Rapid visual detection of Mycoplasma gallisepticum by combining recombinase-aided amplification with lateral-flow dipstick assay

Mycoplasma gallisepticum (MG) is a significant avian pathogen that adversely affects chickens and turkeys globally and is classified as a notifiable disease agent by the World Organisation for Animal Health. Therefore, rapid and accurate diagnosis is a critical factor in controlling MG infections. This study established a simple, rapid, and visual detection method for MG using recombinase-aided amplification (RAA) combined with a lateral flow dipstick (LFD). Following primer and probe screening, the RAA-LFD method was optimized for primer concentration, probe concentration, reaction temperature, and time, and its specificity and sensitivity were analyzed. The results demonstrated that under isothermal conditions at 39°C, RAA could amplify the target gene within 20 minutes, and the products could be visualized via LFD within 5 minutes. The method showed no cross-reactivity with Mycoplasma synoviae, Mycoplasma bovis, Pasteurella multocida, Mannheimia haemolytica, Staphylococcus aureus, Salmonella spp., Escherichia coli, Listeria monocytogenes, Enterococcus faecium, or Enterococcus faecalis. Furthermore, the RAA-LFD method exhibited high sensitivity and good reproducibility, with a detection limit of 10 copies/μL. A total of 100 clinical samples suspected of MG infection were tested using RAA, PCR, and real-time fluorescence quantitative PCR (qPCR). The concordance rates between the RAA detection method and conventional PCR or qPCR were 96% and 98%, respectively. These results indicate that the established RAA-LFD method is easy to operate, highly specific, and sensitive, making it suitable for grassroots-level field diagnosis of MG infections.