Detection primer for methylation degree of related genes of cervical cancer, probe, kit and application thereof

A technology for detection kits and detection primers, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of waste of medical resources, excessive treatment, etc. highly specific effect

Inactive Publication Date: 2017-10-24
广州中心法则生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the current method of judging the risk of cervical cancer by detecting HPV virus or even by pathological analysis of cervical intraepithelial neoplasia will lead to clinical overtreatment and waste a lot of medical resources

Method used

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  • Detection primer for methylation degree of related genes of cervical cancer, probe, kit and application thereof
  • Detection primer for methylation degree of related genes of cervical cancer, probe, kit and application thereof
  • Detection primer for methylation degree of related genes of cervical cancer, probe, kit and application thereof

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Experimental program
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Embodiment 1

[0050] The present invention is aimed at the CpG islands of the methylation sites of the four genes of human PAX1, FAM19A4, PHACTR3, and DAPK and simultaneously designs the C bases (after sulfite treatment, the C bases of the methylated CpG islands still maintain C bases) methylated probes and U bases (after sulfite treatment, the C bases of unmethylated CpG islands become U bases) unmethylated probes and specific primers . A pair of primers are designed for each gene, a methylated probe and an unmethylated probe, the methylated probe detects methylated sites, and the unmethylated probe detects unmethylated sites.

[0051] 1. PCR primer design:

[0052] Table 1. PCR primer sequences for four genes

[0053]

[0054] Note: F means upstream primer, R means downstream primer

[0055] 2. Probe design:

[0056] The present invention designs a methylation probe and a non-methylation probe for each gene. The sequences of the 8 probes are shown in Table 2.

[0057] Table 2. Fo...

Embodiment 2

[0072] According to the method of embodiment 1, extract the genomic DNA of the cervical smear samples (cervical cancer group) of 20 cervical cancer patients and the cervical smear samples (non-cervical cancer group) of 20 non-cervical cancer control groups, and then extract the genome The DNA was sulfite-converted, and the sulfite-converted DNA was used as a template, the primer pair PAX1-F / PAX1-R and the probes PAX1-M and PAX1-U for the PAX1 gene, and the primer pair FAM19A4 for the FAM19A4 gene -F / FAM19A4-R and probes FAM19A4-M and FAM19A4-U, primer pair PHACTR3-F / PHACTR3-R and probes PHACTR3-M and PHACTR3-U, primer pair DAPK-F for the DAPK gene / DAPK-R and probes DAPK-M and DAPK-U (Table 1 and Table 2), according to the qPCR reaction system and reaction conditions of Example 1, were subjected to qPCR amplification (ABI7500 fluorescence quantitative instrument from ThermoFisher Company). According to the peak Ct values ​​of the methylated probes and unmethylated probes of th...

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Abstract

The invention discloses a detection primer for methylation degree of related genes of cervical cancer, a probe, a kit and an application thereof. The invention finds that the methylation degree of promoter zones of four genes, namely, PAX1, FAM19A4, PHACTR3 and DAPK in cervical tissue is obviously related to the cervical cancer; the detection primer can be used as a biomarker for assisting in cervical cancer diagnosis; a methylation probe and a non-methylation probe are designed, so that the influence of non-methylation DNA is avoided and the result is more reliable. Compared with the conventional cervical cancer diagnostic method, the method provided by the invention has the advantages of high speed, high sensitivity, high specificity, and the like, can timely diagnose human cervical carcinoma in early stage, can early accurate diagnose the cervical cancer and can avoid the waste of medical resources.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection primer, a probe, a kit and an application thereof for detecting the degree of methylation of a cervical cancer-related gene. Background technique: [0002] Cervical cancer is the most common reproductive system malignancy in women worldwide, and its incidence rate is second only to breast cancer. In 2008, the number of new cases of cervical cancer in my country was 12.19 / 100,000, ranking the sixth among new female malignant tumors; the death rate of cervical cancer was 3.90 / 100,000; cervical cancer in my country showed an obvious upward trend opposite to developed countries. Since existing studies have confirmed that high-risk human papillomavirus (HPV) infection is closely related to the incidence of cervical cancer, the prevention of cervical cancer is generally carried out from the perspective of detecting whether women are infected with high-risk HPV viru...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/154
Inventor 陈敏汪定亮
Owner 广州中心法则生物科技有限公司
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