The Development of a SYBR Green I Multiple Real-time Fluorescence PCR Assay for Detection of Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida

Actinobacillus pleuropneumoniae, Haemophilus parasuis, and Pasteurelle multocida are common pathogens of respiratory diseases in the pig industry, and they may cause secondary infections and serious economic losses to the pig industry. The clinical symptoms caused by these three pathogens are difficult to distinguish with the naked eye, and mix infections bring difficulties to the diagnosis of diseases. In this study, specific primers were designed on the basis of A. pleuropneumoniae Apx IV, H. parasuis Omp P2 and P. multocida PlpE gene. The expected amplified products ofA. pleuropneumoniae, H. parasuis, and P. multocida were 157, 120 and 305 bp, respectively. After the amplified fragment was cloned into a vector, a standard plasmid was constructed. By using the standard plasmid as template, a fluorescence quantitative PCR method for simultaneous detection ofA. pleuropneumoniae, H. parasuis, and P. multocida multiple SYBR Green I was established. Combined with melting curve analysis, the sensitivity, specificity, and repeatability were also evaluated. The results showed that the sensitivity of the method for detecting the three pathogens were 147, 145, and 61 copies/mu L. On the same melting curve that produced three specific Tm peaks, no cross reaction with other bacteria was observed, and the method demonstrated good specificity and repeatability. This method could be used for the simultaneous detection of the three pathogens, thus providing an effective detection tool for disease prevention and treatment.