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Primers and probes and kit for detecting human PDGFRA gene D842V mutation

A technology for detecting probes and kits, which can be used in DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc. It can solve the problems of long detection time, low detection efficiency, and high false positive rate, and achieve rapid detection, High sensitivity and good specificity

Active Publication Date: 2016-04-06
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 low sensitivity, long detection time, low detection efficiency, easy contamination of samples and high false positive rate in existing detection technologies, the present invention provides primers and probes for detecting the D842V mutation of human PDGFRA gene with high sensitivity, Strong specificity, effectively avoiding false positives

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  • Primers and probes and kit for detecting human PDGFRA gene D842V mutation
  • Primers and probes and kit for detecting human PDGFRA gene D842V mutation
  • Primers and probes and kit for detecting human PDGFRA gene D842V mutation

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Embodiment 1

[0054] 1. Mutation detection primers and probes, quality control primers and probe sequence design:

[0055] According to the wild-type sequence of PDGFRA gene (NCBIReferenceSequence: NM_006206.4) published in the NCBI database, with the D842V mutation site in exon 18 (see Table 1) as a reference, specific mutation primers were designed with PrimerPremier5 and PrimerExpress3.0 bioinformatics software and probes (see Table 2). The mutant plasmid constructed by genetic engineering (namely PM1 plasmid, inserted gene fragment>gi|568815594:54285801-54286200, and D842V base change occurred) and wild-type plasmid (insert gene fragment>gi|568815594:54285801-54286200) were used as templates , established a real-time fluorescent PCR mutation (Mutation) detection system to achieve high-sensitivity and high-specificity detection of the PDGFRA gene D842V mutation site.

[0056] Table 2: Specific Primer and Probe Combinations for Detection of Human PDGFRA Gene D842V Mutation

[0057] ...

Embodiment 2

[0098] The human PDGFRA gene D842V mutation detection kit of the present invention is used to detect the clinically collected tissue samples.

[0099] In this example, paraffin-embedded tissue samples from patients diagnosed as gastrointestinal stromal tumor (GIST) by clinicopathology were collected, and genomic DNA was extracted therefrom. Use the human PDGFRA gene D842V mutation detection kit to detect whether there is a PDGFRA gene D842V mutation in the sample to be tested. Specific steps are as follows:

[0100] (1) Extraction of sample genomic DNA

[0101] For the extraction of paraffin-embedded tissue samples, QIAampDNAFFPETissueKit (CatNo.56404) was used to extract the genomic DNA of the samples to be tested according to the instructions. The extracted DNA samples were tested for concentration and DNA quality with a UV spectrophotometer. The concentration of DNA should be ≥10ng / μL, the quality OD260 / 280 of DNA is between 1.8 and 2.0. The genomic DNA that meets the r...

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Abstract

The invention discloses primers and probes and a kit for detecting human PDGFRA gene D842V mutation. The primers and probes comprise the mutation upstream ARMS primer PM1-F which is specially combined with a D842V (2525A>T) locus mutation sequence to selectively amplify the mutation sequence, the mutation downstream primer PW1-R which is combined with a conserved sequence of the PDGFRA gene, the detection probe PW1-P of which the 5' end is marked with an FAM signal and the 3' end is marked with MGB and the blocking probe PW1-B of which the 5' end is modified through double deoxidation and the 3' end is marked with the MGB, wherein the detection probe PW1-P can be combined with an amplified fragment, and the blocking probe PW1-B can be specially combined with a D842V locus wild type sequence to inhibit wild type nonspecific amplification. The primers and probes are high in specificity and good in sensitivity, and the detectability reaches up to 1%; the kit composed of the primers and probes is accurate and readable in detection result, easy and rapid to operate and wide in application range.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a primer, a probe and a kit for detecting the D842V mutation of human PDGFRA 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. Mainly occurs in the gastrointestinal tract, omentum and mesentery, accounting for 0.2% of all gastrointestinal tumors, accounting for 80% of gastrointestinal sarcomas, C-kit receptor (also known as CD117) gene is usually positive. For the vast majority of patients, gene mutation is the key to tumor formation, among which CKIT gene mutation accounts for 80-85%, PDGFRα mutation accounts for 7%, and wild-type (no mutation is 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 after long-term ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2545/101C12Q2545/113C12Q2563/107
Inventor 朱峰杨惠娟段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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