肠道病毒71型环介导等温扩增检测方法的建立  

Simple,economic detection of EV71 with reverse transcription loop- mediate amplification

作　　者：戴颖[1] 刘峰涛[2] 何书[1] 汪奇伟[1] 

机构地区：[1]河南大学淮河医院儿科,开封475003 [2]河南大学医学院免疫学研究所,开封475004

出　　处：《中国免疫学杂志》2014年第2期240-243,共4页Chinese Journal of Immunology

摘　　要：目的:采用一步逆转录-环介导等温扩增(Reverse Transcription Loop-Mediated Isothermal Amplification,RTLAMP)肠道病毒71型(EV71)特异性基因,以建立一种快速、敏感、经济的EV71检测方法。方法:针对EV71病毒VP1基因特异性序列的8个区域设计6条环介导等温扩增引物,Bst(Bacillus stearothermophilus)DNA聚合酶63℃扩增目的基因1 h,GoldView染色,日光下观察结果,并评价其特异性和灵敏度。结果:该方法与柯萨奇病毒A16型、轮状病毒、诺如病毒无交叉反应发生。建立的RT-LAMP检测方法灵敏,最低检测限为50拷贝/管,比定量PCR灵敏10倍。93份咽拭子标本中,RTLAMP检测示46份阳性,实时荧光定量PCR检测示44份阳性,经卡方检验分析,两者间无显著统计学差异。结论:RT-LAMP是一种快速、敏感、特异、经济的方法,适用于基层医疗机构临床检测普及。Objective：To establish a reverse transcription loop-mediated isothermal amplification （RT-LAMP） assay for rapid and sensitive detection of enterovirus 71 （EV71）.Methods：Six primers which recognized 8 distinct regions on the VP1 gene of EV71 were designed for RT-LAMP assay.RT-LAMP assay was optimized to amplify VP1 gene in the presence of a specific primer set and Bst DNA polymerase at an isothermal 63℃ for 1 h.The amplified products were observed with GoldView staining,and the sensitivity and specificity were also evaluated.Results：There was no cross reaction with other virus.The detection limit of RT-LAMP assay was 50 copies/tube which was 10-fold higher sensitive than real-time RT-PCR.Of 93 swab specimens,46 were EV71 positive by RT-LAMP assay,while 44 were EV71 positive by fluorescence quantitative PCR.Two methods had no significant difference.Conclusion：RTLAMP is a rapid,sensitive,specific and economic method for detection of EV71 in clinical specimens,and especially adapts to primary health care agencies.

关 键 词：逆转录-环介导等温扩增技术 肠道病毒71型 手口足病 

分 类 号：R392[医药卫生—免疫学]