Loop-mediated isothermal amplification combined with a Pyrococcus furiosus argonaute system for the rapid detection of goose astrovirus.

Abstract

Goose astrovirus (GAstV) is a newly emerged viral pathogen in goose, characterized by a high incidence and significant mortality rates. This etiology has repeatedly occurred in coastal areas of China, rapidly spreading to inland provinces in recent decade, and thus imposed huge economic losses on China's goose industry. Therefore, it is essential to establish a rapid, accurate and sensitive method for GAstV diagnosis. In this study, we employed reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay combined with a Pyrococcus furiosus Argonaute (PfAgo) system, providing a simple and precise approach for GAstV. Specific primers and guide DNA (gDNA) were designed to target conserved regions of the GAstV genome. The assay achieved a detection limit of 10 copies/μL in preliminary validation assays when targeting conserved regions of the viral genome with optimized reaction conditions. Importantly, the assay exhibited no cross-reactivity with other viruses, including Goose parvovirus (GPV), Goose circovirus (GoCV), Tembusu Virus (TMUV), Muscovy Duck Reovirus (MDRV), and Escherichia coli (E. coli). Detection results from 59 clinical samples demonstrated complete concordance in positive rates between the LAMP-PfAgo and qPCR methods.