Development and application of a quadruple RT-qPCR assay for simultaneous detection of GPV, GPMV, NGRV, and TMUV

To achieve rapid and simultaneous detection of Goose Parvovirus (GPV), Goose Paramyxovirus (GPMV), Novel Goose Reovirus (NGRV), and Tembusu Virus (TMUV), the specific primers and TaqMan probes were designed and synthesized based on the VP3 gene of GPV, the F gene of GPMV, the σC gene of NGRV, and the E gene of TMUV, respectively, and a quadruplex real-time fluorescent RT-PCR (RT-qPCR) assay was successfully developed through optimization of the reaction system and conditions. The developed quadruplex RT-qPCR assay enabled the specific detection of GPV, GPMV, NGRV, and TMUV without cross-reactivity with other common avian pathogens tested in this study. With the plasmid DNA containing specific amplification sequences of GPV and the in vitro transcribed RNA mixtures of GPMV, NGRV, and TMUV as templates, the developed assay demonstrated a minimum detection limit of 10² copies/reaction. The intra-assay and inter-assay coefficients of variation were below 2.65%, indicating a good reproducibility. Utilizing the developed quadruplex RT-qPCR assay, 306 liver samples and 46 cloacal swab samples were tested. The positive rates of GPV, GPMV, NGRV, and TMUV were 38.07% (134/352), 20.74% (73/352), 17.33% (61/352) and 18.46% (65/352), respectively. In which, 79 samples (22.44%, 79/352) exhibited the mixed viral infections, with positive rates of 10.80%, 6.25%, and 8.24% for dual, triple, and quadruple co-infections, respectively. The developed quadruplex RT-qPCR assay in this study performed well on the clinical samples, thereby providing robust technical support for the epidemiological investigation of GPV, GPMV, NGRV, TMUV infections and disease prevention and control in the goose farming industry.