CRISPR/Cas14a Combined with RPA for Visual Detection of Marek’s Disease Virus

Marek’s disease (MD), a highly contagious avian immunosuppressive disorder caused by the α-herpesvirus MDV-1, poses a significant threat to poultry health. The development of rapid visual detection methods capable of distinguishing epidemic MDV-1 strains from vaccine strains is crucial for early disease warning, vaccine efficacy evaluation, and precise disease control. We developed a novel isothermal detection system that integrates recombinase polymerase amplification (RPA) with CRISPR/Cas14a technology for the visual identification of epidemic MDV-1 strains. This method operates at a constant temperature of 37°C and allows for either real-time analysis or endpoint visual readout without the need for complex instrumentation. Our results showed no cross-reactivity with Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), MDV-1 vaccine strains, or herpesvirus of turkeys (HVT). Plasmid DNA standards were used to determine the sensitivity of the assay and the detection limit was 24.6 copies/μL. Clinical evaluation using 24 field samples confirmed that the method successfully identified all MDV-positive cases, demonstrating its diagnostic reliability. In conclusion, we have developed a rapid, instrument-free, and highly specific nucleic acid detection platform for MDV-1 by combining the sensitivity of RPA with the specificity of CRISPR/Cas14a technology, offering promising potential for field-based diagnostics and disease surveillance.