Human egfr gene mutation detection kit and application thereof

A kit and human detection technology, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of low accuracy, insufficient types of EGFR mutations, false positives, etc.

Active Publication Date: 2018-04-10
苏州华益美生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, the inventors found that the above-mentioned PCR-based detection technology either detects not enough types of EGFR mutations, or the detection accuracy is not high, especially the primers for detecting mutations are also easy to amplify and detect wild-type EGFR, false positive result
Since the proportion of wild-type EGFR samples accounts for the vast majority in actual detection work, if false positive results cannot be avoided, the detection technology has almost no prospect of practical application

Method used

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  • Human egfr gene mutation detection kit and application thereof
  • Human egfr gene mutation detection kit and application thereof
  • Human egfr gene mutation detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] The extraction of embodiment 1 nucleic acid

[0063] The test samples containing human EGFR wild-type and mutant genes can be purchased from the Clinical Inspection Center of the Ministry of Health of the People's Republic of China, and they are all standard products for nucleic acid extraction.

[0064] The extraction of nucleic acid is carried out according to the conventional magnetic bead extraction method. In order to adapt to the nucleic acid extraction of a large number of human EGFR gene mutants, the improvement is as follows: add 90uL nucleic acid extraction solution to 1uL standard (the formula and final concentration are: guanidine isothiocyanate 1.2 M, sodium edetate (pH8.0) 10mM, Tween-202% (W / W), sodium perchlorate 1M, ethanol 40% (V / V), Tris-HCl (pH8.0) 10mM ), incubate at 42°C for 10min, then add 10uL D-Beads DNA magnetic bead suspension (50mg / mL, can be purchased from Beijing Aibigen Biotechnology Co., Ltd.), oscillate and mix evenly, put it on a magn...

Embodiment 2

[0065] Embodiment 2 People's EGFR real-time PCR test

[0066] 1. Primer and probe sequences and fluorescent labels

[0067] Commission the synthesis of primers with nucleotide sequences as shown in SEQ ID NOs: 1-69 and probes with nucleotide sequences as shown in SEQ ID NOs: 70-78.

[0068] At the 5' ends of the probes whose nucleotide sequences are shown in SEQ ID NOs: 70-77, respectively entrust the synthesis of labeled fluorescent marker FAM, and mark the quenching group BHQ at the 3' end; at the nucleotide sequences such as SEQ ID The 5' end of the probe shown in NO:78 is entrusted with the synthesis of a fluorescent marker VIC, and the quencher group BHQ is marked at the 3' end; in the nucleotide sequence such as SEQ ID NOs: 2, 4, 6, 8, 10 , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 41, 44, 47, 49, 51, 54, 56, 58, 61, 64 The 3' end of the primer is labeled with the quencher group BHQ; other nucleic acids or parts are not labeled.

[0069] 2. PCR reaction ...

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Abstract

The invention provides a detection method for multiple mutation of human EGFR genes. The detection method includes the steps that a nucleic acid extracting solution, DNA polymerase, dNTP, primers and probes are mixed in eight PCR reaction tubes respectively; real-time PCR is carried out, and whether target nucleic acid exists in samples or not is judged according to the Ct value obtained through calculation according to fluorescence detection results. In addition, the invention relates to a nucleic acid combination and the like capable of being used for a detection kit.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection. Specifically, the invention relates to a detection kit and a detection method for multiple mutations of the human EGFR gene. In addition, the present invention also relates to nucleic acid combinations and the like that can be used in the detection kit. Background technique [0002] Epidermal growth factor receptor (EGFR) mutations are weakly associated with some diseases (eg, non-small cell lung cancer) or their efficacy. Although they cannot be used to directly determine the corresponding disease or its efficacy, they can be used as a small part of the overall diagnosis. The corresponding results are interpreted comprehensively with other test results, so as to finally obtain the diagnosis result. [0003] There are many mutations of EGFR, mainly concentrated in exons 18, 19, 20 and 21 of the gene, especially in exons 19 and 21, and new mutations are constantly being discovered ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6827C12N15/11
CPCC12Q1/6827C12Q2561/113C12Q2561/101C12Q2531/113
Inventor 郭志武李振勇付昕李秀冬曹喜佳顾晓雅
Owner 苏州华益美生物科技有限公司
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