Establishment of a RPA Method for Detecting Mycoplasma ovipneumoniae

  Objective  To develop a rapid detecting method for Mycoplasma ovipneumoniae (Mo).

  Method  Based on the P80 gene sequence, specific primers were designed using Oligo 7 software. Conditions of the recombinase polymerase amplification (RPA) method were optimized for the application.

  Result  The new assay detected P80 gene in Mo specifically, not any other common pathogens of sheep and goats. The detection sensitivity was 70 fg·μL^-1, which was the same as provided by conventional PCR. The inter-and intra-batch tests showed that the modified method could amplify the bands on Mo-positive samples, not on Mo-negative specimens, indicating an acceptable repeatability of the methodology. Furthermore, the RPA assay and conventional PCR methods were simultaneously used on 186 clinic samples, as well as the Mycoplasma isolation and identification on 40 lung samples, to show that the RPA assay positively identified all 13 Mo-positive samples detected by Mycoplasma isolation and identification and 95 positive samples detected by the conventional PCR.

  Conclusion  The newly developed RPA method had desirable specificity and repeatability on Mo detection. It could be applied for rapid detecting and epidemiological studies on Mo.