Kit for detecting colorectal cancer susceptibility gene mutation and detection method thereof

Abstract

The invention discloses a kit for detecting colorectal cancer susceptibility gene mutation and a detection method thereof. The kit contains specific primers, universal primers, PCR (Polymerase Chain Reaction) mixing reagents, positive controls and magnetic beads aiming at PIK3CA, KRAS and BRAF gene mutation sites. The method comprises the following steps: extracting genome DNA of a sample to be detected, combining the specific primers with target template DNA sequences, amplifying a target area of the sample to be detected by the universal primers, purifying a library by the magnetic beads, performing high-throughput sequencing on the obtained library, and analyzing mutation conditions. The method and the kit disclosed by the invention can simultaneously detect a plurality of susceptibility genes, simplify experimental procedures and simultaneously improve the detection sensitivity, are suitable for performing early screening and postoperative diagnosis of the colorectal cancer, and provide guide for individualized medication.

Description

technical field [0001] The invention relates to the field of gene mutation detection in biotechnology, and more specifically relates to a kit for detecting colorectal cancer susceptibility gene mutation and a detection method thereof. Background technique [0002] Colorectal cancer (CRC) is the general term for colon cancer and rectal cancer. Colorectal cancer is a common malignant tumor of the digestive system, and its morbidity and mortality rank third among malignant tumors in the world, and it is showing an increasing trend year by year. Early symptoms of colorectal cancer, such as increased stool frequency, mucus and pus and blood in the stool, are easily misdiagnosed as dysentery, enteritis or hemorrhoids, and the misdiagnosis rate is as high as 30%. [0003] According to statistics, the 5-year survival rate of colorectal patients without invasion and metastasis can be as high as 90%, the 5-year survival rate of patients with local metastasis is 68%, while the 5-year ...

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Problems solved by technology

At present, the main screening methods for colorectal cancer all have different degrees of defects: (1) routine blood tests, which are not specific and sensitive, and are invasive to the body; (2) imaging studies, such as colonoscopy, soft Type sigmoidoscopy, CT colonography, colonic barium enema, etc., the invasiveness and complications of these methods limit their application in general screening; (3) Fecal occult blood detection, such as fecal occult blood test , fecal immunochemical test, etc., which are convenient and non-invasive, but the specificity and sensitivity are not strong, and the consistency of the test results of different methodologies is poor; (4) tumor marker detection, the positive rate is low, and the specificity is also lacking

The ARMS method can simultaneously detect a variety of known common mutations in tumor tissue, and has the advantages of high sensitivity (mutations as low as 1% can be detected), simple operation, and rapid detection; however, for blood samples that are convenient to sample such as circulating tumors The DNA content of cells and tumors is too low, and the ARMS-PCR method is not enough to detect them. At the same time, the high cost of such kits limits their clinical promotion and application.

The direct sequencing method is the gold standard for gene mutation and diversity detection. It has the advantages of mature technology, accurate and comprehensive results, etc. Compared with the ARMS method, it is cheaper to detect and can detect unknown mutations. The main disadvantage of the current direct sequencing method is the operation. It is complex and the detection sensitivity is too low. Only when the mutation reaches more than 10% or even 20% can it be reliably detected. It is not suitable for clinical application and analysis of a large number of clinical samples

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