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

作者：Di Sun, Yukun Zhu, Mingshu Wang, Jingming Wang, Wenze Cheng, Zheng Li, Yuan Deng, Xumin Ou, Renyong Jia, Shun Chen, Dekang Zhu, Mafeng Liu, Xinxin Zhao, Qiao Yang, Ying Wu, Shaqiu Zhang, Juan Huang, Yu He, Zhen Wu, Anchun Cheng · 发表于：Poultry Science · 年份：2025 · DOI：10.1016/j.psj.2025.105316 · 被引用次数：4 · 研究领域：Viral Infections and Immunology Research、CRISPR and Genetic Engineering、Animal Virus Infections Studies

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-tim...