桃拉综合征病毒的环介导等温扩增联合横向流动试纸条(LAMP-LFD)检测方法的建立  

Establishment of detection method for Taura syndrome virus by loopmediated isothermal amplification combined with lateral flow dipstick(LAMP-LFD)

作　　者：李伟哲 刘露[2] 李丽娜[2] 肖勤[2] LI Weizhe;LIU Lu;LI Lina;XIAO Qin(College of Life Sciences,Hebei Agricultural University,Baoding 071001,China;College of Ocean,Hebei Agricultural University,Qinhuangdao 066003,China)

机构地区：[1]河北农业大学生命科学学院,河北保定071001 [2]河北农业大学海洋学院,河北秦皇岛066003

出　　处：《河北农业大学学报》2020年第3期79-85,共7页Journal of Hebei Agricultural University

基　　金：留学人员科技活动择优资助项目(CN201602).

摘　　要：桃拉综合征病毒(Taura syndrome virus, TSV)是威胁虾类养殖业的主要病毒之一。本试验建立了一种快速检测TSV的环介导等温扩增联合横向流动试纸条(LAMP-LFD)方法,并与荧光定量PCR(qPCR)进行了对比。基于TSV的衣壳蛋白VP2核酸序列,设计3对特异性引物(外引物:FIP/BIP,内引物:F3/B3,环引物:LF/LB),进行LAMP反应体系的优化。其中引物LF以BIO标记,LB以FAM标记,用于横向流动试纸条(LFD)的检测。结果表明:LAMP最佳反应可在63℃恒温条件下40 min内完成。整个扩增反应到LFD结果判定可在45 min内完成。LAMP-LFD可特异性检出TSV特异性片段,未与其它病毒和细菌发生交叉反应,且其检测限为22.9 fg。与qPCR相比,本研究所建立的LAMP-LFD检测体系虽然检测限高于qPCR 10倍,但比qPCR检测时间缩短近1 h,且LAMP-LFD特异性强、操作简单,无需特殊仪器设备,可快捷、方便地检测出TSV,满足TSV基层快速检测的需要。Taura syndrome virus(TSV) is one of major infectious virus for shrimp, threatening prawn culture. In this study, a novel loop-mediated isothermal amplification combined with lateral flow dipstick(LAMP-LFD) method was developed in order to rapidly detect TSV. Three pairs of specific primers(FIP/BIP, F3/B3, LF/LB) were designed based on the nucleic acid sequence of viral coat protein 2(VP2) of TSV for LAMP assay. The primer of LF was labeled with biotin(BIO), and LB was labeled with fluorescein amidite(FAM) for lateral flow dipstick(LFD). The results showed that LAMP reaction for TSV was carried out at 63 ℃ for 40 min. The whole detection period was about 45 min from LAMP(40 min) to LFD(5 min). LAMP-LFD could detect the specific fragment of TSV without cross reaction with other viruses and bacteria. The detection limit of LAMP-LFD was 22.9 fg. Compared to fluorescence quantitative PCR(qPCR), although the detection limit of LAMP-LFD was 10 times higher than that of qPCR, the reaction time was nearly 1 h shorter than that of qPCR. Moreover, LAMP-LFD had high specificity and simple operation without special instruments. It could detect TSV quickly and conveniently, which meets the need of field–based detection.

关 键 词：横向流动试纸条 环介导等温扩增 桃拉综合征病毒 荧光定量PCR 

分 类 号：S942.3[农业科学—水产养殖]