CRISPR/Cas12a-Based One-Tube RT-RAA Assay for PoRV Genotyping

Porcine rotavirus (PoRV), a primary etiological agent of viral diarrhea in piglets, frequently co-infects with other enteric pathogens, exacerbating disease severity and causing substantial economic losses. Its genetic recombination capability enables cross-species transmission potential, posing public health risks. Globally, twelve G genotypes and thirteen P genotypes have been identified, with G9, G5, G3, and G4 emerging as predominant circulating strains. The limited cross-protective immunity between genotypes compromises vaccine efficacy, necessitating genotype surveillance to guide vaccine development. While conventional molecular assays demonstrate sensitivity, they lack rapid genotyping capacity and face technical limitations. To address this, we developed a novel diagnostic platform integrating reverse transcription recombinase-aided amplification (RT-RAA) with CRISPR-Cas12a. This system employs universal primers for the simultaneous amplification of G4/G5/G9 genotypes in a single reaction, coupled with sequence-specific CRISPR recognition, achieving genotyping within 50 min at 37 °C with 10⁰ copies/μL sensitivity. Clinical validation showed a high concordance with reverse transcription quantitative polymerase chain reaction (RT-qPCR). This advancement provides an efficient tool for rapid viral genotyping, vaccine compatibility evaluation, and optimized epidemic control strategies.