Establishment of a method for detecting EGFR gene mutations and its preliminary application
10.3969/j.issn.1671-8348.2014.11.024
- VernacularTitle:一种 EGFR基因突变检测方法的建立及初步应用
- Author:
Qian WANG
;
Kai LUO
- Publication Type:Journal Article
- Keywords:
receptor,epidermal growth factor;
mutation;
gene sequencing;
real-time polymerase chain reaction
- From:
Chongqing Medicine
2014;(11):1351-1353,1356
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a method for detecting the EGFR gene mutations by the real-time fluorescence quantification PCR combined with Sanger sequencing and to preliminarily explore its clinical application value .Methods With EGFR gene hotspot mutations region exon 19 and 21 as the research locus ,the specific amplification and the sequencing primer were designed ,the known wild-type and mutant samples were utilized to construct the corresponding plasmid as the standard substance by the TA clone technique .Then the EGFR gene mutation detection method by the real-time fluorescence quantification PCR combined with Sanger sequencing was established and the methodological and the application evaluation were performed .Results The wild-type and mutant standard plasmids of the EGFR gene exon 19 and 21 were constructed successfully .The EGFR gene mutations detection method of the real-time fluorescence quantification PCR combined with Sanger sequencing was established ,which had high sensitivi-ty(101copies/μL)andgoodrepeatability(intra-assayCVandinter-assayCVofthereal-timefluorescencequantificationPCRofex-on 19 and 21 were 1 .42% /3 .52% and 0 .97% /2 .44% ,respectively ) .20 clinical samples were simultaneously detected by this method and the traditional Sanger sequencing ,the results were completely consistent .Conclusion The EGFR gene mutations detec-tion method of the real-time fluorescence quantification PCR combined with Sanger sequencing is successfully established ,which can be used in the clinical sample detection .
