A RT-ERA-CRISPR/Cas12a assay for rapid point-of-care duck hepatitis A virus detection.

Duck hepatitis A virus (DHAV) is a severe pathogen that threatens the duck industry. DHAV is transmitted primarily through the respiratory and gastrointestinal tracts. Therefore, developing accurate and rapid diagnostic technologies is crucial to prevent the spread of this infectious disease. Currently, the most widely used DHAV detection methods in clinical diagnosis include PCR, real-time PCR (RT-PCR) and ELISA, which require specialized equipment or trained professionals. Isothermal amplification technologies are often combined with lateral flow assays or visual readout methods for detecting pathogen nucleic acids in non-laboratory settings. CRISPR nucleases have accelerated the development of nucleic acid detection, increasing the sensitivity to a higher degree. Here, we applied reverse transcription-enzymatic recombinase amplification (RT-ERA) assisted by a Cas12a-fluorescence assay and a Cas12a-lateral flow assay for the detection of DHAV-1. Based on the sequence of DHAV-1, RT-ERA primers and crRNAs were designed, and different concentrations of ssDNA/Cas12a/crRNA were established to optimize the CRISPR reaction. The LoD for the Cas12a-fluorescence assay was 10 copies/μL, and this assay effectively differentiated DHAV-1 from other avian pathogens, exhibiting high sensitivity and specificity. Additionally, the Cas12a-lateral flow assay is user-friendly and can achieve point-of-care detection. Sixty-four clinical samples were tested and compared with quantitative real-time PCR (RT-PCR). This accurate and rapid point-of-care assay has significant potential for detecting DHAV-1 in clinical applications, especially for duck farms in rural areas.