Development and preliminary validation of a rapid on-site detection method for Schmallenberg virus using RT-RAA-LFD.

Abstract

Schmallenberg virus (SBV) is an emerging arbovirus that causes fever, diarrhea, abortion, and congenital malformations in ruminants. The disease spreads rapidly and poses significant control challenges. Upon its identification, SBV disseminated across Europe, causing major economic losses in livestock production and international trade. Although vaccines exist in certain regions, effective treatment options remain limited, highlighting the need for rapid, early, and accurate detection. In this study, we report the development and preliminary validation of a rapid point-of-care detection method for SBV and closely related viruses based on reverse transcription recombinase-aided amplification (RT-RAA) combined with lateral flow dipstick (LFD) technology. A conserved S gene region was selected as the target, and a single-nucleotide modification was introduced into the primer-probe set to reduce false-positive signals. Due to the absence of naturally infected samples in China, sheep fetal tissues spiked with defined amounts of plasmid standards were used to simulate clinical specimens, and in vitro-transcribed SBV RNA was incorporated to verify performance at the RNA level. After optimization, the method achieved a sensitivity of 5 copies/μL. Compared with a commercial qPCR kit, the assay is faster, simpler, and does not require specialized laboratory equipment, making it well suited for on-site testing in farms, quarantine stations, and regional laboratories. As the assay detects both SBV and closely related viruses, positive results require confirmation by specific PCR or sequencing to accurately identify the virus. These results provide proof-of-concept for the assay and support its potential application in rapid SBV and related virus monitoring and control.