Establishment and Application of a Triple TaqMan qPCR Detection Method for IMNV, DIV1, and CMNV

The escalating incidence of emerging pathogens and their propensity for co‐infection pose a severe threat to the sustainable intensification of Penaeus vannamei aquaculture. To address this challenge, a novel triplex TaqMan quantitative PCR (qPCR) assay was developed for the simultaneous detection of three high‐risk agents: Infectious Myonecrosis Virus (IMNV), Decapod Iridescent Virus 1 (DIV1), and Covert Mortality Nodavirus (CMNV). This study not only establishes a robust multiplex platform but also systematically optimises its performance, achieving superior analytical sensitivity and reproducibility compared to existing single‐target or duplex methods. The assay demonstrated a limit of detection as low as 10 1 copies/μL for all targets, with excellent inter‐ and intra‐assay coefficients of variation (CVs) consistently below 3%. Notably, the method effectively overcomes the technical hurdles associated with detecting both RNA (IMNV, CMNV) and DNA (DIV1) viruses within a single reaction, eliminating cross‐reactivity and ensuring high specificity. Validated against clinical samples, the established protocol exhibits a concordance rate exceeding 95% with current standard methods, while significantly streamlining the diagnostic workflow. This advancement provides a powerful tool for routine biosecurity monitoring, seed stock certification, and the comprehensive management of emerging shrimp diseases.