A streamlined visual RT-RAA-CRISPR/Cas12a assay for rapid detection of Schmallenberg virus

Abstract

Schmallenberg virus (SBV), an emerging Culicoides‑borne arbovirus, is responsible for febrile illness and reduced milk production in adult ruminants and can induce congenital malformations in fetuses, representing a substantial concern for livestock health worldwide. Consequently, rapid, field‑adaptable diagnostic approaches are urgently needed. In this study, we developed a visual nucleic acid detection assay for SBV that combines reverse transcription recombinase‑aided amplification (RT‑RAA) with the CRISPR/Cas12a system, targeting conserved regions of the SBV S gene. Following systematic optimization of the reaction conditions, the assay was completed within 50 min, with a sensitivity of 8.6 copies/μL for the SBV-S plasmid and 8.6 × 101 copies/μL for the RNA transcripts. The assay exhibited high specificity, with no cross‑reactivity observed against a panel of relevant pathogens, including Seoul orthohantavirus (SEOV), infectious bovine rhinotracheitis virus (IBRV), Rift Valley fever virus (RVFV), Crimean‑Congo hemorrhagic fever virus (CCHFV), and bovine viral diarrhea virus (BVDV). When evaluated using simulated clinical samples, the sensitivity of the method was superior to that of conventional real-time fluorescent reverse transcription‒polymerase chain reaction (RT‑qPCR). Importantly, the entire reaction is performed in a single closed-tube format, significantly reducing the risk of cross-contamination and false-positive results. In summary, this method demonstrates favorable analytical performance for the detection of SBV-S plasmid-spiked nasal swab samples and holds promise for further clinical validation, although its diagnostic utility in authentic clinical specimens remains to be confirmed in future investigations.