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Diagnosis kit and method for detecting human c-kit gene exon 9 mutation

A kit and human technology, applied in the directions of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problem of inability to evaluate multiple mutation sites in exon 9, time-consuming, and detection of sites. Single point and other problems, to achieve the effect of good clinical application prospects, rapid detection, and high sensitivity

Active Publication Date: 2016-08-17
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 9 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 site of exon 9 is detected, and the detection site is single, and it is impossible to evaluate multiple mutation sites of exon 9, which cannot meet the actual situation. application needs

Method used

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  • Diagnosis kit and method for detecting human c-kit gene exon 9 mutation
  • Diagnosis kit and method for detecting human c-kit gene exon 9 mutation
  • Diagnosis kit and method for detecting human c-kit gene exon 9 mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 The present invention detects the kit and detection method of exon 9 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

MgCl 2 (25mM)

0.75μl

UTP PLUS

0.5μl

Primer solution

1μl

UNG

0.2μl

Taq

0.2μl

overall system

20μl

[0038] Wherein "Primer solution" is prepared as follows:

[0039] C9F (100nM / ml)

3.125μl

C9F2 (100nM / ml)

3.125μl

C9R (100nM / ml)

3.125μl

C9P (100nM / ml)

0.625μl

H2O

15μl

total

25μl

[0040] The amplification primers and detection probes for mutations in exon 9 of the c-kit gene are as follows:

[0041]

[0042] External control primers and probes:

[0043]

[0044] 2. Using the kit of the present invention to detect mutations in exon 9 of the C-Kit ...

experiment example 1

[0080] Experimental example 1 The accuracy and specificity detection of kit of the present invention and method

[0081] 1. Sample to be tested

[0082] One case of clinical GIST paraffin tissue samples with known mutation type of exon 9 mutation of c-kit gene (respectively Y503_F504insAH, A501_Y502dup, A502_Y503dup mutation) was selected (for the sequencing results of Y503_F504insAH mutation, see figure 1 ) and a known wild-type clinical GIST paraffin tissue sample to detect the mutation type of human c-kit gene.

[0083] 2. Detection method

[0084] The method of the present invention: adopt the kit of embodiment 1, detect according to the method of embodiment 1.

[0085] Sample DNA extraction method: extract with Qiagen kit:

[0086] 1) Use a scalpel to remove the useless paraffin at the tissue border;

[0087] 2) Cutting the paraffin-embedded tissue into 4 μm thick slices;

[0088] 3) Quickly take 2-6 pieces with sterilized tweezers and put them into DNase / RNase Free ...

experiment example 2

[0107] Experimental example 2 sensitivity analysis of kit of the present invention and method

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Abstract

The invention discloses a kit for detecting human c-kit gene mutation type. The kit comprises a primer pair disclosed as SEQ ID NO.1-3 and a probe disclosed as SEQ ID NO.4. The invention also discloses a method for detecting human c-kit gene exon 9 mutation. The kit and method disclosed by the invention can be used for accurately detecting Y503_F504insAH, A501_Y502dup or A502_Y503dup mutation of human c-kit gene exon 9, thereby providing instructions for medication of patients with GIST (gastrointestinal stromal tumor), and having favorable clinical 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 9 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 o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/118C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2545/113
Inventor 叶丰陈卉娇步宏
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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