Methylation quantitative detection method of RASSFIA gene in human plasma

Abstract

The invention relates to a methylation quantitative test method of RASSF1A gene in the DNA of human blood plasma. The methylation quantitative test method obtains venous blood and the blood plasma, prepares a DNA standard curve sample of the blood plasma, extracts the DNA of the blood plasma to be detected, the DNA of a standard blood plasma and the DNA of healthy human blood plasma, realizes chemical modification of the DNA of the blood plasma, the standard blood plasma and the healthy human blood plasma, designs a fluorescent quantitative MSP augmenting architecture of particularity primers and Taqman fluorescent probes according to 565 CpG locus in a human RASSF1A gene sub-promoter, builds an external standard curve according to the augmentation result of the DNA of the standard blood plasma, and implements quantitative result analysis of the sample to be detected. The methylation quantitative test method of the RASSF1A gene solves the defects that the disease course of most tumor patients that receive an operation lies in the mid and late periods, and consequently the obtaining damage of operation samples is large and the detectable rate of methylation is relatively lower, etc., has high sensitivity, the lowest detection limit of 1.5 multiplied by 10<2> copy per milliliter, the detection aimed at the 565 CpG locus in the RASSF1A gene sub-promoter, strong particularity, easy obtaining and small damage, and can quantitatively detect the RASSF1A gene methylation in the DNA of the blood plasma.

Description

technical field [0001] The invention relates to a biological macromolecule detection method, in particular to a quantitative detection method for RASSF1A gene methylation in human plasma DNA. Background technique [0002] In recent years, with the in-depth study of the formation mechanism of tumor epigenetics, a large number of results have shown that the hypermethylation inactivation of tumor suppressor gene promoters occurs in the early stages of cancer development, which provides an opportunity for finding new early Molecular diagnostic biomarkers of tumors provide an objective basis. [0003] RAS-associated domain family 1A (RASSF1A) is a tumor suppressor gene cloned from the short arm of chromosome 3 (3p21.3) in 2000. Most studies have shown that RASSF1A can play a role in both conduction pathways and cell division. CpG island methylation in the promoter region is the most important mechanism of RASSF1A gene inactivation. Low expression or no expression of RASSF1A ge...

AI Technical Summary

Problems solved by technology

Ordinary MSP avoids some problems of restriction endonuclease method and is widely used, but its disadvantages are low detection sensitivity and environmental pollution by electrophoresis. Currently, it is only used for qualitative research on DNA methylation of tumor tissue cells. For The detection and diagnosis of early malignant tumors have no actual clinical application value

[0009] In addition, there are some methods, such as DNA microarray (chip), which will have false positive problems; the methylation-sensitive single-strand conformation analysis method has the disadvantage that it can only distinguish fragments with high methylation levels, which is sensitive and accurate. low sex

[0010] So far, most of the methylation detection of the RASSF1A gene promoter is based on the tumor specimens of surgical patients as clinical samples. The disadvantage is that most of the surgically treated tumor patients are already in the middle and late stages of the disease, so the detection results are not accurate for early stages. The diagnosis is meaningless; at the same time, the acquisition of surgical specimens is invasive, which is not conducive to the widespread promotion of effective detection methods

Using bronchoalveolar lavage fluid as a specimen is only suitable for the diagnosis of central lung cancer, and may easily lead to missed diagnosis of peripheral lung cancer

It is relatively easy to obtain sputum as a test sample, but it is not easy to obtain it for patients with less sputum. The most important thing is that the detection rate of methylation is relatively low (only 5%)

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