Identification of MDV and REV Using RPA-CRISPR/Cas12a Combined with Fluorescence and Lateral Flow Assays

Marek's disease virus (MDV) and reticuloendotheliosis virus (REV) are two major immunosuppressive oncogenic viruses that severely threaten the global poultry industry. Field co-infection of these two pathogens is prevalent, accompanied by highly similar pathological lesions, making conventional diagnostic technologies incapable of rapid on-site differential identification. Recombinase polymerase amplification (RPA) coupled with the CRISPR/Cas12a system represents an innovative platform for rapid nucleic acid detection. In this study, target-specific crRNAs were designed against the conserved unique genes of MDV and REV, respectively, to establish a CRISPR-based diagnostic system capable of differentiating the two viruses for the first time. Comprehensive optimization of RPA reaction composition, buffer formulation and incubation temperature improved the overall detection performance. Three independent visual signal readout modalities were supported by this assay, including real-time fluorescence quantification, naked-eye fluorescence visualization under blue light, and lateral flow assay (LFA). Sensitivity evaluation demonstrated that the limit of detection (LOD) reached 1×10⁰ copies/μL for MDV and 1×10¹ copies/μL for REV. Specificity assays verified that the system only generated specific positive signals in response to target viruses without cross-reactivity against other common avian oncogenic viruses and their subgroups. Blind testing of 30 clinical chick fecal specimens revealed that the assay achieved a 100% positive detection rate for both viruses when referenced to qPCR and RT-qPCR, with outstanding diagnostic efficacy and perfect detection consistency. Collectively, the developed RPA-CRISPR/Cas12a assay overcomes the limitations of traditional diagnostic methods and holds great promise for on-site routine screening in poultry farms and epidemiological surveillance of MDV and REV.