肠道病毒71型RT-LAMP快速检测方法的建立及初步应用  

Establishment and preliminary application of RT-LAMP rapid detection method of entervirus 71

作　　者：李莎[1] 张如胜[1] 陈静芳[1] 

机构地区：[1]湖南省长沙市疾病预防控制中心,410000

出　　处：《国际检验医学杂志》2017年第4期470-472,共3页International Journal of Laboratory Medicine

摘　　要：目的建立手足口病肠道病毒71型(EV71型)逆转录环介导等温扩增(RT-LAMP)快速检测方法,并对其进行应用评价。方法利用软件针对EV71型病毒特异性基因6个区域设计4条LAMP引物,在普通恒温水箱内65℃保温约1 200min完成RT-LAMP扩增,扩增结果通过电泳和肉眼来判断。利用RT-LAMP和逆转录聚合酶链反应(RT-PCR)方法同时检测70份EV71型肠道阳性标本;将EV71型的RNA做一系列10倍稀释后用RT-LAMP和RT-PCR方法进行检测来比较两者敏感度。结果 EV71型出现LAMP特征性梯状条带,肉眼可以判断结果;RT-LAMP与RT-PCR方法检测100份EV71型标本的检出率比较差异无统计学意义(P>0.05);RT-LAMP方法的敏感度(10.0pg/μL)与RT-PCR方法相同。结论建立了一个可以在普通恒温水箱内进行核酸扩增的EV71型RT-LAMP检测方法,初步应用证实该方法有一定的应用前景。Objective To establish a reverse transcription loop-mediated isothermal amplification RT-LAMP rapid detection method of enterovirus 71（EV71）in hand,foot and mouth disease（HFMD）,and to evaluate its application.Methods Four types of LAMP primers were designed by using the software needle based on the 6distinct regions of the specific gene of EV71.The process of amplification was completed in the ordinary thermostatic water container by 65 ℃ for 1 200 min.The results of amplification were judged by electrophoresis and naked-eye.Seventy EV71 type intestinal positive specimens were simultaneously detected by RT-LAMP and RT-PCR methods.EV71 type of intestinal virus RNA were made a series.After 10 times of dilution,the RT-LAMP and RT-PCR methods were used to conduct the detection for comparing their sensitivities.Results The LAMP characteristic ladder bands of EV71 appeared,then the results could judged by the naked-eye.The detection rate in 100EV71 samples had no statistical difference between RT-LAMP and RT-PCR methods（P〉0.05）.The sensitivity（10.0pg/μL）of RT-LAMP was same to that of RT-PCR method.Conclusion The RT-LAMP detection method for EV71 was established,which can be used for nucleic acid amplification in the ordinary thermostatic water container.The preliminary application verifies that this RT-LAMP assay has a certain application prospect.

关 键 词：环介导等温扩增 肠道病毒71型 检测 

分 类 号：R725.1[医药卫生—儿科] R440[医药卫生—临床医学]