Abstract：Abstract Orf virus (ORFV) is the pathogen of Contagious ecthyma (CE). Mycoplasma ovipneumoniae (Mo) is the main pathogen of Mycoplasma pneumonia of goats and sheep (MPGS). In recent years, the mixed infection of ORFV and Mo is increasing. Currently, the main detection method is single PCR, it is imperative to develop a method for rapid and simultaneous detection of ORFV and Mo in clinics. Thus, two pairs of specific primers were designed according to the major envelope protein B2L gene of ORFV and membrane protein P80 gene of Mo. The results showed that the assay could amplify 402 bp for ORFV and 700 bp for Mo simultaneously, but other common pathogen's DNA could not amplify, indicating good specificity. The detection limits of the method was 1.1×103 copies/μL for ORFV and 3.47×103 copies/μL for Mo, respectively, and these limits were 10 times more sensitive than the single PCR assay. The duplex PCR and single PCR were simultaneously performed to detect 83 clinical samples from Fujian, and the results showed that the positive rate of ORFV and Mo were 33.7% and 39.8%, respectively, co-infection rate of ORFV and Mo was 10.8%. The date indicated that the duplex PCR established in this study can be used for clinical rapid detection of ORFV and Mo and epidemiological investigation of CE and MPGS.
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