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Probe, primer and kit for detecting 7 mutations of human CKIT gene

A technology for detecting probes and kits, which is applied in the biological field and can solve problems such as low detection efficiency, long detection time, and poor accuracy

Active Publication Date: 2016-03-30
武汉海吉力生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the problems of the existing CKIT gene mutation detection methods, such as low detection efficiency, easy sample contamination, long detection time, poor accuracy, and difficulty in discrimination, the present invention provides primers and probes for detecting seven mutations of human CKIT gene

Method used

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  • Probe, primer and kit for detecting 7 mutations of human CKIT gene
  • Probe, primer and kit for detecting 7 mutations of human CKIT gene
  • Probe, primer and kit for detecting 7 mutations of human CKIT gene

Examples

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Effect test

Embodiment 1

[0084] 1. Primers and probes of the detection kit for detecting 7 mutations of human CKIT gene:

[0085] According to the wild-type sequence of the CKIT gene (NCBIReferenceSequence: NM_000222.2) published in the NCBI database, the mutation site of exon 13 V654A (CM6), the mutation site of exon 14 T670I (CM7), and the mutation site of exon 17 D816V (CM1 ), D816H (CM2), N822K (CM3 and CM4) and Y823D (CM5) mutation sites as reference, design specific CM1 mutation primers and probes (see Table 2), specific CM2 mutation primers and probes (see Table 3), specific CM3 mutation primers and probes (see Table 4), specific CM4 mutation primers and probes (see Table 5), specific CM5 mutation primers and probes (see Table 6), specific CM6 mutation primers and probes (see Table 7) and specific CM7 mutation primers and probes (see Table 8). Using the mutant plasmid and wild-type plasmid constructed by genetic engineering as templates, a real-time fluorescent PCR mutation detection system wa...

Embodiment 2

[0168] Example 2 The human CKIT gene mutation detection kit of the present invention is used to detect clinically collected tissue samples.

[0169] In this example, tissue samples from patients diagnosed as gastrointestinal stromal tumors were collected and genomic DNA was extracted from them. The human CKIT gene mutation detection kit was used to detect the mutation status of the CKIT gene in the samples to be tested. The specific process is as follows:

[0170] (1) Sample genomic DNA extraction

[0171] Paraffin-embedded tissue samples were extracted using QIAampDNAFFPETissueKit (CatNo.56404). Extract the genomic DNA of the sample to be tested according to the instructions, and use the ultraviolet spectrophotometer to detect the concentration and DNA quality of the extracted DNA sample. The concentration of DNA is required to be ≥10ng / μL, and the quality of DNA OD260 / 280 is between 1.8 and 2.0. Genomic DNA that meets the requirements was diluted to 2-10 ng / μL with TE (10...

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Abstract

The invention discloses a probe, a primer and a kit for detecting 7 mutations of a human CKIT gene. 7 groups of primers and probes CM1 to CM7 are provided; in each group, a mutation forward primer can be combined with a CKIT gene conserved sequence, and a mutation reverse ARMS primer can be specifically combined with a corresponding mutation sequence so as to amplify the mutation sequence; the detection probes can be combined with amplified fragments, and blocking probes can be specifically combined with a wild-type sequence corresponding to a mutation site so as to inhibit wild-type non-specific amplification. According to the probe, the primer and the kit which are disclosed by the invention, a specific mutation primer and probe blocking technology is adopted; 7 mutations of the CKIT gene can be accurately detected; the established real-time fluorescence PCR (Polymerase Chain Reaction) amplified reaction system kit is convenient to rapidly detect the mutations of the CKIT gene, is simple to operate, and is easy in reading of a result; a detection method is high in sensitivity, and 50-copy mutants can be stably detected out; specificity is good, 20ng wild-type genome DNA has no phenomenon of nonspecific amplification, and detectability reaches 1 percent.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a probe, primer and kit for detecting seven mutations of human CKIT gene. Background technique [0002] Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract, and its incidence is increasing year by year. It mainly occurs in the gastrointestinal tract, omentum and mesentery, accounting for 0.2% of all gastrointestinal tumors and 80% of gastrointestinal sarcomas, and C-kit (CD117) is usually positively expressed. For the vast majority of patients, gene mutation is the key factor for tumor formation, among which CKIT gene mutation accounts for 80-85%, PDGFRα mutation accounts for 7%, and wild type (no mutation detected) accounts for 10-15%. GIST patients are prone to recurrence after surgical resection, with a median recurrence time of about 2 years, and only 10% of patients have no recurrence in long-term follow-up. [0003] CKIT is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/106C12Q2600/156C12Q2531/113C12Q2535/137C12Q2537/163C12Q2561/101C12Q2561/113
Inventor 朱峰裴冰雪段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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