Development of rapid detection for white spot syndrome virus by loop-mediated isothermal amplification

Abstract

A rapid,sensitive and simple method for white spot syndrome virus(WSSV) detection was established.Loop-mediated isothermal amplification(LAMP) assay is a novel method of gene amplification with high specificity,sensitivity and rapidity,which can be applied for disease diagnosis in shrimp aquaculture.The method is performed under isothermal conditions with a set of four specially designed primers that recognize six distinct sequences of the target.In the present study,a set of four specific primers were designed based on the conservative sequence of envelope protein gene VP28 using Primer Explorer v4 software,and a rapid detection of WSSV was established by using LAMP assay.The parameters of reaction time and temperature were optimized,and its specificity and sensitivity were assessed.The reactions were carried out at 58,60,62,63,64,65,66,67,68 ℃ for different time(15 -75 min).A plasmid pMDWSSV carrying target sequence of LAMP detection was constructed.Ten-fold diluted pMDWSSV(107-100 copies/ μL) was used as template for LAMP assay to investigate the detection limit.To determine the specificity,LAMP assays were carried out with DNA templates from other pathogens(Infectious Hypodermal and Hematopoietic Necrosis Virus IHHNV,Taura Syndrome Virus TSV,A.hydrophila,V.alginolyticus,V.parahaemolytious,E.coli).The results showed the optimum LAMP assay for the rapid detection of WSSV was performed at 64 ℃ for 60 min.The LAMP assay had an unequivocal detection limit of 100 copies/μL,and it was 100 times lower than that of the nested PCR.The nucleic acids of other pathogens were not amplified by this LAMP system with the specific primers,which showed a good specificity.The resulting amplicons were detected using visual observation after the addition of SYBR Green I and gel electrophoresis.We investigated the efficacy of uracil-N-glycosylase(UNG) in preventing contamination in the LAMP assay procedure and explored its effect on the amplification efficiency.Products of LAMP with dUTP adding could be lysed by UNG.UNG could prevent LAMP products contamination effectively,but its effect on amplification efficiency was visible.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.Clinical suspected WSSV-infected diseased shrimp samples were detected by both LAMP and PCR assay,which indicated LAMP could be more sensitive for WSSV detection.