Development of Reverse Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of Batai Virus in Cattle and Mosquitoes.

Abstract

Batai virus (BATV) is an arthropod-borne single-stranded RNA virus belonging to the genus Orthobunyavirus of the family Bunyaviridae that is primarily transmitted by mosquitoes. Methods for detecting BATV are currently limited to serological surveillance, virus isolation, and conventional reverse transcription-polymerase chain reaction (RT-PCR) assay. In this study, we sought to develop a BATV detection assay that needs no specialized equipment and is highly specific, sensitive, and simple. We first developed and optimized a reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of BATV that uses two pairs of primers to amplify a conserved region of the BATV M gene. The optimal reaction conditions for this RT-LAMP BATV detection assay were 40 min at 65°C. The amplification products could be visualized directly for color changes. This RT-LAMP method has a detection limit of 2.86 copies/μL and a sensitivity that was approximately 10- and 100-fold greater than real-time and conventional RT-PCR, respectively. RT-LAMP for BATV detection showed no cross-reactivity with other viruses and its sensitivity was validated with cattle blood and mosquito specimens. Our results suggest that this RT-LAMP method was simpler and faster than conventional RT-PCR or real-time RT-PCR. Moreover, RT-LAMP represents a potential tool to test for BATV in clinical and mosquito samples, especially in rural areas of China. This method also shows promise as a diagnostic tool due to its rapid and sensitive detection without the need for sophisticated equipment or complicated protocols.