Detection of duck adenovirus 3 using RAA-CRISPR/Cas12a based lateral flow dipstick method.

Duck adenovirus 3 (DAdV-3) was firstly identified in Muscovy ducklings exhibiting slight pericardial effusion, swelling, and hemorrhaging in the kidneys, which is a poorly characterized duck virus. This study established a CRISPR/Cas12a coupled with a Lateral flow dipstick (RAA-CRISPR/Cas12a-LFD) detection platform for DAdV-3. The core mechanism of this platform involves a three-step cascade reaction pathway. First, the target sequence of the DAdV-3100 k gene is amplified via RAA isothermal amplification. Second, the amplified products activate the trans-cleavage activity of Cas12a. Finally, signal output is achieved by cleaving probes on the Lateral flow dipstick. This method exhibits high sensitivity, with a minimum detection limit of 2.87 × 102 copies/μL for the DAdV-3 positive plasmid. It also demonstrates high specificity for DAdV-3, showing no cross-reactivity with other common duck-origin viruses such as avian influenza virus (AIV), Newcastle disease virus (NDV), duck Tembusu virus (DTMUV), duck plague virus (DPV), Muscovy duck parvovirus (MDPV), duck hepatitis A virus type 1 (DHAV-1), and duck reovirus (DRV). The method enables on-site detection of DAdV-3 within 1 h and 20 min. It does not rely on standardized molecular laboratories or professional operation, provides visualized detection results, allows rapid detection at the grassroots level, and has low detection costs, making it suitable for promotion in small-scale farms. This approach provides a technical means for the detection and epidemiological investigation of DAdV-3.