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A DNA point mutation quantitative detection kit

A detection kit and quantitative detection technology, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of low sensitivity, high detection cost, incomplete PNA-LNA clamp PCR clamp, etc.

Active Publication Date: 2021-05-11
THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • A DNA point mutation quantitative detection kit
  • A DNA point mutation quantitative detection kit
  • A DNA point mutation quantitative detection kit

Examples

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Embodiment 1

[0039] 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.

[0040] 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 ...

Embodiment 2

[0050] The kit 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 are compared and tested. The difference between the two methods in terms of amplification sensitivity and mutation detection sensitivity was mainly compared.

[0051] 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;

[0052] 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);

[0053] 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 the method of...

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Abstract

The invention provides a DNA point mutation quantitative detection kit, comprising the following components: 2 outer primers, the deoxynucleotide sequences of which are shown in SEQ ID No.1 and 2, and 2 inner primers, the deoxynucleotide The nucleotide sequence is shown in SEQ ID No.3 and 4 respectively; 1 PNA probe, its deoxynucleotide sequence is shown in SEQ ID No.5; 1 LAN probe, its deoxynucleotide sequence is shown in SEQ ID No.5 As shown in ID No.6; 1 internal standard fluorescent probe, the deoxynucleotide sequence of which is shown in SEQ ID No.7. The kit of the present invention suppresses the wild-type template twice by the PNA probe, greatly suppresses the amplification of the wild-type template, and solves the problem of incomplete clamping of the PNA.

Description

technical field [0001] The invention relates to the field of biological technology, in particular to the technical field of biological gene detection, in particular to a DNA point mutation quantitative detection kit. 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 ampl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12Q1/6848
CPCC12Q1/6848C12Q1/6858C12Q1/6886C12Q2600/156C12Q2600/166C12Q2525/107C12Q2525/113C12Q2537/159C12Q2537/163C12Q2549/119
Inventor 陈之遥缪丽燕赵军黄晨蓉程宗琦朱建国边诣聪李畅张诗超刘林生丁肖梁
Owner THE FIRST AFFILIATED HOSPITAL OF SOOCHOW UNIV
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