer, and provides a simple, convenient and easy method for early detection and treatment of the human colorectal cancer.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.
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Claims (8)
1. A nucleic acid composition for detecting methylation of a colorectal cancer-associated gene, comprising: methylation specific primers and probes of Septin 9 gene target sites, methylation specific primers and probes of MGMT gene target sites, methylation specific primers and probes of VIM gene target sites, methylation specific primers and probes of SDC2 gene target sites, and specific primers and probes of ACTB reference genes;
the methylation specific primers and probes of the Septin 9 gene target site comprise:
a forward primer: 5 '-CGCGATTCGTGTTTATTAG-3' SEQ ID 01,
reverse primer: 5 '-CACCTTCGAAATCGAAA-3' SEQ ID 02,
detecting a probe: 5 'tag-CGCGTTACGCGAAATCCGACATAAT-3' tag SEQ ID 03; the methylation specific primers and probes of the MGMT gene target site comprise:
a forward primer: 5 '-GATATGTTGGATAGTTCGC-3' SEQ ID 04,
reverse primer: 5 '-GCACTCTCCGAAACGAAACG-3' SEQ ID 05,
detecting a probe: 5 'tag-CGTTTTGCGTTGACGTTCGTAGGTTTTCGCGGT-3' tag SEQ ID 06;
the methylation specific primers and probes of the VIM gene target site comprise:
a forward primer: 5 '-GGTCGAGTTTTATGGAGTTACGT-3' SEQ ID 07,
reverse primer: 5 '-CCCGAAAACAAACTAAAAACTA-3' SEQ ID 08,
detecting a probe: 5 'tag-CGTATTTAAGTTGGGTAGCGC-3' tag SEQ ID 09;
methylation specific primers and probes for target sites of the SDC2 gene comprise:
a forward primer: 5 '-AAATTAATAATGGAGGGCGTCGC-3' SEQ ID 10,
reverse primer: 5 '-CGAAAACCGAACTGAAACTCGA-3' SEQ ID 11,
detecting a probe: 5 'tag-TTTCGGGCGTAGTTGCGGCGGCGGGA-3' tag SEQ ID 12;
the ACTB reference gene specific primers and probes comprise:
a forward primer: 5 '-GTGATGAGGAGGTTTGTAAGTT-3' SEQ ID 13,
reverse primer: 5 '-GCACTCTCCGAAAACGAACG-3' SEQ ID 14,
detecting a probe: 5 'tag-ACCACACCCAACACACAATACAAACACA-3' tag SEQ ID 15.
2. A kit for detecting methylation of a colorectal cancer-associated gene, comprising: PCR reaction solution, negative quality control product, positive quality control product and no-template control; the PCR reaction solution comprises: methylation specific primers and probes of Septin 9 gene target sites, methylation specific primers and probes of MGMT gene target sites, methylation specific primers and probes of VIM gene target sites, methylation specific primers and probes of SDC2 gene target sites, specific primers and probes of ACTB reference genes, 2X PCR buffer solution, dNTP, nuclease-free water, genome DNA samples and Taq enzyme;
the methylation specific primers and probes of the Septin 9 gene target site comprise:
a forward primer: 5 '-CGCGATTCGTGTTTATTAG-3' SEQ ID 01,
reverse primer: 5 '-CACCTTCGAAATCGAAA-3' SEQ ID 02,
detecting a probe: 5 'tag-CGCGTTACGCGAAATCCGACATAAT-3' tag SEQ ID 03;
the methylation specific primers and probes of the MGMT gene target site comprise:
a forward primer: 5 '-GATATGTTGGATAGTTCGC-3' SEQ ID 04,
reverse primer: 5 '-GCACTCTCCGAAACGAAACG-3' SEQ ID 05,
detecting a probe: 5 'tag-CGTTTTGCGTTGACGTTCGTAGGTTTTCGCGGT-3' tag SEQ ID 06;
the methylation specific primers and probes of the VIM gene target site comprise:
a forward primer: 5 '-GGTCGAGTTTTATGGAGTTACGT-3' SEQ ID 07,
reverse primer: 5 '-CCCGAAAACAAACTAAAAACTA-3' SEQ ID 08,
detecting a probe: 5 'tag-CGTATTTAAGTTGGGTAGCGC-3' tag SEQ ID 09;
methylation specific primers and probes for target sites of the SDC2 gene comprise:
a forward primer: 5 '-AAATTAATAATGGAGGGCGTCGC-3' SEQ ID 10,
reverse primer: 5 '-CGAAAACCGAACTGAAACTCGA-3' SEQ ID 11,
detecting a probe: 5 'tag-TTTCGGGCGTAGTTGCGGCGGCGGGA-3' tag SEQ ID 12;
the ACTB reference gene specific primers and probes comprise:
a forward primer: 5 '-GTGATGAGGAGGTTTGTAAGTT-3' SEQ ID 13,
reverse primer: 5 '-GCACTCTCCGAAAACGAACG-3' SEQ ID 14,
detecting a probe: 5 'tag-ACCACACCCAACACACAATACAAACACA-3' tag SEQ ID 15.
