数字PCR检测cfDNA中KRAS突变的灵敏度研究

Study on Sensitivity of Digital PCR for Detection of KRAS Mutation in cfDNA

目的该文旨在研究一种适用于数字PCR在KRAS基因突变液态活检中的灵敏度评价方法 ,并通过临床样本的检测结果验证灵敏度算法的可行性。方法 2016年11月基于微滴式数字PCR平台设计引物探针建立KRAS检测方法,确定其检测下限、定量下限、变异系数。2017年1月比较了数字PCR 3种不同检测下限统计算方法。结果成功建立微滴式数字PCR对KRAS突变检测方法。在3种检测下限计算方法结果对比后选择了保守性居中的算法。结论数字PCR是一种灵敏精确的基因突变检测方法,其检测下限可使用基于假阳性率的泊松分布算法进行量化评价,且综合检测性能适用于临床液态活检的需求。

Objective This paper tries to study a sensitivity evaluation method suitable for digital PCR in KRAS gene mutation liquid biopsy, and verify the feasibility of sensitivity algorithm by the test results of clinical samples. Methods In November 2016, a primer probe was designed based on the microdroplet digital PCR platform to establish a KRAS detection method to determine the lower limit of detection, the lower limit of quantitation, and the coefficient of variation. In January 2017, the calculation methods of three different detection lower limits of digital PCR were compared. Results The method of microdroplet digital PCR for KRAS mutation detection was successfully established. A conservative central algorithm was chosen after comparing the results of the three detection lower limit calculation methods. Conclusion Digital PCR is a sensitive and accurate method for detecting gene mutations. The lower limit of detection can be quantified using poisson distribution algorithm based on false positive rate, and the comprehensive detection performance is suitable for clinical liquid biopsy.