Development and validation of three duplex real-time PCR assays for simultaneous detection of major haemorrhagic disease-associated pathogens in cultured eels

Haemorrhagic disease causes significant losses in global eel aquaculture. To enable concurrent diagnosis of its diverse etiological agents, we developed three duplex TaqMan real-time PCR (qPCR) assays targeting: (1) Anguillid herpesvirus (AngHV) DNA polymerase gene ( pol ) and Eel circovirus (EeCV) capsid protein gene ( cp ); (2) Aeromonas cytotoxic enterotoxin ( act ) and heat-stable cytotonic enterotoxin ( ast ) genes; (3) Aeromonas heat-labile cytotoxic enterotoxin ( alt ) and hemolysin ( hlyA ) genes. Each assay was rigorously optimized and validated. Optimized performance was achieved using primer and probe concentrations of 0.2 μM and 0.1 μM, respectively, and a uniform annealing temperature of 60 °C for 20 s. All assays exhibited high specificity, efficient amplification (92.5–99.8 %), excellent linearity (R 2 ≥ 0.998), and high sensitivity, with limits of detection (LOD) of 6.2 copies/μL for pol and cp , and 3.6 copies/μL for the Aeromonas toxin genes ( act, ast, alt and hlyA ). Validation with fourteen Aeromonas strains demonstrated perfect concordance with singleplex qPCR. Applied to 54 clinical eel samples, mixed infections (56 %, 30/54), predominated over single infections (22 %, 12/54). Prevalence was EeCV (68.5 %), AngHV (55.6 %), and virulent Aeromonas spp. (44.4 %). Key co-infection patterns were AngHV + EeCV + Aeromonas spp. (35.2 %), AngHV + EeCV (11.1 %), and AngHV + Aeromonas spp. (9.3 %). Collectively, this study established the first duplex qPCR platform for simultaneous detection of AngHV, EeCV, and virulent Aeromonas spp. (via key toxin genes) associated with eel haemorrhagic disease, providing a powerful, high-throughput tool for rapid diagnosis and epidemiological surveillance. • Three duplex qPCR assays were developed for detection of multiple pathogens in eels. • Simultaneous detection of AngHV, EeCV, and Aeromonas in haemorrhagic disease in eels. • These assays demonstrated good-specificity, high-sensitivity, and rapid-processing. • Pathogens varied across farms; mixed infections (56 %) surpassed single (22 %).