Establishment of a rapid detection method for Tembusu Virus based on MIRA-PfAgo

In this study, a novel nucleic acid detection method based on multienzyme isothermal rapid amplification (MIRA) combined with the Pyrococcus furiosus Argonaute (PfAgo) cleavage system (MIRA-PfAgo) was developed for the detection of Tembusu virus (TMUV), an important pathogen threatening the waterfowl industry. The method targets the highly conserved NS5 gene of TMUV. Target nucleic acids are rapidly enriched by MIRA under isothermal conditions at 39 °C, followed by PfAgo-mediated cleavage guided by sequence-specific guide DNA (gDNA), enabling visual detection through fluorescence signal output. Systematic optimization identified 0.5 μM gDNA, 1 μM PfAgo, and 5 mM Mn²⁺ as the optimal reaction conditions. The assay exhibited excellent specificity for TMUV, with no cross-reactivity observed with common waterfowl pathogens, including H9 subtype avian influenza virus (AIV-H9), duck enteritis virus (DEV), goose parvovirus (GPV), Muscovy duck reovirus (MDRV), Newcastle disease virus (NDV), and duck circovirus (DuCV). The sensitivity reached as low as 10⁰ copies/μL. In the analysis of 21 clinical samples, the results of the MIRA-PfAgo assay were fully consistent with those obtained by RT-PCR. This method does not require complex thermal cycling equipment and combines high sensitivity, strong specificity, and operational simplicity, providing a reliable and practical technical tool for rapid on-site monitoring of TMUV.