Multiplex fluorescence quantitative PCR for the detection of eight bacterial pathogens of porcine respiratory disease complex (PRDC)

As rapid and reliable tools for diagnosis of porcine respiratory disease complex (PRDC) bacterial pathogens are getting more important over time, we established a new multiplex fluorescence quantitative polymerase chain reaction (mFQ-PCR) system with a similar symptom. Our results showed that the system could accurately and simultaneously detect 8 pathogens within 1.5 h. The detection limits were 2-5 standard DNA copies. The correlation coefficient of the standard curve was more than 0.998, with a well dynamic range from 101 to 109, and the intra- and intervariation coefficients (CVs) were less than 2.06%. After setting the positive threshold of Ct value, the coincidence rate between mFQ-PCR and conventional PCR (cPCR) could reach 92%-100%, the specificity and sensitivity of mFQ-PCR exceeded 93.5% and 90.8%, respectively. We also found that the positive rates of 8 bacteria were 12.5%-68.2%, while the rates of co-infection were 90%-100% in the clinical samples. The co-infection among Streptococcus suis (Ss), Haemophilus parasuis (Hps), and Mycoplasma hyorhinis (Mhr) accounted for 78.3%, 91.4%, 100%, respectively of all the positive samples, among Actinobacillus pleuropneumoniae (App) and Mycoplasma pneumoniae (Mhp) are 45.5% and 35.7%, among Bordetella bronchis (Bb), Pasteurella multocida (Pm) and toxic Pasteurella multocida (T$^{+}$Pm) are 52.2%, 41.4%, and 100%, respectively. The results demonstrated that co-infection of 2-4 pathogenic bacteria with similar symptoms was a common situation for PRDC in south of China; our method has the potential to become a reliable detection tool.