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Primer, probe and kit for detecting cis-trans mutation of EGFR genes C797S and T790M

A technology of T790M and C797S, which is applied in biochemical equipment and methods, measurement/inspection of microorganisms, DNA/RNA fragments, etc., can solve the problems that result interpretation depends on special software, expensive instruments, reagents, and complicated detection steps, etc., to achieve The effect of fast detection speed, short cycle and strong detection specificity

Active Publication Date: 2019-07-26
JIAXING ACCB DIAGNOSTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection sensitivity of the NGS method can reach 0.2%, but its detection steps are complicated, the cost of reagents is expensive, and the result interpretation depends on bioinformatics analysis and calculation, and the detection cycle is long; the detection sensitivity of the digital PCR method can reach 0.1%, but there are many detection steps, The cost of reagents is much more expensive than that of real-time fluorescent PCR, and the interpretation of results also depends on special software, and the division of negative / positive intervals mainly depends on the subjective judgment of graphics
NGS and digital PCR instruments and reagents are expensive, so there are high requirements for operators

Method used

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  • Primer, probe and kit for detecting cis-trans mutation of EGFR genes C797S and T790M
  • Primer, probe and kit for detecting cis-trans mutation of EGFR genes C797S and T790M
  • Primer, probe and kit for detecting cis-trans mutation of EGFR genes C797S and T790M

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Embodiment 1 detects the analytical sensitivity (minimum detection limit) of EGFR gene C797S mutation

[0095] (1) Preparation of detection limit reference substance

[0096] a) Design and construct a corresponding mutant plasmid according to the sequence of the mutant gene by the gene cloning technique routinely used in the art, see Table 3. The sequence of the constructed plasmid containing the EGFR mutant gene sequence (hereinafter referred to as "mutant plasmid") was verified to meet the design requirements by Sanger sequencing.

[0097] Table 3 mutant plasmid information

[0098]

[0099] b) Select the human lung cancer cell line A549 (where the EGFR gene is wild-type) for in vitro culture, collect the cells and use a cell genome DNA extraction kit to extract the DNA of the A549 cell line, and use a micro-spectrophotometer to measure the concentration of the DNA, according to Determination of concentration DNA was diluted to 4 ng / μl with TE (Tris-HCl EDTA pH 8...

Embodiment 2

[0106] The analytical specificity of embodiment 2 detection reagents

[0107] (1) Preparation of wild type reference product

[0108] a) Select the human lung cancer cell line A549 (where the EGFR gene is wild-type) for in vitro culture, collect the cells and use a cell genome DNA extraction kit to extract the DNA of the A549 cell line, and use a micro-spectrophotometer to measure the concentration of the DNA, according to Determination of concentration DNA was diluted with TE (Tris-HCl EDTA pH 8.0) to 2, 5, 20, 50, 100 ng / μl respectively.

[0109] b) Using an ultrasound machine (Covaris M220) and the parameter configuration in the instruction manual, the genomic DNA can be ultrasonically broken into fragmented DNA with a length of 150±50bp, which is almost the same length as the plasma free DNA (cfDNA), and can be used as a cfDNA reference product use.

[0110] (2) Preparation of strong positive reference substance

[0111] With reference to the method in Example 1, prepar...

Embodiment 3

[0115] Example 3 Changes in the detection primers and probe sequences will affect the analytical sensitivity and / or analytical specificity, so that the minimum detection limit cannot reach 0.1%, and / or there is cross-recognition. The presence of cross recognition in reagent A means that the reagent falsely detects wild-type DNA as positive, or the false detection of DNA containing only T790M or C797S single mutation as positive; the presence of cross recognition in reagent B means that the reagent falsely detects wild-type DNA It was positive, or the T790M single mutation DNA was falsely detected as positive.

[0116] Therefore, the primers and probe sequences of the present invention are all optimal combinations, which can ensure high sensitivity and high specificity in the detection of EGFR gene C797S, and C797S and T790M cis double mutations.

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Abstract

The invention discloses a primer, probe and kit for detecting cis-trans mutation of EGFR genes C797S and T790M. The primer, probe combination and kit can accurately detect cis-double mutations of EGFRgenes C797S and T790M based on a real-time fluorescent PCR technology platform, have the advantages of fast detection speed, easiness in result interpretation, low detection reagent cost, high detection sensitivity and high specificity, and are suitable for detecting tissue sample DNAs and plasma free DNAs.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a primer, a probe combination and a kit for detecting cis-trans mutations of EGFR gene C797S and T790M. Background technique [0002] The incidence of cancer has been increasing year by year in recent years, and the number of new cancer patients in my country reaches more than 3.5 million every year. Lung cancer is one of the most common cancers worldwide. In my country, lung cancer has become the first cause of cancer death, and the 5-year survival rate is about 15%. With the development of tumor molecular biology, targeted drug therapy has been widely used clinically, and selecting appropriate patients for targeted therapy is the key to its success. [0003] EGFR (Epidermal Growth Factor Receptor) is currently an important target for lung cancer targeted therapy. The EGFR gene is located on the short arm of human chromosome 7 (7p12), encoding a transcellular membrane ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2563/107C12Q2545/101C12Q2561/113
Inventor 陈钊莫敏俐刘玉忠李东霞刘阳张艳
Owner JIAXING ACCB DIAGNOSTICS
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