对虾白斑综合征病毒LAMP检测方法的建立

Establishment of a loop-mediated isothermal amplification(LAMP) assay for detection of shrimp white spot syndrome virus(WSSV)

根据对虾白斑综合征病毒(WSSV)囊膜蛋白VP28的基因序列设计合成4条特异性引物,以pMDT-VP28重组质粒为标准模板,通过优化反应体系和反应条件,建立了WSSV的环介导等温扩增(LAMP)检测方法,测定LAMP对WSSV-DNA的最低检测限,并与巢式PCR进行了比较。结果显示,该LAMP方法的最佳反应温度为65℃,反应时间为60min;LAMP对WSSV的最低检测限为10copies/μL,而巢式PCR为100copies/μL,表明该LAMP方法的敏感性显著高于巢式PCR检测方法。因此,WSSV的LAMP检测方法比PCR更为简便、快速、灵敏,且无需昂贵的变温仪器,更适应水产养殖的现地检测,本研究为WSSV早期感染的快速检测提供了新方法。

Following the LAMP protocol,a set of four specific primers was designed based on the sequence of gene encoding envelope protein 28（VP28） of WSSV.Using the recombinant plasmid of pMDT-VP28 as a standard template,the LAMP method for WSSV detection was developed by optimizing its reaction system and conditions.Its detection limit for WSSV was determined by detecting the ten-fold diluted WSSV-DNA（107 to 10-3 copies/μL） and compared with nested PCR.The optimized reaction temperature and time of WSSV-LAMP were at 65 ℃ for 60 min,respectively.Detection limit of the LAMP method for the WSSV-DNA was determined to be 10 copies/μL,whereas that of nested PCR was 100 copies/μL,suggesting the LAMP method was much more sensitive than the nested PCR methods for WSSV detection.In conclusion,the LAMP method for WSSV detection is more convenient,rapid,and sensitive than the nested PCR,and do not need the expensive sophisticated instrument.Therefore,it is very suitable for detection in the fields that would provide a novel rapid detection approach for early WSSV infection in aquaculture.