Rapid extraction-free RT-RAA assay for on-site detection of goose astrovirus type 1 in goslings

Goose astrovirus type 1 (GAstV-1) is a major pathogen threatening the goose industry in China, frequently causing severe digestive tract lesions and growth retardation in goslings. In recent years, the average annual incidence in farms has remained at 15-25%, leading to substantial economic losses. To enable rapid, accurate, and on-site detection, this study developed a nucleic acid extraction-free, visual detection method for GAstV-1 based on reverse transcription recombinase-aided amplification (RT-RAA). Targeting a conserved region of the GAstV-1 ORF1a gene, we designed and screened primers and probes with high sequence conservation and specificity. The RT-RAA assay demonstrated high specificity for GAstV-1, with no cross-reactivity observed against other common avian viruses. In terms of sensitivity, the limit of detection (LOD) for the real-time fluorescent RT-RAA assay was 199.55 copies per reaction (95% confidence interval, CI), while the visual RT-RAA had an LOD of 1150.85 copies per reaction. Using reverse transcription quantitative polymerase chain reaction (RT-qPCR) as a reference standard, the assay was validated using 122 clinical samples from suspected cases. The real-time fluorescent RT-RAA showed 100% agreement with RT-qPCR and a Kappa value of 1. The visual RT-RAA exhibited a sensitivity of 92.98%, a specificity of 100%, and a Kappa value of 0.93, indicating excellent clinical agreement. This method eliminates the need for sophisticated instruments and nucleic acid extraction steps. When combined with a portable blue-light imager, the results can be readily interpreted, making it suitable for rapid preliminary screening and epidemiological surveillance at farms and primary quarantine sites. This technique provides an efficient and scalable on-site detection tool for the early diagnosis of GAstV-1, epidemiological warning, and precise control, promising to significantly enhance biosecurity and disease prevention capabilities in the global goose industry.