Research Note: Real-time fluorescence-based recombinase-aided amplification for rapid detection of Mycoplasma synoviae

Mycoplasma synoviae ( MS ) is an essential pathogenic mycoplasma in poultry worldwide, posing a serious threat to the poultry industry 's health. Timely detection is imperative for early diagnosis, prevention, and control of MS infection. Current laboratory methods for MS detection are generally complicated, time-consuming, and require sophisticated equipment. Therefore, a simple and rapid method is urgently needed. This study developed a novel real-time fluorescence-based recombinase-aided amplification ( RF- RAA ) technique for detecting MS nucleic acids, enabling target gene amplification within 20 min at 39 degrees C. The RF-RAA outcomes are interpretable in 2 modalities: real-time fluorescence monitoring employing a temperature-controlled fluorescence detector or direct visual inspection facilitated by a portable blue light transilluminator. This method exhibits robust specificity, demonstrating no cross-reactivity with various common poultry pathogens, and achieves high sensitivity, detecting as low as 10 copies/ mL for the standard plasmid. Seventy-one clinical samples of chicken throat swabs were detected by RF-RAA and realtime fluorescence quantitative polymerase chain reaction ( qPCR ) methods. The diagnostic coincidence rates of qPCR with RF-RAA (fluorescence monitoring) and RF-RAA (visual observation) were determined to be 100% and 97.2% (69/71), respectively. In conclusion, the RF-RAA method developed in this study provides a rapid and visually observable approach for MS detection, offering a novel technique to diagnosing MS infection, especially in resource-limited settings.