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Background  Mycoplasmal pneumonia is an important infectious disease that threatens sheep and goat production worldwide, and  Mycoplasma ovipneumoniae  is one of major etiological agent causing mycoplasmal pneumonia. Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technique, and RPA-based diagnostic assays have been described for the detection of different types of pathogens.    Results  The RPA assays using real-time fluorescence detection (real-time RPA) and lateral flow strip detection (LFS RPA) were developed to detect  M. ovipneumoniae  targeting a conserved region of the 16S rRNA gene. Real-time RPA was performed in a portable florescence scanner at 39 °C for 20 min. LFS RPA was performed in a portable metal bath incubator at 39 °C for 15 min, and the amplicons were visualized with the naked eyes within 5 min on the lateral flow strip. Both assays were highly specific for  M. ovipneumoniae , as there were no cross-reactions with other microorganisms tested, especially the pathogens involved in respiratory complex and other mycoplasmas frequently identified in ruminant s . The limit of detection of LFS RPA assay was 1.0 × 10 1  copies per reaction using a recombinant plasmid containing target gene as template, which is 10 times lower than the limit of detection of the real-time RPA and real-time PCR assays. The RPA assays were further validated on 111 clinical sheep nasal swab and fresh lung samples, and  M. ovipneumoniae  DNA was detected in 29 samples in the real-time RPA, 31 samples in the LFS RPA and 32 samples in the real-time PCR assay. Compared to real-time PCR, the real-time RPA and LFS RPA showed diagnostic specificity of 100 and 98.73%, diagnostic sensitivity of 90.63 and 93.75%, and a kappa coefficient of 0.932 and 0.934, respectively.    Conclusions  The developed real-time RPA and LFS RPA assays provide the attractive and promising tools for rapid, convenient and reliable detection of  M. ovipneumoniae  in sheep, especially in resource-limited settings. However, the effectiveness of the developed RPA assays in the detection of  M. ovipneumoniae  in goats needs to be further validated.

Development and validation of the isothermal recombinase polymerase amplification assays for rapid detection of Mycoplasma ovipneumoniae in sheep