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.

Keywords

Schmallenberg virus / CRISPR-Cas12a system / Reverse Transcription Recombinase-Aided Amplification (RT-RAA) / Viral nucleic acid detection

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Funding

National Key Research and Development Program of China(No.2021YFF0703600)

Jilin Provincial Department of Science and Technology Project(No.20250601001RC)

RIGHTS & PERMISSIONS

The Author(s)