Objective To establish a method about detecting KRAS gene mutations through Sanger sequencing combined with Real-time PCR and evaluate its clinical value. Methods A KRAS gene mutations detection method through Sanger sequencing combined with Real-time PCR was established with specific primers targeting the hotspot mutation region (codon12 and codon13), and wild-type plasmid and mutant plasmid were constructed as the standard samples. Then the performance and application of method were assessed. Results The standard plasmids were constructed successfully. A KRAS gene mutations detection method through Sanger sequencing combined with Real-time PCR was established successfully which owned high sensitivity (9.39×101 copies/uL) and good repeatability(intra-assay CV and inter-assay CV of the Real-time PCR were 1.60%and 2.54%, respectively). There was no difference between traditional Sanger sequencing and Sanger sequencing combined with Real-time PCR to detect KRAS gene mutations in 40 clinical samples. Conclusion The established mutation detection method through Sanger sequencing combined with Real-time PCR can be used in clinical samples to detect KRAS gene mutations.% 目的建立一种KRAS基因实时荧光定量PCR-Sanger测序突变检测方法，并初步探讨其临床应用价值.方法以KRAS基因热点突变区域12、13密码子为研究位点设计特异性扩增、测序引物，利用已知野生型、突变型样品以TA克隆技术构建相应质粒作为标准品，建立KRAS基因实时荧光定量PCR-Sanger测序突变检测方法，并进行方法学和应用评估.结果成功构建了KRAS基因12、13密码子野生型、突变型质粒.建立了KRAS基因实时荧光定量PCR-Sanger测序突变检测方法，该方法灵敏度高(9.39×101 copies/uL)，重复性好(实时荧光定量PCR部分批内、批间变异系数分别为1.60%、2.54%).该法与传统Sanger测序法同时检测40例临床样品，结果符合率为100%.结论本研究成功建立了可用于临床样品检测的KRAS基因实时荧光定量PCR-Sanger测序突变检测方法.
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