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Diagnostic kit and method for detecting human C-Kit gene exon 17 mutations

A kit and exon technology, which is applied in the field of diagnostic kits for detecting mutations in human C-Kit gene exon 17, can solve the problem of inability to evaluate multiple mutation sites in exon 17, unfavorable clinical detection, and detection sites. Single point and other problems, to achieve the effect of good clinical application prospect, good specificity and high sensitivity

Inactive Publication Date: 2016-10-12
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 17 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 17 is detected, and the detection site is single, and it is impossible to evaluate multiple mutation sites of exon 17, which cannot meet the actual situation. application needs

Method used

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  • Diagnostic kit and method for detecting human C-Kit gene exon 17 mutations
  • Diagnostic kit and method for detecting human C-Kit gene exon 17 mutations
  • Diagnostic kit and method for detecting human C-Kit gene exon 17 mutations

Examples

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Effect test

Embodiment 1

[0034] Example 1 The present invention detects the kit and detection method of exon 17 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] C17F (100nM / ml)

12.5μl

C17F2 (100nM / ml)

12.5μl

C17F3 (100nM / ml)

12.5μl

C17R (100nM / ml)

12.5μl

C17P (100nM / ml)

2.5μl

H2O

47.5μl

total

100μl

[0040] Amplification primers and detection probes for c-kit gene exon 17 mutation:

[0041]

[0042] External control primers and probes:

[0043]

[0044]

[0045] 2. Using the kit of the present invention to detect mutations in exon...

experiment example 1

[0082] Experimental example 1 The accuracy and specificity detection of kit of the present invention and method

[0083] 1. Sample to be tested

[0084] One case of clinical GIST paraffin tissue samples with known mutation types of c-kit gene exon 17 mutation (respectively D816V, D820H, N822I, N822K mutation) were selected (for the sequencing results of D816V mutation, see figure 1 ) and a known wild-type clinical GIST paraffin tissue sample to detect the mutation type of human c-kit gene.

[0085] 2. Detection method

[0086] The method of the present invention: adopt the kit of embodiment 1, detect according to the method of embodiment 1.

[0087] Sample DNA extraction method: extract with Qiagen kit:

[0088] 1) Use a scalpel to remove the useless paraffin at the tissue border;

[0089] 2) Cutting the paraffin-embedded tissue into 4 μm thick slices;

[0090] 3) Quickly take 2-6 pieces with sterilized tweezers and put them into DNase / RNase Free EP tubes (each spreading ...

experiment example 2

[0109] 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 the human c-kit gene mutation type. The kit comprises primer pairs shown in SEQ ID NO.1-4 and a probe shown in SEQ ID NO.5. The invention further discloses a method for detecting human c-kit gene exon 17 mutations. The kit and method can accurately detect D816V, D820H, N822I and N822K mutations of the human c-kit gene exon 17, provide a medicine guide for GIST patients, and are good in clinic 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 17 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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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/106C12Q2600/118C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2545/113
Inventor 叶丰肖林陈卉娇
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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