Establishment and application of multiplex real-time qPCR detection for Tembusu virus and goose astrovirus genotypes 1 and 2.

The concurrent infection and epidemic of goose astrovirus type 1 (GoAstV-1), goose astrovirus type 2 (GoAstV-2), and Tembusu virus (TMUV) in geese have resulted in substantial economic losses to the goose industry. Therefore, establishing a rapid and accurate diagnostic method for these three viruses is imperative. In the present study, we designed specific primers targeting the Cap gene of GoAstV and the E gene of TMUV and developed a SYBR Green I-based multiplex real-time qPCR method for the simultaneous detection of GoAstV-1, GoAstV-2, and TMUV. The results revealed that the R2 values for the standard curve of the multiplex real-time qPCR of GoAstV-1, GoAstV-2 and TMUV reached 0.998, 0.998, and 0.994, respectively. In addition, the limits of detection for GoAstV-1, GoAstV-2 and TMUV were 1.08×102 copies/μL, 2.63×102 copies/μL and 3.07×102 copies/μL, respectively. Moreover, specificity test results indicated that the multiplex real-time qPCR system is highly specific for these three target pathogens, and no cross-reactions with other goose-origin viruses, such as Newcastle disease virus (NDV), goose reovirus (GRV), avian influenza virus (AIV), or goose parvovirus (GPV), were observed. Moreover, the established multiplex real-time qPCR method had excellent repeatability, with intra- and interassay coefficients of variation ranging from 0.23 to 0.91 and 0.71 to 1.75, respectively. The present study provides a rapid and efficient tool that is both specific and sensitive for detecting GoAstV-1, GoAstV-2, and TMUV and is helpful for determining the epidemiology of these viruses in geese.