环介导等温扩增联合横向流动试纸条检测草鱼呼肠孤病毒方法的建立

Development of a loop-mediated isothermal amplification combined with Lateral flow dipstick(LAMP-LFD) method for detection of Grass carp reovirus

将环介导等温扩增技术(LAMP)与横向流动试纸条(LFD)联合建立一种快速检测草鱼呼肠孤病毒(GCRV)的LAMP-LFD检测方法,并与实时荧光定量PCR(qPCR)技术进行对比。以GCRV S8片段为检测靶标,设计6条特异性引物(FIP/BIP,F3/B3,LF/LB),其中上游引物FIP以生物素(Biotin,BIO)标记,进行LAMP反应条件优化,同时设计1条羟基荧光素(Fluorescein amidite,FAM)标记的探针,用于LAMP反应产物的LFD检测。结果表明:LAMP最佳反应温度为63℃,反应时间40min。从LAMP扩增到LFD结果判读共需50min,比qPCR检测缩短近1h。LAMP-LFD与qPCR均可特异性检出GCRV,针对同一重组质粒的检测,LAMP-LFD的检测限为970fg,qPCR的检测限为97fg,虽然LAMP-LFD的检测灵敏度低于qPCR 10倍,但该方法操作简单,仪器设备依赖性低,可快速、特异地检测出GCRV,有望成为GCRV现场快速检测的常规技术手段。

A novel loop-mediated isothermal amplification(LAMP)combined lateral flow dipstick(LFD)detection method(LAMP-LFD)was developed and compared with quantitative real-time PCR(qPCR)assay in order to achieve Grass carp reovirus(GCRV)field-based detection.A set of six specific primers(FIP/BIP,F3/B3,LF/LB)were designed based on the GCRV S8 sequence for LAMP assay,and FIP was labeled with biotin(BIO).A hybrid primer(HP)labeled with(Fluorescein amidite)FAM was also designed.The results showed that LAMP reaction parameter for GCRV was at 63℃for 40 min.The whole detection time,from LAMP to LFD visualization was about 50 min,which was nearly 1hour shorter than that of qPCR.Furthermore,the specificity and sensitivity were also evaluated.LAMP-LFD was similar to qPCR,which discriminated GCRV from Cyprinid herpesvirus 3(Cy HV-3)and viremia of carp virus.As for pure plasmid of GCRV gene,the detection limit of LAMP-LFD was 970 fg,and qPCR was 97 fg.Although detection limit of LAMP-LFD was 10 times lower that of qPCR,LAMP-LFD method was suitable for field-based detection of GCRV in the study.The study provides a valuable data and robust method for virus molecular detection techniques on-site testing.