Development and application of an economical real-time PCR for WSSV detection and quantification in ridgetail white prawns(Exopalaemon carinicauda)

Abstract

White spot syndrome virus(WSSV)can infect many kinds of crustaceans and thus has caused huge economic losses to the crustacean farming.In order to detect WSSV precisely and sensitively,quantify WSSV conveniently,and further investigate the WSSV multiplication patterns in crustaceans,a practical real-time PCR for the quantification and diagnosis of WSSV is required to be developed.However,currently,the harvested quantitative real-time PCR for WSSV was either costly for those which were developed based on the Taqman probe technology,or insensitive for a latest method which was set up with the SYBR green dye.In this study,a practical,sensitive,and specific quantitative real-time PCR for WSSV based on SYBR Green was developed,and the tissue distributions and the profiles of WSSV proliferation in ridgetail white prawns(Exopalaemon carinicauda)were examined with this method.The details were as follows.First,a pair of primers(F1,R1)was designed to amplify the target fragment,which would be used to construct WSSV standard plasmid,and then another pair of primers(RF2,RR2)located inside the above target fragment was synthesized to produce amplicons in real-time PCR reaction,thereby developing a new diagnostic and quantitative real-time PCR for WSSV with SYBR Green dye.The results demonstrated that this quantitative method for WSSV was specific,sensitive,and precise.Subsequently,we further investigated WSSV distribution and proliferation pattern in ridgetail white prawns.The results of WSSV distribution showed that WSSV infected all detected tissues,among which,the connective tissues under the carapace possessed the highest virus load(1.1×107 copies/μg tissue),and the second highest tissue were gills with the virus load of 4.1×106 copies/μg tissue;The data of WSSV proliferation pattern illustrated that the copies of WSSV began to increase dramatically at 48 h after challenge,but before that there was no obvious change for virus copies.With the increase of infection time,the copies of WSSV increased more and reached the highest numbers at 120h post infection.In addition,we found that during this process the mortality of ridgetail white prawns caused by WSSV was approximately 20%.All these results suggest that WSSV is a deadly pathogen to ridgetail white prawns,and the new quantitative real-time PCR for WSSV is practical.