Rapid and Sensitive One-Tube Detection of Getah Virus Using RT-LAMP Combined with Pyrococcus furiosus Argonaute.

Abstract

Getah virus (GETV) is a mosquito-borne virus that poses a significant threat to both animal and public health. Traditional diagnostic methods for GETV, such as RT-PCR and RT-qPCR, require expensive equipment and complex procedures, making them unsuitable for rapid, on-site detection. The combination of RT-LAMP and PfAgo offers a novel approach for nucleic acid detection, providing high specificity and effective without the need for sophisticated instruments. Herein, we developed a RT-LAMP combined with PfAgo assay for GETV detection. The RT-LAMP assay was conducted at 60~65 °C, and then the RT-LAMP product was cleaved, together with a fluorescent probe, mediated by PfAgo at 95 °C. After optimizing the primary reaction conditions, the detection limit of the RT-LAMP-PfAgo assay was 100 copies/µL. Importantly, there was no cross-reactivity with other viruses, including PEDV, PDCoV, PoRV, PRRSV, and CSFV. Compared to qPCR, analysis of 86 clinical samples showed that LAMP-PfAgo had a consistent positive rate with the qPCR method. In conclusion, we developed a valuable diagnostic tool for the rapid detection of GETV, enabling timely surveillance and control measures to mitigate the impact of GETV outbreaks.