Quantitative detection method of DNA point mutation

A quantitative detection method and detection method technology, which are applied in the direction of determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of low sensitivity, incomplete PNA-LNA clamp PCR clamp, and easy contamination of two-step nested PCR.

Active Publication Date: 2018-06-15
THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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Problems solved by technology

[0004] The purpose of the present invention is to solve the problems of low sensitivity, high detection cost, and difficulty in quantification of commonly used clinical gene mutation detection methods, and also to solve the problems of incomplete clamping of PNA-LNA clamped PCR and easy contamination of two-step nested PCR

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  • Quantitative detection method of DNA point mutation
  • Quantitative detection method of DNA point mutation
  • Quantitative detection method of DNA point mutation

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Embodiment 1

[0056] The present invention establishes a highly sensitive point mutation detection method, in order to evaluate the detection of clinical samples by the method. We applied this method to the detection of EGFR L858R mutation sites related to targeted drugs in plasma and tissue samples of non-small cell lung cancer.

[0057]EFGR is a common driver gene in non-small cell lung cancer, and its mutations mainly occur in exons 18 / 19 / 20 / 21. EGFR L858R (NCBI accession number: NG_007726.1) is the 858th amino acid encoded by exon 21 of the EGFR gene, which is changed from leucine to arginine. This mutation can cause the activation of the downstream pathway of EGFR, and then lead to the occurrence of tumors . Iressa (Iressa) and Tarceva (Tarceva) are common EGFR tyrosine kinase inhibitors, and are currently commonly used targeted drugs for the treatment of non-small cell lung cancer. A large number of studies have shown that EGFR L858R is one of the sensitive mutations of Iressa and T...

Embodiment 2

[0066] The method of the present invention is the development and improvement of the PNA-LNA clamped PCR technique, so the method of the present invention and the PNA-LNA clamped PCR technique have been compared with each other. The difference between the two methods in terms of amplification sensitivity and mutation detection sensitivity was mainly compared.

[0067] 1. The primers and sequences of the experimental group and the matched group 1 of the present invention are the same as in Example 1;

[0068] 2. Preparation of standard substances with known content: NCI-H1975 cells containing mutant EGFR L858R and H293T cells containing wild-type EGFR L858R were purchased from American Type Culture Collection (Rockville, USA);

[0069] The EGFR L858R wild-type and mutant DNA in the cells were quantified by digital PCR, and used as standards of known content of the wild-type and mutant DNA, respectively. The EGFR L858R mutant cell DNA of known concentration was diluted to 10 by...

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Abstract

The invention provides a quantitative detection method of DNA point mutation. A PNA probe is used for inhibiting a wild-type template for two times, so that amplification of the wild-type template isgreatly prevented, and the problem of incomplete clamping of PNA is solved. At the same time, difference of Tm values of amplimers is utilized, and the annealing temperature during amplification is controlled, so that nested PCT can be carried out in a single tube, and the amplification intensity and amplification specificity are improved. Therefore, amplification of a clinic sample with extremelylow copy number is possible. Finally, through real-time fluorescent closed-tube detection, real-time detection is achieved, cross contamination of samples is prevented, and quantification for mutation is achieved. Furthermore, according to the method, only a common real-time fluorescent PCR apparatus is needed, and the detection result can be obtained in 2 h. The reagent is low in cost, and the samples can be fast detected at low cost. In general, through successful establishment of the method, a new detection method which is high in sensitivity and specificity, is simple and fast, and is free of pollution and low in cost is provided for clinic DNA point mutation.

Description

technical field [0001] The invention relates to the field of biological technology, in particular to the technical field of biological gene detection. Background technique [0002] Methods commonly used in clinical detection of gene mutations mainly include Sanger sequencing, pyrosequencing, restriction fragment length polymorphism (Restriction Fragment Length Polymorphism, RFLP), etc. These methods have very low sensitivity and can only detect more than 5%~ 20% of mutation samples are difficult to meet the detection requirements of clinical samples. Real-time fluorescent quantitative PCR (Real-time PCR, RT-PCR), amplification retardation mutation system (Amplification Refractory Mutation System, ARMS), high-resolution melting curve (High-resolution Melting, HRM) method, mutation enrichment PCR (Mutant Enrichment Compared with the Sanger sequencing method, the sensitivity of methods such as multiplex PCR amplification method (Coamplification at lower denaturation temperatur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12Q1/6848
CPCC12Q1/6848C12Q1/6858C12Q1/6886C12Q2600/156C12Q2600/166C12Q2525/107C12Q2525/113C12Q2537/159C12Q2537/163C12Q2549/119
Inventor 陈之遥缪丽燕赵军张华包健安陈蓉刘筱雪张诗超丁成马晟薛领
Owner THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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