摘要
以探针熔解曲线技术为平台,结合锁核酸(LNA)修饰探针建立熔解曲线分析(MCA)体系(MCA-LNA体系),实现一管同时检测Kras基因中7个热点突变;并通过对探针的改良达到富集突变型的效果,使其分析灵敏度得到明显提升,达0.05%~0.5%.利用MCA-LNA体系对72份临床结直肠癌标本进行Kras突变检测,共检出24份阳性标本,与普通探针MCA体系相比多检出5份突变阳性标本,经扩增阻碍突变系统(ARMS)验证为突变型.由上述结果可见MCA-LNA体系能提高检测限和阳性标本检出率,更有利于精准基因筛查和指导靶向治疗.
We established a melting curve analysis(MCA)system using locked nucleic acid(LNA)-modified probe,which was referred to as MCA-LNA assay.This approach could detect seven Kras hotspot mutations simultaneously in a single PCR reaction.Owing to the LNA probe,the mutation types was enriched and the sensitivity of detection was improved evidently to 0.05%0.5%.When tested with clinical colorectal cancer specimens,Kras mutations were detected in 24 of total 72 specimens using MCA-LNA assay,in which five more positive samples of Kras mutant were identified compared to the MCA assay with a non-modified probe,and all the five samples were further confirmed by amplification refractory mutation system.Therefore,the MCA-LNA assay improved the limit of detection and the detection rate of positive sample,which is profitable for accurate genetic screening and guiding targeted therapy.
作者
倪润芳
黄秋英
钱思雨
李腾文
苏国强
李庆阁
NI Runfang;HUANG Qiuying;QIAN Siyu;LI Tengwen;SU Guoqiang;LI Qingge(Engineering Research Centre of Molecular Diagnostics,Ministry of Education,School of Life Sciences,Xiamen University,Xiamen 361102,China;Department of Gastrointestinal Surgery,The First Affiliated Hospital of Xiamen University,Xiamen 361005,China)
出处
《厦门大学学报（自然科学版）》
CAS
CSCD
北大核心
2019年第6期817-824,共8页
Journal of Xiamen University：Natural Science
基金
福建省高校杰出青年科研人才培育计划
关键词
熔解曲线分析
Kras突变
锁核酸
melting curve analysis
Kras mutation
locked nucleic acid
