Research on Digital PCR Reference Measurement Method of PIK3CA Gene Mutation
WANG Lei-lei1,2,WANG Xia2,XING De-chun1,DONG Lian-hua2,YANG Jing-ya1
1. College of Food Sciences & Technology, Shanghai Ocean University, Shanghai 201306, China
2. Frontier Metrology Science Center, National Institute of Metrology, Beijing 100029, China
Abstract：PIK3CA mutation detection is an important predictor for realizing individualized treatment of tumors. Therefore, using PIK3CA as the target gene, three hotspot mutations, E542K, E545K, and H1047R, were selected to establish a droplet digital polymerase chain reaction (ddPCR) method for accurate and quantitative detection. The annealing temperature, primer probe concentration and other conditions were optimized, and the specificity, linear range and repeatability of the method were investigated. The results show that the established ddPCR detection method has good precision, the relative standard deviation value is between 0.392%~14.031% in the abundance range of 0.05%~82.75%, and the accuracy is high. The correlation coefficient with gravimetric method reach 99.91%, 99.98%, 99.94%, when the mutation abundance is above 0.2%, the repeatability of the method can reach within 5%, and when the mutation abundance is below 0.2%, the repeatability remains below 15%. The limit of blank of the three hot spot mutations in the 20μL reaction system are 0.03%, 0.04%, 0.04%, and the lower limit of detection and lower limit of quantification are both 0.05%. Therefore, the ddPCR reference measurement of PIK3CA gene E542K, E545K and H1047R mutations has high sensitivity and good repeatability, and has good applications in cancer diagnosis, individualized medication guidance and prognosis.
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2023, Vol. 44 
