Multiplex MALDI-TOF MS for simultaneous detection of 13 goose viruses.

The global waterfowl industry faces escalating economic losses from complex polymicrobial infections, yet conventional diagnostics remain shackled to single-pathogen workflows that falter under mixed infection scenarios. Here, we present a high-throughput multiplex MALDI-TOF MS platform enabling simultaneous detection of 13 critical goose-origin viruses through targeted interrogation of 16 conserved genomic signatures, including GAstV-1/2 (ORF1b), DRV (p10/L1), AIV (NP), TMUV (E), FAdV (hexon), GPV (VP1), DPV (UL6), NDV (L), GoCV (Rep), REV (LTR), MDPV (VP1), IBDV (VP2), and ALV (env/p27). The assay exhibited exceptional analytical performance, achieving limits of detection ranging from 2.87 to 29 copies/μL, absolute specificity, and robust reproducibility (intra- and inter-assay coefficients of variation ≤ 3.3%, corresponding to precision ≥ 96.7%). Clinical validation using 74 field specimens, encompassing oropharyngeal/cloacal swabs, environmental swabs, and tissue samples, demonstrated 98.3% concordance with qPCR, with markedly superior capability in resolving complex co-infections that confound conventional methods. The platform accommodates diverse clinical matrices, processing 384 samples per run within a < 10-hour workflow, thereby overcoming the throughput limitations of single-target PCR and the resource intensity of next-generation sequencing. By enabling comprehensive pathogen profiling in a single analytical run, this technology offers a pragmatic, scalable solution for rapid outbreak investigation, routine surveillance, and evidence-based disease management in intensive waterfowl production systems.