DEVELOPMENT OF A MULTIPLEX REAL-TIME PCR ASSAY FOR DETECTION OF WSSV AND IHHNV

Abstract

White spot syndrome virus(WSSV) and Infectious hypodermal and haematopoietic necrosis virus(IHHNV) are responsible for significant economic loss in the shrimp industry.In order to simultaneously and massively identify WSSV and IHHNV,two pairs of primers and two TaqMan probes were designed and synthesized according to the conserved gene sequences of WSSV(AF369029) and IHHNV(AF218226) in GenBank.The reaction parameters such as the concentration of two pair of primers,two TaqMan probes and the reaction buffer were optimized to develop a multiplex real-time PCR assay for the rapid detection of WSSV and IHHNV.The multiplex real-time PCR assay was found to be specific and be able to detect and differentiate WSSV and IHHNV,and no positive results were observed when nucleic acid from Vibrio,Taura Syndrome Virus and Streptococcus were used as multiplex real-time PCR templates.The developed multiplex real-time PCR assay was compared with that of routine PCR.The sensitivity of multiplex real-time PCR assay was 2 and 20 template copies for WSSV and IHHNV respectively,and its sensitivity was 103 and 102 times higher than that of the routine PCR.The samples were examined using the multiplex real-time PCR repeatedly and the results indicated that the multiplex real-time PCR was reproducible.Different concentrations of WSSV and IHHNV could be identified when mixed together,which implied the assay could be applied to clinical confirmation for simultaneous infection of WSSV and IHHNV.The multiplex real-time PCR results of 30 routine PCR positive samples showed that one specific amplified curve was displayed when shrimp was infected by only one of these two viral pathogens,whereas two specific amplified curves were displayed when shrimp was infected by two viral pathogens.The result indicated that multiplex real-time PCR was able to detect and differentiate the presence of each pathogen in infected clinical shrimp.This multiplex real-time PCR assay is a quick,sensitive,specific and quantitative tool for detection of WSSV and IHHNV,and it will be useful for the control of WSS and IHHN in shrimp.