Colon cancer microsatellite instability detection kit based on next generation sequencing platform

Abstract

The invention discloses a colon cancer microsatellite instability detection kit based on a next generation sequencing platform. The colon cancer microsatellite instability detection kit comprises 4.5-6.5mu l of a primer panel solution, wherein the concentration of solute in the primer panel solution is 45-55 mmole / L; the primer panel solution comprises primers of 10 microsatellite loci; the kit further comprises 8-10mu l of Master Mix and 1.3-1.7ml of non-enzyme water; Master Mix comprises 3-7mu l of 10*LA Taq Buffer and 3-5mu l of 2-4mmol / L dNTPs, and 0.5-1mu l of LA Taq enzyme. By adopting the kit disclosed by the invention, all MSI loci of multiple samples can be simultaneously detected by sequencing once, influence caused by artificial operation can be reduced, and the detection result reliability can be improved.

Description

technical field [0001] The invention relates to a detection kit for microsatellite instability of colon cancer, in particular to a detection kit for detecting microsatellite instability of colon cancer patients based on a next-generation sequencing platform. Background technique [0002] The incidence rate of colorectal cancer ranks the third among all kinds of cancers in my country and the fifth among the causes of cancer death. Its 5-year survival rate after radical resection is about 50%. Postoperative recurrence and metastasis are the main causes of its death. reason. Patients with early colon cancer can usually be resected by surgery. Once it metastasizes, there are not many treatment options, and the five-year survival rate of patients is not ideal. Studies have found that genome instability is closely related to the pathogenesis of colorectal cancer. Genome instability includes chromosome instability (chromosomalinstability, CI) and microsatellite instability (microsa...

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Problems solved by technology

[0010] 1) Immunohistochemical method, this method is only used for MLH1, MSH2, MSH6 and PMS2 protein binding in tumor cells, if no protein is bound, it proves that MSI has occurred; although the sensitivity of this method is close to 90%, it is specific The performance and repeatability are low, and the operation is more complicated;

[0011] 2) Capillary electrophoresis method, this method is mainly aimed at the simple repeating structure on the MSI-related gene sequence, and the number of repeating bases can be judged by fragment analysis, so as to perform MSI genotyping; this method is relatively mature, and the injection volume is small , high resolution and high degree of automation, however, due to the small amount of sample injection, the preparation ability is poor; the small diameter of the capillary results in a short optical path, and when some detection methods (such as ultraviolet absorption spectroscopy) are used, the sensitivity is low; in addition, the electric Osmolarity can vary with sample composition, affecting separation reproducibility;

[0012] 3) PCR method, this method is by selecting specific primers, with self normal tissue as contrast, carry out PCR amplification to microsatellite site in vitro, the product after amplification undergoes polyacrylamide gel electrophoresis, through autoradiography etc. to analyze whether there is a change in mobility; this method is currently a commonly used detection method and has been proven to be the most effective primary screening method, but the PCR method only detects five microsatellite genes, and the product is subjected to gel electrophoresis. cumbersome, high cost and many other defects;

[0014] 5) Direct sequencing method, this method performs sanger sequencing on the amplification products of each region of the MMR gene (MLH1, MSH2, MSH6 and PMS2, etc.) to determine its MSI status; the detection cycle of this method is long and the cost is relatively high

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