首页|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

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

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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 应用新型位点一参考探针法检测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.
Optimization of EGFR gene Ex19del and L858R mutation detection based on Droplet Digital Polymerase Chain Reaction
Objective To establish and optimize a method for detection of EGFR Ex19del and L858R mutations by using droplet digital PCR(ddPCR).Methods A new Target-Reference probe method was applied for detection of the Ex19del mutation,whereas the traditional Wild-Mutation probe method was used for L858R mutation detection analysis.The an-nealing 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 Ex19del mutations was successfully achieved,with an optimal annealing temperature of 60.1 ℃,as well as the favorable specificity,and a minimum detection limit of 0.5％for sensitivity analysis.The annealing temperature of the L858R muta-tion detection method was set at 58.2 ℃,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 Ex19deland L858R muta-tions is successfully established and optimized based on ddPCR.

epidermal growth factor receptor geneEx19delL858Rtarget-reference probe methodwild-mutation probe methoddroplet digital PCR

丁姗姗、宋兴国

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山东省肿瘤防治研究院(山东省肿瘤医院)检验科,山东第一医科大学(山东省医学科学院),山东济南 250117

表皮生长因子受体基因 Ex19del L858R 位点-参考探针法 野生-突变探针法 微滴式数字PCR

山东省自然科学基金创新发展联合基金山东省中医药科技项目泰山学者青年专家项目山东第一医科大学附属肿瘤医院"青苗"计划

ZR2023LZL011M2023-013tsqn202312366CH-SFMU-QM-20210005

2024

10.16073/j.cnki.cjcpt.2024.17.02

中华肿瘤防治杂志
中华预防医学会 山东省肿瘤防治研究院

中华肿瘤防治杂志

CSTPCD北大核心
影响因子：1.292
ISSN：1673-5269
年,卷(期)：2024.31(17)