Bovine contagious pleuropneumonia and Mycoplasma bovis pneumonia are important bovine infectious diseases with respiratory symptoms that are difficult to distinguish from clinically.To establish a duplex TaqMan real-time PCR method for the differential diagnosis of these diseases,primers and probes were designed according to the conserved specific regions of the genomes of Mycoplasma mycoides subsp.mycoides(GenBank:u61140.1,from nt826 to nt1742),and Mycoplasma bovis(GenBank:afl30119.1,from nt189 to nt618).By optimizing the reaction system and conditions,a duplex TaqMan real-time PCR was established to detect these pathogens simultaneously.The specificity test results showed that the method was specific for Mycoplasma mycoides subsp.mycoides and Mycoplasma bovis without cross-reaction with other related pathogens,including Pasteurella,infectious bovine rhinotracheitis virus,Mycoplasma agalactia,Mycoplasma capricolum subsp.capripneumoniae,Mycoplasma mycoides subsp.capri,and Mycoplasma leachii strain,indicating strong specificity.The sensitivity test results showed that the detection limit of this method for Mycoplasma mycoides subsp.mycoides and Mycoplasma bovis recombinant plasmid standards both were 1.0×101 copies/μL,representing a high sensitivity.The repeatability test showed that the coefficient of variation in intra-and inter-batch was less than 2.5％,suggesting excellent repeatability.This method detected 112 clinical samples in our laboratory,which were identified as negative by routine PCR for Mycoplasma mycoides subsp.mcoides and Mycoplasma bovis.The results showed that the detection results for Mycoplasma mycoides subsp.mycoides were negative.However,Mycoplasma bovis was detected in 4 positive samples,which were confirmed to be authentic positive samples by sequencing.This study established a duplex TaqMan real-time PCR to detect Mycoplasma mycoides subsp.mycoides,and Mycoplasma bovis simultaneously.The method showed strong specificity,high sensitivity,and excellent repeatability.It can detect various clinical samples and provide a technical tool for rapidly detecting and epidemiological investigating these pathogens.
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