CAJ | 학술논문

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.
목적건립일충KRAS기인실시형광정량PCR-Sanger측서돌변검측방법,병초보탐토기림상응용개치.방법이KRAS기인열점돌변구역12、13밀마자위연구위점설계특이성확증、측서인물,이용이지야생형、돌변형양품이TA극륭기술구건상응질립작위표준품,건립KRAS기인실시형광정량PCR-Sanger측서돌변검측방법,병진행방법학화응용평고.결과성공구건료KRAS기인12、13밀마자야생형、돌변형질립.건립료KRAS기인실시형광정량PCR-Sanger측서돌변검측방법,해방법령민도고(9.39×101 copies/uL),중복성호(실시형광정량PCR부분비내、비간변이계수분별위1.60%、2.54%).해법여전통Sanger측서법동시검측40례림상양품,결과부합솔위100%.결론본연구성공건립료가용우림상양품검측적KRAS기인실시형광정량PCR-Sanger측서돌변검측방법.
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.