Rapid nucleic acid detection of Mycoplasma synoviae using dual-mode RAA–CRISPR/Cas12a system

Mycoplasma synoviae is a significant avian pathogen implicated in diverse clinical manifestations, including air sacculitis, infectious synovitis with arthritis, and eggshell apex abnormalities, which significantly threaten the poultry industry. To enable the rapid and accurate detection of M. synoviae, we developed a dual-mode diagnostic platform that combines recombinant enzyme-assisted amplification (RAA) with CRISPR/Cas12a-based fluorescence and lateral flow dipsticks (LFD). The Qsep100 automatic nucleic acid analysis system was used to screen for RAA primers by evaluating the amplicon size and concentration. Fluorescence monitoring revealed that the optimal reaction time for the RAA-CRISPR/Cas12a system was 28 min. After optimizing the concentrations of LbaCas12a, crRNA, and the ssDNA reporter, the assay achieved a sensitivity of 5.2 copies/µL. Cross-reactivity testing with seven common avian pathogens confirmed the high specificity of the established method for detecting M. synoviae. In the clinical validation, the method perfectly matched the results of quantitative real time polymerase chain reaction (qPCR). Furthermore, an epidemiological investigation revealed that chickens had the highest positivity rate for M. synoviae among the chickens, ducks, and pigeons. In summary, we developed a rapid, accurate, and portable diagnostic platform for M. synoviae detection, which provides a valuable tool for disease prevention and control in resource-limited settings.