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Kit for detecting EGFR T790M locus mutation

An EGFRT790M, site mutation technology, applied in the field of molecular biology, can solve the problem of not being found, and achieve the effects of rapid detection, good stability and high sensitivity

Inactive Publication Date: 2019-04-19
WUHAN CMLABS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This patent uses the combination of RNase H2 and PCR technology to develop a detection for the EGFR T790M mutation site gene. At present, there is no related technology that applies this method to the EGFR T790M site gene detection.

Method used

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  • Kit for detecting EGFR T790M locus mutation
  • Kit for detecting EGFR T790M locus mutation

Examples

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Embodiment 1

[0038] In this embodiment, primers and probe sequences for the detection of gene mutations at the EGFR T790M site were designed. The probe sequence for mutation detection is SEQ ID NO.3, and the specific sequence is as follows:

[0039] 5'-CCGAAGGGCATGAGCTGCaTGAT-x, the sequence is shown in SEQ ID NO.1;

[0040] 5'-CCGAAGGGCATGAGCTTCA-3', the sequence is shown in SEQ ID NO.2;

[0041] FAM-CACGGTGGAGGTGAGGCAGATG-BHQ1, the sequence is shown in SEQ ID NO.3.

[0042] The difference between the above primers and ordinary primers is that the patent of the present invention adopts the dual-enzyme system amplification method, that is, the RNase H2 and Taq DNA polymerase dual-enzyme system, so there are changes on the basis of the ordinary primers, that is, the primer The 3' end of the sequence SEQ ID NO.1 is connected with a spacer x, so that the primer can specifically bind to the template sequence, and can be specifically amplified after RNaseH2 shearing; The optimized design, an...

Embodiment 2

[0044] In this example, a kit for detecting the mutation of the EGFR T790M locus gene is designed. In order to ensure the reliability of the detection system, the patent of the present invention introduces positive and negative quality control products.

[0045] The specific design of the kit is as follows:

[0046] (1) PCR reaction master mix: components include PCR buffer, 4 kinds of dNTPs, probe SEQ ID NO.3 and general human internal standard probe, primers consist of SEQ ID NO.1 and SEQ ID NO.2 and general Internal standard primers are mixed;

[0047] (2) Enzyme mixture: the components include RNase H2 and Taq DNA polymerase, the enzyme activity concentration ratio of RNase H2 and Taq DNA polymerase is 1:50;

[0048] (3) Negative quality control: a plasmid template negative for the constructed EGFR T790M site mutation;

[0049] (4) Positive quality control: the constructed plasmid template that is positive for the EGFR T790M site mutation;

[0050] The above four reagen...

Embodiment 3

[0052] In this example, a kit for detecting the mutation of the EGFR T790M locus gene is designed. In order to ensure the reliability of the detection system, the patent of the present invention introduces positive and negative quality control products.

[0053] The specific design of the kit is as follows:

[0054] (1) PCR reaction master mix: components include PCR buffer, 4 kinds of dNTPs, probe SEQ ID NO.3 and general human internal standard probe, primers consist of SEQ ID NO.1 and SEQ ID NO.2 and general Internal standard primers are mixed;

[0055] (2) Enzyme mixture: the components include RNase H2 and Taq DNA polymerase, and the enzyme activity concentration ratio of RNase H2 and Taq DNA polymerase is 1:250;

[0056] (3) Negative quality control: a plasmid template negative for the constructed EGFR T790M site mutation;

[0057] (4) Positive quality control: the constructed plasmid template that is positive for the EGFR T790M site mutation;

[0058] The above four r...

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Abstract

The invention provides a novel kit for detecting EGFR T790M locus gene mutation. A double-phase enzyme system RNase H2 and a DNA polymerase technology are utilized; due to the adoption of a double-enzyme system amplification method, change is made on the basis of a common primer, that is, a spacer arm x is connected to the 3' terminal of a primer sequence SEQ ID NO.1, and therefore a primer can bespecifically combined with a template sequence and can be specifically amplified after RNase H2 is sheared; a primer probe for an EGFR T790M mutation locus is designed, and a fluorescent PCR and closed detection are used, so that the probability of later contamination is reduced; the kit is high in sensitivity, quick in detection and good in stability, and the closed detection reduces the probability of later contamination.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a kit for detecting mutations at EGFR T790M sites. Background technique [0002] In recent years, Molecular Targeted Therapy (Molecular Targeted Therapy) with the epidermal growth factor receptor (Epidermal Growth Factor Receptor, EGFR) as the therapeutic target has received widespread attention from the tumor community at home and abroad. Among them, the EGFR tyrosine kinase inhibitor Genfitinib ( Also known as Iressa) and Erlotinib (also known as Tarceva) have been approved by the US Food and Drug Administration (FDA) for the treatment of advanced non-small cell lung cancer (NSCLC), and Gefitinib has also been approved by 27 countries including Japan and Australia. my country also approved Genfitinib in February 2005 to enter clinical practice. [0003] Data show that a large number of clinical studies have confirmed that EGFR mutation positive patients have an effective rate of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/106C12Q2600/156
Inventor 徐志勇秦伟
Owner WUHAN CMLABS CO LTD
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