Mycoplasma ovipneumoniae (Mo) is a common pathogen prevalent worldwide that causes pneumonia and leads to significant economic losses in sheep farming. Early testing of nasal swabs from diseased sheep in Xinjiang yielded a 51.5% positivity rate, indicating severe infection within the sheep population. Existing detection methods are time-consuming and rely on large-scale equipment, making them unsuitable for frontline clinical testing. Rapid, accurate, and on-site pathogen detection is essential for the early prevention and control of infectious diseases. Conventional nucleic acid detection methods are limited by long reaction duration, sophisticated instruments, and strict operating requirements, which restrict their application in grassroots rapid diagnosis scenarios. Herein, we established a combined Mo-RPA integrated CRISPR/Cas12a detection assay, which realized constant-temperature nucleic acid detection without large-scale laboratory equipment. The entire detection workflow was completed within 30 min under a constant temperature of 37°C. The optimized assay achieved a limit of detection of 10° copies/μL, with 100% analytical specificity against non-target pathogen strains. In clinical sample verification, the detection consistency between the established method and standard real-time PCR assay reached 100%. The established constant-temperature detection system integrates rapid nucleic acid amplification and CRISPR fluorescent signal output and provides a quantifiable on-site detection scheme for grassroots epidemic surveillance.
