Duplex MIRA assays for simultaneous detection of novel goose astrovirus and goose circovirus

Novel goose astrovirus (NGAstV) and goose circovirus (GoCV) are two major pathogens responsible for disease outbreaks in goslings, causing substantial economic losses to the goose farming industry. In this study, two duplex multienzyme isothermal rapid amplification (MIRA) assays were developed for the simultaneous detection of NGAstV and GoCV, and the specificity and sensitivity of these detection methods were evaluated. Both MIRA assays demonstrated high specificity for NGAstV and GoCV, with no cross-reactivity observed with six waterfowl pathogens, including duck enteritis virus, goose parvovirus, fowl adenovirus serotype 4, H9 subtype avian influenza virus, Muscovy duck reovirus and duck Tembusu virus. The basic duplex MIRA assay completed amplification within 25 min under a constant temperature of 25 °C, with minimum detection limits of 1 × 10 2 copies/µL for NGAstV and 1 × 10 3 copies/µL for GoCV. In contrast, the duplex MIRA-qPCR assay reduced the reaction time to 20 min at 39 °C, and increased the sensitivity to 1 × 10 1 copies/µL for NGAstV and 1 × 10² copies/µL for GoCV. Fluorescence imaging technology enables differentiation of infection types based on color variations: mixed infections appear yellow, single NGAstV infections show green fluorescence, and single GoCV infections exhibit red fluorescence. In the clinical sample testing, the detection rates of the two pathogens were relatively high, with a mixed infection rate of up to 18%. This method significantly improves pathogen detection efficiency and serves as an effective tool for the rapid identification of NGAstV and GoCV.