基于微滴式数字聚合酶链式反应检测EGFR基因Ex19del和L858R突变方法的优化

Optimization of EGFR gene Ex19del and L858R mutation detection based on Droplet Digital Polymerase Chain Reaction

目的采用微滴式数字聚合酶链式反应(ddPCR)技术建立并优化检测EGFR基因敏感突变[19外显子缺失(Ex19del)和21外显子L858R突变(L858R)]的方法。方法应用新型位点-参考探针法检测Ex19del突变,采用传统野生-突变探针法进行L858R突变检测分析。应用温度梯度法优化2种突变检测方法的退火温度,并分析其特异度和灵敏度等。结果实现Ex19del突变多重位点缺失一次性检测,确定最佳退火温度为60.1℃,所建立方法具有良好的特异度,灵敏度分析显示突变检测下限为0.5%;L858R突变检测方法退火温度设定为58.2℃,特异度良好,通过灵敏度检测确定检测下限为0.1%。结论应用ddPCR成功建立并优化EGFR基因Ex19del和L858R敏感性突变的检测方法,为ddPCR的临床应用提供更多参考与支持。

Objective To establish and optimize a method for detection of EGFR Ea19del and L858R mutations by using droplet digital PCR(ddPCR).Methods A new Target-Reference probe method was applied for detection of the Er19del mutation,whereas the traditional Wild-Mutation probe method was used for L858R mutation detection analysis.The annealing temperatures for the two mutation detection methods were optimized using temperature gradient method,and their specificity and sensitivity were analyzed,respectively.Results The one-time detection of multiple site deletions for Er19del mutations was successfully achieved,with an optimal annealing temperature of 60.1 C,as well as the favorable specificity,and a minimum detection limit of 0.5%for sensitivity analysis.The annealing temperature of the L858R mutation detection method was set at 58.2 C,possessing excellent specificity.The detection limit was determined to be 0.1%through sensitivity detection.Conclusion The sensitive method for the detection of EGFR gene Er19deland L858R mutations is successfully established and optimized based on ddPCR.