对虾白斑综合征病毒可视化环介导等温扩增(LAMP)快速检测方法的建立

    Establishment of rapid detection for shrimp white spot syndrome virus (WSSV) by visual loop-mediated isothermal amplification(LAMP)

    • 摘要: 根据对虾白斑综合征病毒(WSSV)基因保守序列,利用PrimerExplorer V4设计合成4套环介导等温扩增(Loop mediated isothermal amplification,LAMP)特异性引物,每套引物包含两条内引物(FIP和BIP)、两条外引物(F3和B3)和一条环引物(LF或LB)。利用钙黄绿素作为检测指示剂,建立了可视化WSSV的环介导等温扩增(LAMP)快速检测方法。以构建的pMD18-WSSV重组质粒为标准模板进行10倍梯度稀释,测定LAMP对WSSV核酸的最低检测限,4套引物组分别建立LAMP方法均在63℃恒温条件反应60 min,结果显示WSSV-2、WSSV-3和WSSV-4的最低检测限分别为2.25~0.225、2.25~0.225和225~22.5 copies/μL。本研究建立的LAMP方法在1 h内即可完成检测,操作简单,无需复杂仪器,肉眼可直接观察检测结果,适用水产养殖的现场检测,为WSSV早期感染的快速检测提供了方法。

       

      Abstract: In the present study,four sets of five specific primers were designed based on the conservative sequence of WSSV genome using PrimerExplorer V4.Five specific primers included one set of inner primers FIP and BIP,one set of outer primers F3 and B3,and one loop primer LF or LB.A visual WSSV-LAMP was developed in which calcein was applied to be detection indicator.Its detection limit for WSSV was determined by detecting the ten-fold diluted recombinant plasmid of pMD18-WSSV.The reaction temperature and time of WSSV-LAMP were at 63℃ for 60 min,respectively.Detection limit of the LAMP method for the WSSV DNA in which WSSV-2,WSSV-3 and WSSV-4 were determined to be 2.25~0.225 copies/μL,2.25~0.225 copies/μL and 225~22.5 copies/μL,respectively.The LAMP assay could be finished within an hour,requiring only a regular laboratory water bath or heat block for reaction and the result could easily be detected using visual observation.Therefore,it was very suitable for detection in the fields that would provide a rapid detection approach for early WSSV infection in aquaculture.

       

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