One-pot LAMP-Argonaute assay with 3′-mismatched gDNA for duplex detection of Nipah and Japanese encephalitis viruses

Nipah virus (NiV) is an emerging zoonotic pathogen with a high mortality rate, posing a serious threat to public health. Its clinical manifestations are often indistinguishable from those of Japanese encephalitis virus (JEV), making rapid and accurate differential diagnosis essential for effective prevention and control. In this study, we developed a one-pot duplex detection assay that integrates loop-mediated isothermal amplification (LAMP) with the Pyrococcus furiosus Argonaute (PfAgo) cleavage, guided by 3′-mismatched gDNA to enhance detection efficiency. The assay exhibited high sensitivity in duplex detection, detecting as few as 1.5 copies per reaction for both NiV and JEV without cross-reactivity to major porcine pathogens. Simulated clinical validation further confirmed its on-site applicability, achieving a sensitivity of 3 copies/μL for NiV using a portable 3D-printed device. This work highlights the utility of an Ago-based detection platform for rapid, sensitive, and field-deployable diagnosis of emerging animal diseases, and offers a promising tool for the detection of other pathogens in resource-limited settings.