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Diagnostic kit for detecting human C-Kit gene number 11 exon mutation and method

A kit and exon technology is applied in the field of diagnostic kits for detecting mutations in human C-Kit gene exon 11, which can solve the problem of inability to evaluate multiple mutation sites in exon 11, unfavorable clinical detection, and detection sites. single problem, to achieve the effect of good clinical application prospect, good specificity and high sensitivity

Active Publication Date: 2016-09-21
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the current detection of mutations in exon 11 of the human C-Kit gene is mainly through traditional sequencing methods, which are time-consuming and costly, and do not use ordinary clinical testing.
Even if there are a few reports using the fluorescent quantitative PCR method, only one of the sites of exon 11 is detected, and the detection site is single, and it is impossible to comprehensively evaluate the multiple mutation sites of exon 11, which cannot meet the actual situation. application needs

Method used

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  • Diagnostic kit for detecting human C-Kit gene number 11 exon mutation and method
  • Diagnostic kit for detecting human C-Kit gene number 11 exon mutation and method
  • Diagnostic kit for detecting human C-Kit gene number 11 exon mutation and method

Examples

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Effect test

Embodiment 1

[0034] Example 1 The present invention detects the kit and detection method of exon 11 mutation of C-Kit gene

[0035] One, the composition of kit of the present invention

[0036] PCR amplification reagent (1 serving):

[0037] water

14.85μl

10XPCR buffer

2.5μl

MgCl2 25mM

0.75μl

UTP PLUS

0.5μl

Primer solution

1μl

UNG

0.2μl

Taq

0.2μl

overall system

20μl

[0038] Amplification primers and detection probes for exon 11 mutation of c-kit gene:

[0039] The primers are as follows: (primers are all in the 5'-3' direction)

[0040] (Note: The serial number consists of three numbers + one letter, where the first two numbers represent the 5XX amino acid; the third number represents the nucleotide number of the amino acid codon; the letter represents the mutation to a certain nucleoside acid.

[0041] For example, 471A represents the mutation of the first nucleotide of the triplet codon ...

experiment example 1

[0112] Experimental example 1 The accuracy detection and specificity detection of kit of the present invention and method

[0113] 1. Sample to be tested

[0114] Select 1 case of clinical GIST paraffin tissue samples whose mutation type is known to be mutation of exon 11 of c-kit gene (a total of 90 cases, involving all amino acids that can be detected by the primers of the present invention; wherein the mutation type of the 547th amino acid is shown in figure 1 ) and a known wild-type clinical GIST paraffin tissue sample to detect the mutation type of human c-kit gene.

[0115] 2. Detection method

[0116] The method of the present invention: adopt the kit of embodiment 1, detect according to the method of embodiment 1.

[0117] Sample DNA extraction method: extract with Qiagen kit:

[0118] 1) Use a scalpel to remove the useless paraffin at the tissue border;

[0119] 2) Cutting the paraffin-embedded tissue into 4 μm thick slices;

[0120] 3) Quickly take 2-6 pieces wi...

experiment example 2

[0139] Experimental example 2 sensitivity analysis of kit of the present invention and method

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Abstract

The invention provides a diagnostic kit for detecting human C-Kit gene number 11 exon mutation. The diagnostic kit comprises primer pairs shown as SEQ ID NO.1-283 and a probe shown as SEQ ID NO.284. The invention further discloses a method for detecting human C-Kit gene number 11 exon mutation. The kit and the method can accurately detect all mutation types of human C-Kit gene number 11 exon, provide guidance of medication usage for GIST patients and have good clinic application prospects.

Description

technical field [0001] The invention belongs to the field of gene detection, and in particular relates to a diagnostic kit and method for detecting the mutation of exon 11 of human C-Kit gene. Background technique [0002] Gastrointestinal stromal tumors (GIST) are a type of tumor originating from the mesenchymal tissue of the gastrointestinal tract, and are the most common mesenchymal tumors of the gastrointestinal tract. difference. Mazur et al. proposed in 1983 that GIST is an independent tumor type different from leiomyoma and neurogenic tumors, and its direct cause is the gain-of-function mutation of oncogene. [0003] Early treatment of GIST mainly relies on surgical resection, but even if the tumor is completely resected, many patients die of tumor recurrence and metastasis, and traditional radiotherapy and chemotherapy have no obvious effect. The emergence of targeted drugs, especially the tyrosine kinase inhibitor imatinib, has fundamentally changed the treatment ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/156C12Q2531/113
Inventor 肖林叶丰陈卉娇
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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