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The assays were validated using bronchoalveolar lavage fluid and nasal swab samples collected from sheep. The positive rate of RPA-LFD (97.4%) was higher than the real-time PCR (95.8%) with DNA as a template purified from the clinical samples. The RPA assay was significantly better at detecting M. ovipneumoniae in clinical samples compared to the real-time PCR when DNA extraction was omitted (50% and 34.4% positive rate for RPA-LFD and real-time PCR respectively). The RPA-LFD developed here allows easy and rapid detection of M. ovipneum