A New Multiplex PCR Assay for Simultaneously Detecting Mycoplasma ovipneumoniae and Corynebacterium pseudotuberculosis in Diseased Goats and Sheep

  Objective   To establish a multiplex PCR method for simultaneous clinic detection of Mycoplasma ovipneumoniae (Mo), one of the main pathogens causing Mycoplasma pneumonia of goats and sheep(MPGS), and Corynebacterium pseudotuberculosis (CP), the pathogen of goat pseudotuberculosis, in the livestock animals infected by both pathogens at a same time.

  Method   Two pairs of specific primers were designed based on the P80 gene of Mo and the PLD gene of CP for the PCR. After optimizing the detection system and reaction conditions, the specificity, sensitivity, and repeatability of the new method were verified for methodology validation. The results obtained using two separate PCR and the multiplex PCR on 70 clinical specimens were comparatively analyzed.

  Result   The newly developed multiplex PCR method amplified a 700 bp fragment from Mo and a 290 bp fragment from CP, with none from other common pathogens of goats and sheep. The method had a detection limit of 1 530 pg·μL⁻¹ on Mo and 3 500 pg·μL⁻¹ on CP with a high repeatability. A test on 70 clinical specimens using the new method yielded a 25.71% positive rate on Mo, 18.57% on CP, and 7.14% on Mo/CP infections. Its coincidence rate with the two single PCR results was 100%.

  Conclusion  The newly established multiplex PCR detection methodology exhibited high specificity, sensitivity, and reproducibility. It appeared to be applicable for rapid and simultaneous clinic detection on Mo and CP as well as epidemiological studies on the disease infected by the pathogens.