Primer probe system for MLH1 (MutL homolog1) gene methylation detection and kit adopting primer probe system

Abstract

The invention relates to a primer probe system for MLH1 (MutL homolog1) gene methylation detection and a kit adopting the primer probe system. The primer probe system comprises a primer probe set X used for determining genomic DNA quality, a primer probe set Y used for determining a methylation conversion rate and a primer probe set Z used for detecting MLH1 gene promoter methylation conditions, wherein the primer probe set X comprises a forward primer a, a reverse primer a and a probe a; the primer probe set Y comprises a forward primer b, a reverse primer b and a probe b; the primer probe set Z comprises the forward primer b, the reverse primer b, a probe c and a probe d; fluorescence report groups are arranged at 5' ends of the probe a, the probe b and the probe c, and fluorescence quenching groups are arranged at 3' ends of the probe a, the probe b, the probe c and the probe d. According to the kit, the implementing scheme is concise, and the sensitivity and the accuracy rate are high.

Description

technical field [0001] The invention relates to a gene mutation detection product, a detection primer and a detection system used in the product, and belongs to the field of biotechnology. Background technique [0002] DNA mismatch repair system (MMR) is a safety guarantee system in human cells that can repair DNA base mismatches and maintain the integrity and stability of genetic material. hMLH1, hMSH2, and hMSH3 are important mismatch repair genes in the MMR system. The reduced expression caused by the hypermethylation of DNA damage repair genes will lead to the decline of DNA damage repair ability and the subsequent gene mutation. [0003] The MLH1 gene (MutLhomolog1) is located at 3p21.3 with a full length of 2439bp. Methylation of the MLH1 gene will lead to the inactivation of the expression of this gene or the inability of the expressed protein to repair the mismatched bases in time, which may lead to the inactivation of tumor suppressor genes and the activation of o...

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Problems solved by technology

The MSP method combined with the nested PCR amplification method can improve the specificity of the single MSP method, but the operation time is long, and compared with the MSP method, there are also false positives caused by incomplete bisulfite treatment. The pH value in the modification process is relatively absolute. Accurate, all reagents require fresh configuration, and need to repeatedly explore to find the appropriate reaction time, resulting in cumbersome detection process, and can not achieve quantitative detection, there is a high false positive rate; there is also excessive sulfite treatment, resulting in amplification 3. Methylation Sensitivity Melting Curve Analysis (MS-High Resolution Melting Curve, MS-HRM), which is cumbersome to operate, has a high false positive rate, and is not suitable for the detection of a large number of samples; 4. Fluorescence method (Methylight) , due to the limitations of the probe, there is generally no suitable control system, and the false positive rate is high; 5. Bisulfite sequencing PCR (BSP), which is a PCR combined with Sanger sequencing technology, has accurate results, but the process is cumbersome. Not suitable for high-volume testing, and too expensive to be widely used

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