Liquid phase chip for detecting PIK3CA (phosphoinositide-3-kinase, catalytic, alpha) gene mutation

Abstract

The invention discloses a liquid phase chip for detecting PIK3CA (phosphoinositide-3-kinase, catalytic, alpha) gene mutation. The liquid phase chip mainly comprises (A) a wild-type and mutant-type allele specific primer extension (ASPE) primer pair designed for mutational sites of a PIK3CA gene respectively, microspheres which are respectively coated with specific anti-tag sequences and have different colors of codes and amplification primers for respectively amplifying the target sequences of the PIK3CA gene with the corresponding mutational sites of the Exon 9 and / or Exon 20, wherein the anti-tag sequences can correspondingly complement and form pair with the selected tag sequences. The liquid phase chip has the following advantages: the coincidence rate of the detection method provided by the invention and a sequencing method is as high as 100%; the prepared liquid phase chip has very good signal to noise ratio; cross reactions do not exist between the designed probes and the anti-tag sequences; and parallel detection of various mutant types and various mutational sites on the same site can be realized.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, and in particular relates to a PIK3CA gene mutation detection liquid phase chip. Background technique [0002] PI3Ks (phosphatidylinositol3-kinases) are a group of protein multimers, which are divided into three classes (ClassI, II and III). Class I proteins may include two subunits: IA and IB. IA is a heterodimer composed of a p110 catalytic subunit and a p85 regulatory subunit. The PIK3CA gene encodes the p110α catalytic subunit of class IA. PI3Ks can be activated by growth factor receptor tyrosine kinases (RTKs). Activation of PI3K produces a variety of biological effects, including regulation of cell proliferation, survival, and cell cycle regulation. [0003] The site of PIK3CA point mutation is found in multiple exons, but mainly occurs in the kinase and helical domains. The most common mutations are E542K and E545K of exon 9 (Exon 9), and H1047R ...

AI Technical Summary

Problems solved by technology

The PCR-sequencing method has the advantage of being able to determine the range and type of mutations, and is currently a widely used detection method, but the sensitivity of the sequencing method is only 20%-25%, which is far from meeting the needs of clinical applications, especially for heterogeneity of tumor somatic mutations, low sensitivity will lead to a large number of missed detections

At the same time, the detection operation of the sequencing method is complicated and the timeliness is poor. For clinical detection that requires high timeliness and high sensitivity, the limitations of the sequencing method have long been highlighted

The real-time fluorescent quantitative PCR detection technology has high detection efficiency and strong timeliness, but its high false positive rate is also criticized by clinical applications.

Based on the problems of the above-mentioned detection technology, the detection method of "PIK3CA gene mutation detection probe, liquid chip and its detection method" (application number: 200810220266.1) successfully developed by the applicant in the early stage is simple to operate, and a large number of The interference caused by the wild-type sequence has good specificity, high sensitivity, and an accuracy of more than 99%. However, this method involves two rounds of PCR operations, which is easy to cause pollution, and the probes in the hybridization detection step are relatively close (only the mutation site is different). ), it is not convenient for parallel detection of multiple gene mutation sites

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