Duplex SYBR Green I real-time RT-PCR for Simultaneous Detection of Goose Astrovirus Genotypes 1 and 2

As the prevalence and complexity of goose astrovirus genotype 1 and 2 (GAstV 1 and GAstV 2) infections continue to rise in China's goose farming industry, a rapid, reliable and cost-effective diagnostic tool is urgently needed. Duplex SYBR Green I real-time RT-PCR (qRT-PCR) method was developed, which is capable of simultaneous detection of GAstV 1 and GAstV 2. We designed two primer pairs, targeting the viruses conserved genomic regions, facilitating the distinction of GAstV 1 and GAstV 2 through unique melting curve peaks. This method exhibited strong linear relationships in standard curves (R2 = 0.99), and detection limits were 3.18 x 102 and 3.16 x 102 copies/mu L for GAstV 1 and GAstV 2, respectively. With intra- and inter-assay variation below 2%, the assay exhibits excellent repeatability. This duplex qRT-PCR, displaying consistent detection rates comparable to a TaqMan-based method, offers a promising, rapid, and cost-effective diagnostic strategy for waterfowl farming.