Establishment of EGFR-T790M mutation detection method for non-small cell lung cancer based on droplet digital PCR
10.3760/cma.j.cn114452-20200827-00692
- VernacularTitle:基于微滴式数字PCR的非小细胞肺癌EGFR-T790M突变检测方法的建立
- Author:
Shanshan DING
;
Hongxin MA
;
Xingguo SONG
;
Xiaohan DONG
;
Li XIE
;
Xianrang SONG
- From:
Chinese Journal of Laboratory Medicine
2021;44(4):335-340
- CountryChina
- Language:Chinese
-
Abstract:
Objective:Use the droplet digital PCR (ddRCR) technology to establish, optimize and evaluate the method of EGFR-T790M mutation detection.Methods:The relevant probes and primers were designed for EGFR-T790M mutations. The ddPCR reaction system was established, the optimal annealing temperature was set and the basic performance of the method was tested. On this basis, from January 2019 to October 2019, 72 cell-free DNA (cfDNA) samples from NSCLC patients were collected from Shandong Cancer Hospital Affiliated to Shandong First Medical University, and clinically verified. The consistency of the gene mutation detections with Bole ddPCR products was analyzed using Kappa test.Results:The ddPCR reaction system was established and optimized. Linear evaluation showed the R2 value was greater than 0.99. Using ddPCR, the blank detection limit was determined to be the numbers of mutant droplets≥2, with excellent specificity. For the sensitivity analysis, the lower limit of mutation detection was determined to be at least 0.05%. In the repeatability and inter-assay precision tests, the results had a coefficient of variation( CV)<20%. The relative deviation of the results was within the range of±10% for the accuracy analysis. Using the established T790M mutation detection method, 72 samples from the NSCLC patients were tested for genetic mutation in cfDNA, and the overall agreement with the Bole ddPCR products was 91.67% (66/72, Kappa=0.749; P<0.001). Conclusion:Using ddPCR, the method of EGFR-T790M mutation detection for NSCLC was successfully established.
