Development of a multiplex PCR system for the simultaneous detection of white spot syndrome virus and hepatopancreatic parvovirus infection

A multiplex PCR kit for simultaneous detection of white spot syndrome virus ( WSSV ) and hepatopancreatic parvovirus ( HPV ) was developed and field testing was conducted. A 604‐bp target sequence was selected from the vp28 gene of WSSV . A primer set was developed to amplify a 338‐bp DNA fragment at the junction of the NS 2 and NS 1 protein genes of HPV after alignment of eight sequences from different strains. Another internal positive control primer set produced a 139‐bp PCR fragment from the β‐actin gene by alignment of this gene from Litopenaeus vannamei , Fenneropenaeus chinensis and Penaeus monodon . The detection limits, tested using purified plasmids, for WSSV and HPV were 21.4 and 19.0 copies respectively. The optimum ratio for HPV , WSSV and β‐actin was 3:1:1, with an optimum annealing temperature of 57°C. Field test of the multiplex PCR with 170  L. vannamei individuals from 17 aquaculture farms showed 41.8% coinfection with WSSV and HPV , and 40.0% and 3.5% single infection with WSSV and HPV respectively. No virus‐free shrimp farm was found. Ten wild catch F. chinensis individuals showed 60% coinfection, and 40% were infected with HPV .