EGFR基因T790M突变COLD-PCR扩增体系的Tc值筛选  

Screening of Tc value for COLD-PCR amplification system of EGFR gene T790M mutation

作　　者：罗凯[1] 王倩[1] 仇秦威[1] 贺智敏[1] 

机构地区：[1]广州医科大学附属肿瘤医院/广州医科大学肿瘤研究所,广州510095

出　　处：《检验医学与临床》2016年第10期1328-1330,1333,共4页Laboratory Medicine and Clinic

基　　金：广东省广州市卫生和计划生育委员会医药卫生科技项目(20141A011096)

摘　　要：目的筛选表皮生长因子受体(EGFR)基因T790M突变PCR扩增体系的Tc值并初步建立具T790M突变富集效果的低变性温度下的复合聚合酶链反应(COLD-PCR)体系,同时探讨Tc值的筛选策略以及注意事项。方法以肺癌MSTO-211H、NCI-H1975细胞为T790M突变野生型和突变型标准样品,通过目标片段关键变性温度筛选实验、COLD-PCR反应模式比较实验以及Tc值的验证实验,确定COLD-PCR体系的Tc值并初步验证其效果。结果本实验体系T790M突变的目的扩增片段关键变性温度低于野生型片段,据此体系反应模式选择为快速COLD-PCR反应模式,同时确定Tc值为89.6℃,验证实验显示本体系可检出1%的T790M突变,较常规PCR提高5倍。至此T790M突变富集COLD-PCR扩增体系已成功建立。结论成功完成体系Tc值的筛选并建立T790M突变富集COLD-PCR扩增体系。Objective To screen the Tc value of COLD-PCR amplification system of the epidermal growth factor receptor（EGFR）gene T790 M mutation,to preliminarily establish the COLD-PCR system with the T790 M mutation enrichment effect under low denatured temperature and to investigate the screening strategy of Tc value and the matter needing attention.Methods The lung cancer cell line MSTO-211 Hand NCI-H1975 were taken as the wildtype and mutation type standard samples respectively.The Tc value of T790 M mutation detection COLD-PCR system was found and verified through screening experiment of key denaturation temperature of target amplicon,reaction pattern comparison experiment and verification test of Tc value.Results The pattern of fast COLD-PCR was chosen as standard reaction pattern because the key denaturation temperature of mutation amplicon was lower than wild type amplicon and 89.6℃ was founded as the Tc value of this system.The T790 M mutation enrichment COLD-PCR system was established,which had higher sensitivity than traditional PCR and could detect 1% of T790 M mutation,and was increased by 5times compared with the conventional PCR.Conclusion The Tc value screening of the system is successfully completed and the T790 M mutation enrichment COLD-PCR system is also established.

关 键 词：表皮生长因子受体基因 Tc值 低变性温度下的复合聚合酶链反应 

分 类 号：R734.2[医药卫生—肿瘤]