Establishment of A triplex TaqMan qPCR assay for EHP, V_pTPD, V_pAHPND with optimized efficacy using methyl cellulose

The frequent occurrence of mixed infections involving emerging pathogens in Penaeus vannamei poses a severe threat to sustainable aquaculture. Consequently, there is an urgent need for the development of highly efficient multiplex detection technologies to enable more rapid and accurate identification of key causative agents. Here, a triple TaqMan fluorescent quantitative PCR assay was developed for the simultaneous detection of Ecytonucleospora hepatopenaei (EHP), V_pTPD, and V_pAHPND. Based on this established method, the interference between multiple targets and the effects of four common macromolecular additives on the amplification efficiency of targeted sequences were systematically evaluated. The results demonstrated high specificity with no cross-reactivity, excellent reproducibility, and a limit of detection of 10¹ copies/μL for each pathogen. Inter-target interference analysis revealed that as the concentration of co-amplified competing targets increased, the Ct values of the target template initially decreased and subsequently rebounded. Notably, this interference was markedly attenuated at higher initial concentrations of the target. This interference was negligible at high target concentrations but enhanced amplification efficiency near the limit of quantification, thereby facilitating reliable qualitative analysis of low-abundance pathogens. Furthermore, methyl cellulose significantly improved amplification efficiency, effectively reducing detection time and rendering the process more rapid and convenient, suggesting its potential as a promising additive for optimizing multiplex PCR detection systems. This study establishes a robust technical foundation for the rapid detection of EHP, V_pTPD, and V_pAHPND, providing valuable methodological insights for the advancement of multiplex TaqMan fluorescent quantitative PCR assay in aquaculture.