A crRNA/Cas12a complex-driven rapid and visual detection method for four porcine diarrhea viruses

BACKGROUND: Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine delta-coronavirus (PDCoV), and porcine rotavirus-A (PoRV) G9 are major swine pathogens primarily responsible for gastrointestinal diseases, particularly affecting lactating piglets and resulting in significant economic losses, especially in China. This study reports a novel CRISPR-based nucleic acid detection method that integrates the high specificity of huLbCas12a with the sensitivity of loop-mediated isothermal amplification (LAMP) technology. Central to this method, the crRNA/Cas12a complex, enhances diagnostic accuracy through targeted gene editing. In this approach, the nucleic acids of the four viruses are amplified in parallel by LAMP and subsequently detected in four singleplex CRISPR–Cas12a reactions performed in separate tubes, with the incorporation of fluorescent reporter probes and a lateral flow dipstick assay establishing a visual detection system capable of separately identifying each of the four viruses. RESULTS: It enables the visual detection of viral genomes from as low as 1 copy/µL without cross-reactivity. In comparative testing of 95 clinical samples, our quadruplex LAMP-CRISPR assay demonstrated 100% concordance with RT-qPCR for the three porcine coronaviruses and 98.9% concordance with RT-qPCR for PoRV G9. CONCLUSIONS: Offering a robust and reliable tool for on-site virus detection, this method significantly aids in the timely prevention of virus spread and mitigates its impact on the pig farming industry, demonstrating its potential role in enhancing biosecurity and disease management in veterinary contexts.