PIK3CA基因突变数字PCR参考测量方法研究

Research on Digital PCR Reference Measurement Method of PIK3CA Gene Mutation

PIK3CA突变检测是实现肿瘤个体化治疗的重要预测因子。以PIK3CA为目的基因,选择E542K、E545K、H1047R三个热点突变,建立对其准确定量检测的微滴式数字聚合酶链式反应(ddPCR)方法。优化退火温度、引物探针浓度等条件,对方法特异性、线性范围、重复性等方面进行考察。结果表明,建立的ddPCR检测方法精密度好,在突变丰度(0.05~82.75)%范围内相对标准偏差值介于(0.392~14.031)%,准确性高,与重量法的相关系数达到99.91%、99.98%、99.94%;在突变丰度0.2%以上,方法的重复性可达5%以内;在突变丰度0.2%以下,重复性保持在15%以下。在20μL反应体系中3个热点突变的空白限分别为0.03%、 0.04%、0.04%,检测下限和定量下限均为0.05%。因此,所建立的PIK3CA基因E542K、E545K及H1047R位点突变的ddPCR参考测量灵敏度高,重复性好,在癌症诊断、个体化用药指导和预后方面具有良好的应用。

The mutation of phosphatidylinositol 3-kinase catalytic subunitɑ(PIK3CA)gene is closely related to the occurrence and development of a variety of tumors.It has a high mutation rate in human tumors and is expected to be a new target for accurate early diagnosis and treatment of tumors.Point,provide a theoretical basis for individualized treatment.At present,PIK3CA mutation detection has become an important predictor of individualized tumor therapy.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.Optimize the annealing temperature,primer probe concentration and other conditions,and investigate the method specificity,linear range,repeatability and other aspects.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.