Accurate and rapid detection of six viral pathogens in penaeid shrimp by using a novel multiplex PCR

Shrimp viral diseases, primarily caused by Taura syndrome virus (TSV), white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV)/Penaeus stylirostris penstyldensovirus 1 (PstDV1), hepatopancreatic parvovirus (HPV), Penaeus monodon-type baculovirus (MBV)/P. monodon nudivirus (PmNV) and yellow head virus (YHV), posed a significant global challenge and resulted in enormous financial losses in the shrimp cultivation industry. In this research, six pairs of specific primers of the above six viruses were designed according to the highly conserved sequences of the viruses to amplify conserved gene segments of each virus and generate the corresponding PCR products of different sizes. Following system optimization, a multiplex PCR method was established to simultaneously detect and differentiate the six viruses mentioned above in penaeid shrimp. The assay exhibited high specificity with no cross-reactivity towards other non-target shrimp viruses or shrimp nucleic acids. The limit of detection (LOD) of the multiplex PCR assay was 5 × 10² copies/μL for TSV, WSSV, IHHNV, HPV and YHV, and 5 × 10³ copies/μL for MBV. Using this assay, 1180 shrimp samples that were divided into 236 pools of five samples each pool were tested for further assessment. The total positive rate for all pools was 30.5 %, with IHHNV at 22.88 %, WSSV at 7.2 %, HPV at 0.42 %, and TSV, MBV, and YHV were undetected. The agreement rates between the multiplex PCR and conventional singleplex PCRs as well as real-time singleplex PCRs were all higher than 98.5 %. Overall, the multiplex PCR developed here is thus a sensitive and cost-effective diagnostic tool for the detection and daily monitoring of TSV, WSSV, IHHNV, HPV, MBV and YHV in shrimps.