CRISPR/Cas14a combined with RPA for visual detection of Marek’s disease virus

Marek's disease, 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, infectious bursal disease virus, MDV-1 vaccine strains, or herpesvirus of turkeys. 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 Marek's disease virus-positive cases, demonstrating its diagnostic reliability. In conclusion, we have developed a rapid, highly specific nucleic acid detection platform for MDV-1 that enables visual readout without complex instrumentation by combining the sensitivity of RPA with the specificity of CRISPR/Cas14a technology, offering promising potential for field-based diagnostics and disease surveillance.IMPORTANCEMarek's disease virus (MDV-1) is a highly contagious and economically important avian pathogen. Existing diagnostic methods are unable to reliably distinguish between epidemic and vaccine strains in field settings, which hampers effective surveillance and evaluation of vaccination programs. To address this challenge, we developed a portable isothermal detection assay that combines recombinase polymerase amplification with CRISPR/Cas14a technology. This approach enables highly sensitive (24.6 copies/μL) and specific visual detection of epidemic MDV-1 strains without cross-reactivity with vaccine strains or related viruses. The assay demonstrated 100% agreement with reference methods when evaluated using clinical samples. As a cost-effective method that avoids the need for complex detection instruments, it offers a practical solution for rapid on-site diagnosis, facilitating enhanced outbreak control and improved poultry health management globally.