Highly sensitive detection and identification of six viruses in shrimps by universal primer based multiplex PCR

The global expansion of shrimp aquaculture faces severe threats from viral disease outbreaks, which cause substantial economic losses and create an urgent demand for rapid, accurate diagnostic tools. To address this need, we developed a universal primer-mediated multiplex PCR (UP-M-PCR) assay for the simultaneous detection of six major shrimp-infecting viruses: Taura syndrome virus (TSV), hepatopancreatic parvovirus (HPV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), Penaeus monodon-type baculovirus (MBV), and yellow head virus (YHV), white spot syndrome virus (WSSV). The assay employed a strategy of combined usage of a universal primer and chimeric primers who had a universal primer tail at the 5'-end of each virus-specific primer to increase analytic specificity and sensitivity. The assay specifically detected the six viruses and demonstrated exceptional sensitivity, with limits of detection (LODs) of 5 copies/μL for TSV, WSSV, and IHHNV, and 50 copies/μL for HPV, MBV, and YHV. In testing of 1180 shrimp samples in 236 pools of 5, the assay detected a calculated overall viral positivity rate of 8.17% at the individual shrimp level, with IHHNV showing the highest prevalence (5.91%), followed by WSSV (1.93%) and HPV (0.08%), and WSSV-IHHNV coinfections were identified in 0.26% of samples. This UP-M-PCR assay allows for the concurrent detection and differentiation of multiple shrimp viral pathogens with excellent analytical and diagnostic performance, supporting its good application potential for viral surveillance in shrimp aquaculture.