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Probes, primers and kits for detecting five mutations of human pik3ca gene

A technology for detecting probes and kits, applied in the biological field, can solve the problems of easy contamination of samples, low detection efficiency, poor accuracy, etc.

Active Publication Date: 2019-04-23
武汉海吉力生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problems of the existing PIK3CA gene mutation detection methods, such as low detection efficiency, easy sample contamination, long detection time, poor accuracy, and difficulty in discrimination, the present invention provides primers and probes for detecting five mutations of human PIK3CA gene

Method used

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  • Probes, primers and kits for detecting five mutations of human pik3ca gene
  • Probes, primers and kits for detecting five mutations of human pik3ca gene
  • Probes, primers and kits for detecting five mutations of human pik3ca gene

Examples

Experimental program
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Effect test

Embodiment 1

[0083] 1. Primers and probes of the detection kit for detecting 7 mutations of human PIK3CA gene:

[0084] According to the wild-type sequence of the PIK3CA gene (NCBI Reference Sequence: NM_006218.2) published in the NCBI database, the E542K (PIM1), E545K (PIM2) and E545D (PIM3) mutation sites in exon 10, and the H1047R (PIM4) in exon 21 ) and H1047L (PIM5) mutation sites as reference, design specific E542K mutation primers and probes (see Table 2), specific E545K mutation primers and probes (see Table 3), specific E545D mutation primers and probes ( See Table 4), specific H1047R mutation primers and probes (see Table 5) and specific H1047L mutation primers and probes (see Table 6). Using the mutant plasmid and wild-type plasmid constructed by genetic engineering as templates, a real-time fluorescent PCR mutation detection system was established to achieve high sensitivity and high specificity detection of PIK3CA gene mutation.

[0085] (1) Specific primer and probe sequence...

Embodiment 2

[0159] The five kinds of human PIK3CA gene mutation detection kits of the present invention are used to detect clinically collected tissue samples.

[0160] In this example, tissue samples from patients diagnosed as breast cancer were collected and genomic DNA was extracted from them. The 5 kinds of PIK3CA gene mutation detection kit was used to detect the mutation status of PIK3CA gene in the samples to be tested. Specific steps are as follows:

[0161] (1) Sample genomic DNA extraction

[0162] Extract the paraffin-embedded tissue samples using QIAamp DNAFFPE Tissue Kit (CatNo.56404), extract the genomic DNA of the sample to be tested according to the instructions, and use the UV spectrophotometer to detect the concentration and DNA quality of the extracted DNA samples, and the DNA concentration requirements ≥10ng / μL, the quality of DNA OD260 / 280 is between 1.8 and 2.0. Genomic DNA that meets the requirements was diluted to 2-10 ng / μL with TE (10 mmol / L, pH 8.0) as a PCR ...

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Abstract

The invention discloses primers and probes and a kit for detecting five mutation types of a human PIK3CA gene. According to each group of the five groups (PIM1-PIM5) of the primers and probes, the mutation primer can be combined with a conserved sequence of the PIK3CA gene, a mutation ARMS primer can be specially combined with a corresponding mutation sequence of the mutation ARMS primer to amplify the mutation sequence, the detection probe can be combined with an amplified fragment, and the blocking probe can be specially combined with a wild type sequence corresponding to a mutation locus to inhibit wild type nonspecific amplification. Accordingly, by adopting a specific mutation primer and probe blocking technology, the five mutation types of the PIK3CA gene can be accurately detected; through the established kit of a real-time fluorescence PCR amplification reaction system, rapid detection on the PIK3CA gene mutation is facilitated, operation is easy, and a result is easy to read; the detection method is high in sensitivity, and 50-copy mutation can be stably detected; the specificity is good, no nonspecific amplification is generated in 20-ng wild type genome DNA, and the detectability reaches up to 1%.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a probe, primer and kit for detecting five mutations of human PIK3CA gene. Background technique [0002] The PIK3CA gene is located at 3q26.3, is 34kb long, and contains 20 exons. play an important role in the process of death and tumor formation. The PI3K-Akt (phosphatidylinositol-3 kinase / serine-threonine protein kinase B) signaling pathway is an important signaling pathway that regulates cell function. When the PIK3CA gene is activated in the form of gene amplification or point mutation, its transcription and Elevated translation levels will overactivate AKT through the PI3K / AKT pathway, promote cell growth and reproduction uncontrollably, and inhibit cell apoptosis, eventually leading to tumorigenesis. [0003] Mutations in the PIK3CA gene are present in a variety of human solid tumors. Mutations of the PIK3CA gene include gene amplification, deletion, and somatic missense mut...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/156C12Q2531/113C12Q2535/137C12Q2537/163C12Q2561/101C12Q2561/113C12Q2565/1015
Inventor 朱峰邹芳段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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