对虾白斑综合征病毒可视化环介导等温扩增(LAMP)检测技术的建立与应用

摘要

针对当前对虾(Penacus orientalis)养殖业亟需研究开发一种简便快速、敏感特异的检测技术来实现对对虾白斑综合征病毒(White spot syndrome virus,WSSV)进行检测监控的现状,本研究根据WSSV基因组ORF120保守区序列,设计合成6条环介导等温扩增(loop mediated isothermal amplification,LAMP)特异性引物,分别为外引物F3/B3、内引物FIP/BIP和环引物LF/LB.利用钙黄绿素/氯化锰作为检测指示剂,通过对反应条件优化,建立了可视化WSSV-LAMP检测方法.在检测中未发生扩增反应时,钙黄绿素被氯化锰螯合,呈淬灭状态.当样品中有WSSV靶基因存在时,大量DNA被合成,并产生同样大量的焦磷酸根离子,此时焦磷酸根离子竞争性与Mn2+结合,形成稳定的不溶性焦磷酸盐沉淀,钙黄绿素被释放而发出肉眼可见的黄绿色荧光.该扩增一次性反应约60 min即可完成对待检样品的检测.经测定,本方法能特异性地检测出阳性样品中的WSSV目的基因,并能通过扩增产物所产生的黄绿色变化与阴性样品区别;本技术对目的基因的最低检测量为1fg.在临床检测试验中,WSSV-LAMP与世界动物卫生组织(OIE)推荐的WSSV-PCR检测方法均能从250份对虾样品中,同时检测到15份编号相同的阳性样品,符合率达100％.除此之外,WSSV-LAMP还能从另外3份PCR检测为阴性的样品中检测到目的基因.比较这两种方法的阳性检出率,WSSV-LAMP为7.2％(18/250),WS SV-PCR为6.0％(15/250),WSSV-LAMP对自然感染的临床样品阳性检出率要比WSSV-PCR高.说明WSSV-LAMP在病毒含量较低的临床样品检测中比OIE推荐的PCR方法具有更高的技术优势,能在对虾养殖生产上的临床诊断、育种及口岸检疫,甚至海洋生态监测中对WSSV进行现场检疫.%Aim to current situation of urgent need to develop a simple,rapid,sensitive and specific technique for White spot syndrome virus (WSSV) detection in shrimp (Penacus orientalis) culture industry.Six specific primers including one set of outer primeres F3 and B3,one set of inner primers FIP and BIP,and one set of loop primers LF and LB were targeted on the conserved region in ORF120 of WSSV genome.These primers were designed and synthesized for amplifying the target gene ORF120 of WSSV in a loop mediated isothermal amplification (LAMP) system.A visual WSSV-LAMP was developed based on the optimized reaction conditions in which calcein was applied to be detection indicator.As none amplification taken place in the detection,calcein was cancellation by chelated with manganese chloride.When WSSV target gene was existed in the detected sample,a large amount of DNA was synthesized,yielding a large pyrophosphate ion byproduct.Pyrophosphate ion combined with metallic ion competitively to form an insoluble pyrophosphate salt.Calcein was emitted visual Kelly fluorescence.It took about 60 min to finish the detection during the one-step amplification reaction.Testing results showed that this WSSV-LAMP assay was able to detect the target gene contained in WSSV positive samples by nake-eye observation through the Kelly fluorescence emited by the amplification production.The lowest detected limitation was lfg of target gene DNA.The preliminary tested results of clinical application showed that fifteen positive samples with same labels were figured out from 250 shrimp clinical samples by WSSV-LAMP and WSSV-PCR recommened by Office International Des Epizooties,2012,respectively.The diagnose accordance rate between WSSV-LAMP and WSSV-PCR was hundred percent.WSSV-LAMP could also detected another three samples as positive,which were confirmed negative by WSSV-PCR.The comparison between these two methods,WSSV-LAMP was higher positive detection rate than that of WSSV-PCR,in which WSSV-LAMP was 7.2％(18/250) and WSSV-PCR was 6.0％(15/250).Results suggest that WSSV-LAMP is with higher technical advantage than WSSV-PCR in the detection of clinical samples with lower virus quantities.It can be used as an on-the-spot detection tool for WSSV in clinical diagnosis,breeding and port quarantine inspection,as well as sea ecological monitoring.