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Kit for detecting PIK3CA gene mutation and detecting method thereof

A kit, GAGCAGGAG-PO4 technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as easy pollution, positive results cannot be confirmed, specific unknowns, etc., to achieve strong specificity and clear interpretation of results Objective, effects that increase the likelihood of contamination

Inactive Publication Date: 2015-07-15
JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method can detect both known and unknown mutation sites, and the sensitivity is about 5%. However, the reaction tube needs to be opened during the detection process, which is easy to cause pollution, and the positive result cannot confirm the specific unknown, and finally needs to be confirmed by sequencing

Method used

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  • Kit for detecting PIK3CA gene mutation and detecting method thereof
  • Kit for detecting PIK3CA gene mutation and detecting method thereof
  • Kit for detecting PIK3CA gene mutation and detecting method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The PIK3CA gene negative quality control product is human blood genomic DNA without PIK3CA gene mutation, and then diluted to a concentration of 10ng / μl (the copy number is about 10 3 ). The PIK3CA gene positive quality control product is connected to the pUC57-Amp vector by the mutation sequence of the 5 mutation sites of the PIK3CA gene, and then transformed into Escherichia coli, extracted and purified, and then diluted to a concentration of 10 3 Copy number (concentration is about 10-5ng / μl) where the mutation site information comes from the Cosmic database, the mutation site is E542K (1624G>A), E545K (1633G>A), E545D (1635G>T), H1047R (3140A >G) and H1047L(3140A>T), the sequence of the mutation site was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. The specific sequence is as follows:

[0037] E542K:

[0038] aaatgtatttgctttttctgtaaatcatctgtgaatccagaggggaaaaatatgacaaagaaagctatataagatattattttattttacagagtaacagactagctagagacaatgaattaagggaaaatgacaaagaacagctc...

Embodiment 2

[0072] The minimum detection limit of PIK3CA gene E542K, E545K, E545D, H1047R, H1047L mutation sites was analyzed by using the optimized detection system and reaction program.

[0073] The research on the minimum detection limit mainly includes the following two aspects: 1) Research on the minimum detection limit of mutation ratio: the ratio of the lowest mutant DNA that can be detected under a certain DNA concentration; 2) Research on the minimum detection limit of DNA concentration: The lowest DNA concentration that can be detected under a certain ratio; 3) The lowest copy number research.

[0074] 1) Research on the minimum detection limit of mutation ratio

[0075] First, accurately dilute the PIK3CA gene E542K, E545K, E545D, H1047R, H1047L mutation positive quality control products and Control quality control products, uniformly diluted to 10 3 For copy number, the dilution is 10 ng / μl of wild-type DNA. After mixing the positive quality control with 10ng / μl negative gen...

Embodiment 3

[0095] Collect 10 colorectal cancer patient tissue samples and matched preoperative plasma samples. After the samples are collected, they are immediately stored in a -80 degree refrigerator. The DNA in the tissue samples and the free DNA (cf-DNA) in the plasma were extracted using Qiagen kits, and subjected to agarose gel electrophoresis and concentration determination.

[0096] Then, the method of Example 1 was used to detect the mutation sites in the tissue samples and plasma samples of 10 cases, and the tissue samples were sequenced at the same time, and the results are shown in Table 7.

[0097] Table 7. Results of colorectal cancer patients detected by the method of the present invention

[0098]

[0099] "-" means that the sample has no amplification curve at this site.

[0100] The result shows that the coincidence rate of the mutation in tissue samples detected by the method of the present invention and that detected by the sequencing method is 100%. The coinciden...

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Abstract

The invention discloses a kit for detecting PIK3CA gene mutation and a detecting method thereof. The kit comprises a special probe which is modified by LNA locked nucleic acid and used for PIK3CA gene mutation sites; the special probe can be combined with wild type DNAs, a sample containing 0.01% PIK3CA gene mutation DNAs can be detected, and the minimum detectability ranges from 2 copies to 5 copies. The detecting method has the advantages that as for PIK3CA gene mutation detection, the specificity is high, the flexibility is high, pollution is small, operation is easy and rapid, and the safety performance is high; the detecting method is particularly suitable for detecting gene mutation from body fluid such as plasma, urine and saliva with low mutation content, and is suitable for early screening and diagnosis of colorectal cancers, and guidance is provided for personalized medicines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit for detecting PIK3CA gene mutation, and also relates to a method for detecting PIK3CA gene mutation in non-disease diagnosis by using the kit. Background technique [0002] In my country, the incidence rate of colorectal cancer has jumped to the fourth place among malignant tumors, and the incidence rate is increasing year by year. It has become one of the main tumors that seriously threaten the health of our people. In recent years, surgery, chemotherapy, and radiotherapy have developed rapidly, but they have failed to significantly improve the 5-year survival of colorectal cancer patients. In recent years, with the rise of molecular targeted therapy represented by anti-epidermal growth factor receptor (EGFR), not only the comprehensive treatment of colorectal cancer has been enriched and developed, but also the treatment of colorectal cancer patients Expectations...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q2600/156
Inventor 王弢李宗飞孙爱娟李杰张丽
Owner JIANGSU MICRODIAG BIOMEDICINE TECH CO LTD
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