Establishment of loop-mediated isothermal amplification (LAMP) assay for visual detection of Bombyx mori nucleopolyhedrovirus

ObjectiveThe present study established loop-mediated isothermal amplification (LAMP) for visual detection of Bombyx mori nucleopolyhedrovirus (BmNPV),in order to provide support for early diagnosis of nuclear polyhedrosis at work field.MethodTaking BmNPV polyhedrin gene polh as amplification target,five groups of inner primer/outer primer and five pieces of loop primer were designed in order to screen the best primer combination based on amplification efficiency.LAMP reaction conditions were optimized and assays on specificity,sensibility as well as clinical sample detection were conducted.The amplification result was observed by colorimetric determination with hydroxynaphthol blue (HNB) staining.The conditions for simplifying treatment were analyzed.ResultWhen using LAMP of loop primer,the lowest detection line of BmNPV genomic DNA could reach 7 fg/μl,the sensitivity of detection was improved effectively and reaction time was reduced.The established LAMP detection specifically amplified the target DNA,and its sample detection rate was superior to conventional PCR.The lowest detection line was as 100 times as that of conventional PCR.The result could be easily judged and the cross-contamination could be reduced by adding HNB before reaction.The hemolymph of infected silkworm boiled at 100 C could be directly applied to LAMP,which could simplify operation steps and reduce detecting cost.ConclusionThe visual LAMP detection targeting BmNPV established in this study is sensitive,quick and reliable,which is appropriate to use for early diagnosis of BmNPV infection at work field.