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Probe, primer and kit for detecting drive mutation of PIK3CA (Phosphatidylinositol-3-kinases) gene

A technology for detection kits and probes, applied in recombinant DNA technology, DNA/RNA fragments, fluorescence/phosphorescence, etc., can solve unreachable problems and achieve high sensitivity, fast detection speed, and strong specificity

Active Publication Date: 2013-07-24
AMOY DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, technologies such as direct sequencing are far from meeting the needs of their practical applications, and there is an urgent need to develop a highly sensitive and specific PIK3CA driver mutation gene detection technology to achieve the detection of PIK3CA gene driver mutations with high sensitivity detection methods , so as to provide scientific reference for clinical individualized drug regimen

Method used

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  • Probe, primer and kit for detecting drive mutation of PIK3CA (Phosphatidylinositol-3-kinases) gene
  • Probe, primer and kit for detecting drive mutation of PIK3CA (Phosphatidylinositol-3-kinases) gene
  • Probe, primer and kit for detecting drive mutation of PIK3CA (Phosphatidylinositol-3-kinases) gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Take the H1047R driver mutation of exon 20 of the PIK3CA gene as an example to detect the real-time fluorescent PCR detection reaction system of the present invention. One plasmid template (including H1047R) and two cell lines were used in the experiment, namely H1650 (wild type PIK3CA gene) and H460 (wild type PIK3CA gene). The method for detecting the H1047R driver mutation of exon 20 of the PIK3CA gene by using the above fluorescent PCR is as follows:

[0042] (1) Sample DNA extraction

[0043] Cut the sample into 5-10μm, add 1mL xylene to dewax, centrifuge to collect the precipitate, add 1mL absolute ethanol to the precipitate, dry at room temperature or 37°C, add proteinase K and Buffer ATL, digest at 56°C for 1 hour, 90°C Incubate for 1h, add 200mL Buffer AL to mix well, then add 200μl absolute ethanol to mix well, carefully transfer the supernatant to a QIA 2mL spin column, centrifuge at 6000×g (8000rpm) for 1min, add 500μl Buffer AW1, 6000×g (8000rpm) ) and ce...

Embodiment 2

[0055] Using the present invention to detect clinical samples, a total of 90 cases of clinical breast cancer samples were taken and sent to our company for detection, including 70 cases of paraffin-embedded tissue samples and 20 cases of blood samples; 42 cases of males and 48 cases of females, aged 35-72 years, The average age is 58 years old. The steps for detecting PIK3CA gene driver mutations in 90 clinical samples by using the double-loop probe and fluorescent PCR system of the present invention are as follows.

[0056] (1) DNA extraction from clinical pathological samples

[0057] For fresh pathological tissue and paraffin tissue samples, cut the sample into 5-10μm, add 1mL xylene to dewax, collect the precipitate by centrifugation, add 1mL absolute ethanol to the precipitate, dry at room temperature or 37°C, add proteinase K and Buffer ATL, Digest and lyse at 56°C for 1 hour, incubate at 90°C for 1 hour, add 200mL Buffer AL and mix well, then add 200μL absolute ethanol...

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Abstract

The invention discloses a probe, a primer and a kit for detecting drive mutation of a PIK3CA (Phosphatidylinositol-3-kinases) gene and relates to detection of gene mutation. The method disclosed by the invention comprises the steps of: (1) providing 6 groups of primers (18 in number) and the probe; (2) extracting a DNA (deoxyribonucleic acid) template of a sample to be detected; (3) preparing a fluorescent PCR (polymerase chain reaction) system for detecting drive mutation of the PIK3CA gene; and (4) detecting the FAM and HEX fluorescent intensity of the reaction system by using the hybridization of a bi-loop probe and the specific primers, and determining a result according to the FAM and HEX fluorescent intensity. The method disclosed by the invention can detect 5 types of drive mutation of the PIK3CA gene, and has the characteristics of high sensitivity, strong specificity, fast detect speed and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a detection probe, primer and kit for PIK3CA gene driver mutation. Background technique [0002] PIK3CA (Phosphatidylinositol-3-Kinases, phosphatidylinositol-3-kinase) is an intracellular phosphatidylinositol kinase, composed of a relative molecular mass of 110×10 3 The catalytic subunit p110 and a relative molecular mass of 85×10 3 The regulatory subunit p85 constitutes. PIK3CA participates in a variety of signaling pathways and regulates major physiological functions such as cell growth and proliferation. The lipid products produced by PIK3CA activation, 3.4-phosphatidylinositol diphosphate and 3.4.5-phosphatidylinositol triphosphate, can act as second messengers to activate various target protein molecules in downstream cells to form a signaling cascade Amplifies the complex, ultimately regulating cell proliferation, differentiation, and migration. [0003] Akt (Protein Kinase...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 江风阁张海龙阮力郑立谋
Owner AMOY DIAGNOSTICS CO LTD
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