Methylated quantitative detection method of GSH2 (glutathione 2) gene

Abstract

The invention belongs to the technical field of biomedical sciences. Abnormal methylation of genes participates in the development of tumors. The invention aims at overcoming the defects of a methylation-sensitive restrictive endonuclease method, a bisulfite sequencing method and a methylation-specific PCR (Polymerase Chain Reaction) method and provides a methylated quantitative detection method of a GSH2 (glutathione 2) gene, which has simple and convenient operation and high senibilitiy. The method comprises the following steps of: taking pancreatic cancer tissues, extracting DNA and preparing an ACTB (Actin, Beta) standard curve sample, and preparing a GSH2 (Glutathione 2) standard curve sample by utilizing a full methylation template; extracting DNA of tissues to be detected, and carrying out chemical modification; designing methylation primers and probes aiming at CPG islands at a human GSH2 gene promoter region, designing BSP (Bisulfite) primers and probes aiming at CPG islands at a human reference gene ACTB promoter region, carrying out real-time quantitive PCR on DNA after bisulfite treatment by adopting a Taqman-MGB real-time quantitive PCR method, and calculating a quantitive value of the GSH2 gene methylation degree. The method is rapid, accurate and high in sensitivity.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for detecting nucleic acid, in particular to a method for detecting methylation of the GSH2 gene. Background technique [0002] The occurrence and development of malignant tumors is a process in which multiple genes interact and influence each other. Abnormal methylation of CpG islands is an important mechanism for the occurrence and development of this gradual process, which includes two types: hypermethylation and hypomethylation. According to literature reports, abnormal methylation of multiple pancreatic cancer-related genes has been detected in pancreatic cancer tissue or pancreatic juice, while the proportion of methylation in normal tissues is very low, so the detection of methylation of pancreatic cancer-related genes , to achieve the purpose of early diagnosis of pancreatic cancer. For example, abnormal methylation of gene P16 (Virmani AK, et al...

AI Technical Summary

Problems solved by technology

The key to reliable MSP lies in the primers. The primer design requires two known regions containing multiple fully methylated or unmethylated CpG sites, but there are not many CpG islands with the above characteristics, which limits the potential of MSP. use

This method has the following disadvantages: (1) This method can only be used for qualitative research, that is, it can only determine whether there is methylation; if quantification is required, other methods need to be used for further detection; Due to the difference in the amount of amplification products between methylated primers and unmethylated primers, it is necessary to strictly control the conditions and cycle number of the PCR reaction.

[0008] Currently, there is no quantitative detection method related to GSH2 gene methylation

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