3. The kit for detecting methylation of genes related to colorectal cancer according to claim 2, wherein the PCR reaction solution comprises the following components in final concentration: 1 XPCR buffer solution, 0.4-0.6 MuM Septin 9 forward primer, 0.4-0.6 MuM Septin 9 reverse primer, 0.2-0.3 MuM Septin 9 detection probe, 0.4-0.6 MuM MGMT forward primer, 0.4-0.6 MuM MGMT reverse primer, 0.2-0.3 MuM MGMT detection probe, 0.4-0.6 MuM VIM forward primer, 0.4-0.6 MuM VIM reverse primer, 0.2-0.3 MuM VIM detection probe, 0.4-0.6 MuM forward primer, 0.4-0.6 MuM SDC2 reverse primer, 0.2-0.3 MuM SDC2 detection probe, 0.1-0.3 MuTB forward primer, 0.1-0.3 MuM reverse primer, 0.05 MuM MACTP sample, 0.05-0.05 MuM Taq DNA/5 MuM DNA.
4. The kit of claim 2, wherein the positive quality control product is genomic DNA of SssI methylase-treated Hct116 cell line.
5. The kit for detecting methylation of genes related to colorectal cancer according to claim 2, wherein the negative quality control product is genomic DNA of Hct116 cell line after knocking out genes DNMT1 and DNMT3 b.
6. A detection method for detecting methylation of a colorectal cancer-associated gene, comprising the steps of:
the method comprises the following steps: extracting DNA of a sample to be detected, and taking the DNA after conversion treatment as a genome DNA sample of a PCR template;
step two: performing PCR amplification on a genomic DNA sample by using a kit for detecting methylation of genes related to colorectal cancer;
the kit comprises: PCR reaction solution, negative quality control product, positive quality control product and no-template control; the PCR reaction solution comprises: methylation specific primers and probes of Septin 9 gene target sites, methylation specific primers and probes of MGMT gene target sites, methylation specific primers and probes of VIM gene target sites, methylation specific primers and probes of SDC2 gene target sites, specific primers and probes of ACTB reference genes, 2X PCR buffer solution, dNTP, nuclease-free water, genome DNA samples and Taq enzyme;
the methylation specific primers and probes of the Septin 9 gene target site comprise:
a forward primer: 5 '-CGCGATTCGTGTTTATTAG-3' SEQ ID 01,
reverse primer: 5 '-CACCTTCGAAATCGAAA-3' SEQ ID 02,
detecting a probe: 5 'tag-CGCGTTACGCGAAATCCGACATAAT-3' tag SEQ ID 03;
the methylation specific primers and probes of the MGMT gene target site comprise:
a forward primer: 5 '-GATATGTTGGATAGTTCGC-3' SEQ ID 04,
reverse primer: 5 '-GCACTCTCCGAAACGAAACG-3' SEQ ID 05,
detecting a probe: 5 'tag-CGTTTTGCGTTGACGTTCGTAGGTTTTCGCGGT-3' tag SEQ ID 06;
the methylation specific primers and probes of the VIM gene target site comprise:
a forward primer: 5 '-GGTCGAGTTTTATGGAGTTACGT-3' SEQ ID 07,
reverse primer: 5 '-CCCGAAAACAAACTAAAAACTA-3' SEQ ID 08,
detecting a probe: 5 'tag-CGTATTTAAGTTGGGTAGCGC-3' tag SEQ ID 09;
methylation specific primers and probes for target sites of the SDC2 gene comprise:
a forward primer: 5 '-AAATTAATAATGGAGGGCGTCGC-3' SEQ ID 10,
reverse primer: 5 '-CGAAAACCGAACTGAAACTCGA-3' SEQ ID 11,
detecting a probe: 5 'tag-TTTCGGGCGTAGTTGCGGCGGCGGGA-3' tag SEQ ID 12;
the ACTB reference gene specific primers and probes comprise:
a forward primer: 5 '-GTGATGAGGAGGTTTGTAAGTT-3' SEQ ID 13,
reverse primer: 5 '-GCACTCTCCGAAAACGAACG-3' SEQ ID 14,
detecting a probe: 5 'tag-ACCACACCCAACACACAATACAAACACA-3' tag SEQ ID 15;
step three: and determining whether the sample to be detected is methylated or not according to the fluorescence Ct values of the amplification results of Septin 9, MGMT, VIM, SDC2 and ACTB genes.
7. The method according to claim 6, wherein in the first step, the reagent used in the conversion treatment is bisulfite or bisulfite.
8. The method according to claim 6, wherein in the second step, the reaction procedure of PCR amplification is: denaturation at 95 deg.C for 10 min; cycles 45cycles, 95 ℃ for 15sec, 60 ℃ for 30sec (fluorescence acquisition).
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| CN113981083B (en) * | 2021-10-29 | 2023-07-11 | 江苏康为世纪生物科技股份有限公司 | Nucleic acid combination and kit for colorectal cancer gene methylation detection |
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