Development and application of a dual RT-qRPA assay for rapid differentiating of goose astrovirus genotypes 1 and 2

Goose astrovirus (GAstV) is an important pathogen causing joint and visceral gout in goslings. Its high incidence and mortality had caused enormous economic losses to the goose farming industry. To achieve the simultaneous and rapid identification and detection of Goose astrovirus genotype 1 (GAstV-1) and genotype 2 (GAstV-2), the present study aimed to design and synthesize specific recombinase polymerase amplification (RPA) primers and exo probes targeting the ORF1b gene of GAstV. Utilizing a portable fluorescence detector (Genie III), the RPA primers and exo probes were optimized for concentration, and the reaction temperature was adjusted to establish a dual real-time reverse transcription recombinase polymerase amplification (RT-qRPA) assay for the simultaneous identification and detection of GAstV-1 and GAstV-2. The specificity and sensitivity of the assay were further evaluated. The results demonstrated that the established dual RT-qRPA assay could complete detection within 20 minutes at 42 °C, the specific amplification curves for GAstV-1 and GAstV-2 were produced in the assay, with no amplifications for other tested common goose pathogens. Utilizing the in vitro transcribed RNA as templates, the detection limits for GAstV-1 and GAstV-2 were determined to be 109 copies/reaction (95 % CI: 87.93-169.73 copies/reaction) and 86 copies/reaction (95 % CI: 65.11-179.02 copies/reaction), respectively. The developed dual RT-qRPA assay was further evaluated on 104 liver and cloacal swab samples from goslings, The GAstV-1 and GAstV-2 were detected in 19 (18.27 %, 19/104) and 69 (66.35 %, 69/104) samples, respectively, in which 16 samples (15.38 %, 16/104) carried both GAstV-1 and GAstV-2 nucleic acids. These results were consistent with those obtained using the dual RT-qPCR assay, showing a 100 % diagnostic concordance. Further analysis indicated a good linear relationship between the TT values from RT-qRPA and the CT values from RT-qPCR, with R² values of 0.965 and 0.948 for GAstV-1 and GAstV-2, respectively. The developed dual RT-qRPA assay in this study performed well on the clinical samples, thereby providing robust technical support for the rapid field differential detection of GAstV infections in goose flocks.