Mycoplasma bovis is a major pathogen of cattle, causing substantial economic losses in the global cattle industry and emerging as a serious threat to North American bison (Bison bison). Effective outbreak management is hindered by the lack of rapid diagnostic methodologies deployable in field settings. Current testing approaches are limited by slow turnaround times for microbial culture and the dependence of qPCR on centralized laboratory infrastructure, both of which are poorly suited for field settings. To address this gap, we developed a highly sensitive, isothermal diagnostic platform coupling Recombinase Polymerase Amplification with CRISPR-Cas12a detection targeting the conserved uvrC gene. The optimized assay requires less than 1 h at 37°-39°C and achieves an analytical limit of detection of approximately 8 copies, comparable to that of the gold-standard quantitative PCR. Clinical validation using 110 samples from bison and cattle demonstrated 100% concordance with PCR and complete differentiation from closely related species, including Mycoplasma canis and Mycoplasma felis. Overall, these data establish the RPA-CRISPR/Cas12a assay as a sensitive and specific isothermal approach for detecting M. bovis in clinical specimens, providing a field-adaptable framework for future instrument-free surveillance in resource-limited agricultural settings and supporting improved herd health management in both commercial cattle and bison conservation operations.
