Establishment and application of a triplex real-time fluorescent quantitative RT-PCR assay for simultaneous detection...

To rapidly monitor clinical infections induced by highly pathogenic avian influenza (HPAI) of H5/H7 subtypes and virulent Newcastle disease virus (NDV), mitigate economic losses in intensive poultry industry and reduce zoonotic spillover risks to human population, a one-tube triplex TaqMan real-time RT-qPCR was developed in this study. Three pairs of subtype-specific primers and corresponding fluorophore-labeled TaqMan probes were rationally designed targeting conserved hemagglutinin (HA) coding regions of H5-AIV, H7-AIV and fusion (F) gene of virulent NDV, respectively, referring to published multiplex RT-qPCR design strategies for avian respiratory viruses. After gradient optimization of primer/probe dosage, reverse transcription condition and thermal cycling parameters via matrix titration assay, standard curves were generated using absolute copy-number quantified viral RNA standards via droplet digital PCR (ddPCR), followed by systematic evaluation of analytical specificity, limit of detection (LOD), intra/inter-assay reproducibility and field diagnostic performance against official Chinese national standard monoplex RT-qPCR protocols. Specificity testing revealed exclusive positive amplification only for target H5-AIV, H7-AIV and virulent NDV, without any cross-reactive fluorescent signals from 10 heterogeneous non-target AIV subtypes (H1-H4, H6, H9-H13), attenuated NDV, infectious bursal disease virus (IBDV), infectious laryngotracheitis virus (ILTV) and infectious bronchitis virus (IBV). The absolute LOD was determined as 4.32 copies/μL (H5-AIV), 20.40 copies/μL (H7-AIV) and 5.14 copies/μL (virulent NDV). Linear regression analysis of standard curves yielded coefficients of determination (R²) of 0.9997, 0.9988 and 0.9997 and corresponding amplification efficiencies (E) of 94.58%, 103.60% and 97.20%. All intra- and inter-assay coefficients of variation (CV) across four concentration gradients were less than 1%, demonstrating robust assay repeatability. The assay accurately quantified co-infections across ratios of 1:9-9:1. The CT value is directly proportional to the target abundance. Even when the content is only 10% in co-infection, it can still be detected, indicating its excellent ability for co-infection detection. For total 384 field poultry oropharyngeal/cloacal swabs, the triplex assay is highly consistent with the national standard methods, with Kappa values exceeding 0.85 for all targets. In summary, this validated triplex TaqMan RT-qPCR features superior specificity, ultrahigh analytical sensitivity and outstanding stability, which is applicable for routine clinical pathogen diagnosis, large-scale live poultry market surveillance and epidemiological tracing of mixed co-infections caused by H5/H7 HPAIV and virulent NDV.