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Diagnostic kit and method for detecting mutation on human C-Kit gene exon 13

A kit and exon technology, which is applied in the field of diagnostic kits for detecting mutations in exon 13 of the human C-Kit gene, can solve the problems of not being able to meet practical applications, single detection sites, and long time-consuming, and achieve clinical Good application prospect, good specificity and low cost effect

Active Publication Date: 2016-10-26
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 13 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 13 is detected, and the detection site is single, and it is impossible to evaluate multiple mutation sites of exon 13, which cannot meet the actual situation. application needs

Method used

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  • Diagnostic kit and method for detecting mutation on human C-Kit gene exon 13
  • Diagnostic kit and method for detecting mutation on human C-Kit gene exon 13
  • Diagnostic kit and method for detecting mutation on human C-Kit gene exon 13

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1 The present invention detects the kit and detection method of exon 13 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] Wherein "Primer solution" is prepared as follows:

[0039] C13F1 (100nM / ml)

3.125μl

C13F2 (100nM / ml)

3.125μl

C13F3 (100nM / ml)

3.125μl

C13R (100nM / ml)

3.125μl

C13P (100nM / ml)

0.625μl

h 2 o

11.875μl

total

25μl

[0040] Amplification primers and detection probes for c-kit gene exon 13 mutation:

[0041]

[0042] External control primers and probes:

[0043]

[0044] 2. Using the kit of the present invention to detect mutations in ex...

experiment example 1

[0080] Experimental example 1 The accuracy 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 13 mutation of c-kit gene (respectively K642E, V654A, N655K mutation) was selected (for the sequencing results of K642E 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 EP tubes (each spreading area is 500 (maximum) ...

experiment example 2

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

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Abstract

The invention provides a kit for detecting mutation on human C-Kit gene exon 13. The kit comprises primer pairs as shown in SEQ ID NO. 1-4 and a probe as shown in SEQ ID NO. 5. The invention also discloses a method for detecting mutation on human C-Kit gene exon 33. The kit and the method are capable of accurately detecting K642E, V654A and N655K mutation of the human c-kit gene exon 13 and providing a medication guide for GIST patients, and has favorable clinical application prospect.

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 13 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): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/118C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2545/113
Inventor 叶丰肖林步宏
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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