Development of a duplex TaqMan real-time RT-PCR assay for simultaneous detection of goose astrovirus genotypes 1 and 2

Goose astrovirus (GAstV) is a novel pathogen that was discovered in 2018. It has two genotypes, GAstV-1 and GAstV-2, and both can cause visceral gout of goslings and result in significant economic losses. The present work aimed to develop a duplex TaqMan real-time quantitative reverse transcription PCR (RT-qPCR) assay to distinguish the two genotypes. MegAlign software was used to design two pairs of primers and a pair of matched probes based on the open reading frame 2 (ORF2) sequence with the greatest difference between GAstV-1 and GAstV-2, and primer and probe concentrations and annealing temperatures were optimised. Fluorescence signals were obtained for GAstV-1 and GAstV-2 in the FAM and VIC channels, respectively, but no fluorescent signal was observed for other pathogens. The detection limit for GAstV-1 and GAstV-2 was 33.3 and 33.7 DNA copies/μL, respectively. Intra- and inter-assay variability tests revealed excellent reproducibility. Furthermore, the assay detected GAstV-1 and GAstV-2 in allantoic fluids (100% positive) spiked with viruses, and 70 clinical gout gosling samples were examined, of which 11.4% were positive for GAstV-1, 74.3% were positive for GAstV-2%, and 5.7% were positive for mixed infection. In summary, the developed duplex RT-qPCR assay has high specificity, sensitivity, and reproducibility, and can be used in the clinic for detection of GAstV-1 and GAstV-2.