Development of a triplex TaqMan real-time PCR for detection of three major bacterial pathogens causing bovine respiratory disease complex
In order to establish a triplex TaqMan real-time PCR assay for detection of the three common bacterial pathogens Pasteurella multocida,Mannheimia haemolytica and Mycoplasma bovis,caus-ing bovine respiratory disease complex(BRDC),specific primers and probes targeting the 16S rRNA gene sequences of the Pas.multocida,Man.haemolytica and Myc.bovis were designed,repectively.After the op-timizing of reaction system and amplification procedure,the triplex TaqMan real-time PCR assay for de-tecting of the three common bacterial pathogens was established.The specificity test showed that the DNA samples of Pas.multocida,Man.haemolytica and Myc.bovis were positive,and the other DNA samples of the 24 common bacterial species in cattle,such as Staphylococcus aureus,Esc.coli and Salmonella en-terica,were negative by using the established assay,indicating the high specificity of the method.The results of the sensitivity test showed that the minimum detection concentrations of the recombinant plasmids pMD-Past,pMD-Mann and pMD-Myco were 38 copies/μL,45 copies/μL and 28 copies/μL,respective-ly,indicating the high sensitivity of the method.The results of repeatability test showed that the co-efficient variations were ≤2.15％,indicating the good repeatability of the method.The established assay was compared with the documented TaqMan real-time PCR assay by testing forty-three DNA samples of cattle with BRDC in parallel,and the results indicated that the total compliance rates of the two as-says for Pas.multocida,Man.haemolytica and Myc.bovis detections were 100％,97.7％and 100％,respec-tively.These results indicated that the established triplex TaqMan real-time PCR assay provided a ef-fective technical support for the high-throughput and rapid detection of the three common bacterial pathogens causing BRDC.
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