PIK3CA gene mutation fluorescence quantitative PCR genotype detection kit and detection method

Abstract

The invention discloses a PIK3CA gene mutation fluorescence quantitative PCR genotype detection kit comprising a PCR mixed reaction liquid, a positive control, and a fluorescent probe used for detecting PIK3CA gene mutant genotypes. The PCR mixed reaction liquid comprises PCR primers used for amplifying PIK3CA gene segments where the mutation sites exist. The invention also discloses a PIK3CA gene mutation fluorescence quantitative PCR genotype detection method. According to the method, PIK3CA gene mutant genotypes are subjected to fluorescence quantitative PCR detections by using the kit provided by the invention. With the technical scheme provided by the invention, detections can be carried out upon tissue PIK3CA gene mutations, and especially rapid, accurate, and high-sensitively detections upon PIK3CA gene 542,545 and 1047 nucleotide mutations can be realized.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a fluorescent quantitative PCR kit for detecting gene mutations and a detection method thereof, in particular to a fluorescent quantitative PCR typing detection kit for PIK3CA gene mutations and a detection method thereof. Background technique [0002] PIK3CA (phosphatidylinositol 3-kinase catalytic alpha) was detected by Volinia et al. in 1994 using in situ hybridization. It is a newly discovered oncogene with somatic mutations, which may be regulated by participating in the PIK3 / AKT cell signal transduction pathway. Cell growth and apoptosis. Studies have shown that PIK3CA is related to a variety of malignant tumors, such as colorectal cancer, cervical cancer, breast cancer, lung cancer and so on. At present, the relationship between oncogene PIK3CA and tumors and its mechanism of tumorigenesis have become a hot spot in tumor research. The occurrence of human tumors is a mult...

AI Technical Summary

Problems solved by technology

[0007] 1. The detection cycle is long, and it takes about 48-72 hours at the shortest from the time the specimen is submitted for inspection to the result;

[0008] 2. The operation process is complicated and involves the operation after PCR amplification, so it is easy to be contaminated, resulting in unsatisfactory results (see item F for details);

[0009] 3. The interpretation of sequencing results is complex and time-consuming (this is especially prominent when the sample size is large, see item F for details);

[0010] 4. The detection sensitivity is not high enough. Katsuhiko et al. reported in "Lung Cancer" published in August 2005 that if the content of the mutated gene accounts for less than 10% of the total genomic DNA, it cannot be detected by direct sequencing the presence of mutant samples;

[0011] 5. The sample size of an experimental test is limited, at most 8-24 cases;

[0012] 6. It is not safe. The whole experiment process uses a variety of toxic substances, such as EB, acrylamide, etc., which are harmful to the operator and the environment;

[0013]7. The cost is relatively high (about 300 yuan per site)

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