Mycoplasma synoviae (MS) is an important avian pathogen that has brought substantial economic losses to the global poultry industry. Fast and accurate diagnosis is one of the critical factors for the control of MS infection. This study established a simple, rapid and visual detection method for MS using a recombinase-aided amplification (RAA) combined with a lateral flow dipstick (LFD). The reaction temperature and time of the RAA-LFD assay were optimized after selecting the primers and probe, and the specificity and sensitivity rates were analyzed. The results showed that RAA could amplify the target gene in 20 min at a constant temperature of 38°C, and the amplification products could be visualized by LFD within 5 min. There was no cross-reaction with Mycoplasma gallisepticum (MG), Pasteurella multocida ( P. multocida ), Escherichia coli ( E. coli ), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), and avian reovirus (ARV). Furthermore, the RAA-LFD assay exhibited high sensitivity with a detection limit of 10 copies/μL. A total of 128 clinical samples with suspected infection of MS were tested by RAA-LFD, PCR, and real-time fluorescence quantitative PCR (RFQ-PCR). The coincidence rate of the detection results was 95.3% between RAA-LFD and PCR, and 98.4% between RAA-LFD and RFQ-PCR. These results suggested that the RAA-LFD method established in the present study was easy to use and was associated with strong specificity and high sensitivity. This method was very suitable for the rapid detection of MS in clinical practice.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases that has brought significant economic losses to the swine industry worldwide. Rapid and accurate PRRS virus (PRRSV) detection is one of the key factors for PRRS prevention and control. This study developed a real-time fluorescence-based reverse transcription recombinase-aided amplification (RF-RT-RAA) method for type 2 PRRSV (PRRSV-2) detection. The RF-RT-RAA assay could be performed at 42 °C for 20 min with the optimal primers and a probe. RF-RT-RAA results could be monitored using real-time fluorescence read-out or visually observed with the naked eye using a portable blue light transilluminator. The method had a strong specificity; no cross-reaction was identified with the detected common swine viruses. Moreover, the technique yielded high sensitivity with the lowest detection limit of 10¹ copies/μL and exhibited good repeatability and reproductively with the coefficients of variation (CV) less than 10%. Eighty-seven clinical samples were tested using RF-RT-RAA and a commercial PRRSV-2 RT-qPCR detection kit. The coincidence rate was 100% between RF-RT-RAA (real-time fluorescence read-out) and RT-qPCR, and 97.7% between RF-RT-RAA (visually observed) and RT-qPCR. The RF-RT-RAA assay provides a new method for rapid and visual detection of PRRSV-2.

African swine fever (ASF) is one of the most severe diseases caused by the ASF virus (ASFV), causing massive economic losses to the global pig industry. Serological tests are important in ASF epidemiological surveillance, and more antigen targets are needed to meet market demand for ASFV antibody detection. In the present study, ASFV p15 protein was fusion-expressed in Escherichia coli (E. coli) with elastin-like polypeptide (ELP), and the ELP-p15 protein was purified using a simple inverse transition cycling (ITC) process. The ELP tag was cleaved off using tobacco etch virus protease (TEVp), resulting in a tag-free p15 protein. Western blot analysis demonstrated that the p15 protein reacted strongly with ASFV-positive serum. The p15 protein was used as a coating antigen in an indirect ELISA (iELISA) for detecting ASFV antibodies. The p15-iELISA method demonstrated high specificity to ASFV-positive sera, with a maximum detection dilution of 1:1600. Moreover, the method exhibited good reproducibility, with less intra-assay and inter-assay CV values than 10%. Therefore, p15-iELISA offers a novel approach for accurately detecting ASFV antibodies with significant clinical application potential.