Development of a Triplex qPCR Assay Based on the TaqMan Probe for the Detection of Haemophilus parasuis, Streptococcus suis Serotype 2 and Pasteurella multocida

Porcine respiratory disease is a significant economic problem for the global swine industry. <i>Haemophilus parasuis</i> (<i>H. parasuis</i>), <i>Streptococcus suis</i> (<i>S. suis</i>), and <i>Pasteurella multocida</i> (<i>P. multocida</i>) are three important pathogenic bacteria of the swine respiratory tract. Notably, the three pathogens not only frequently manifest as mixed infections, but their striking clinical similarities also present difficulties for pig populations in terms of disease prevention and treatment. Thus, we developed a triplex real-time quantitative polymerase chain reaction (qPCR) assay based on a TaqMan probe for the detection of <i>H. parasuis</i>, <i>S. suis</i> serotype 2, and <i>P. multocida</i>. Primers and probes were designed to target the conserved regions of the <i>H. parasuis OmpP2</i> gene, the <i>S. suis</i> serotype 2 <i>gdh</i> gene, and the <i>P. multocida Kmt1</i> gene. By optimizing the reaction system and conditions, a triplex qPCR method for simultaneous detection of <i>H. parasuis</i>, <i>S. suis</i> serotype 2, and <i>P. multocida</i> was successfully established. The amplification efficiencies of the standard curves for all three pathogens were found to be highly similar, with values of 102.105% for <i>H. parasuis</i>, 105.297% for <i>S. suis</i> serotype 2, and 104.829% for <i>P. multocida</i>, and all R² values achieving 0.999. The specificity analysis results showed that the triplex qPCR method had a strong specificity. The sensitivity test results indicated that the limit of detection can reach 50 copies/μL for all three pathogens. Both intra- and inter-assay coefficients of variation for repeatability were below 1%. This triplex qPCR method was shown to have good specificity, sensitivity, and reproducibility. Finally, the triplex qPCR method established in this study was compared with the nested PCR as recommended by the Chinese national standard (GB/T34750-2017) for <i>H. parasuis</i>, the PCR as recommended by the Chinese national standard (GB/T 19915.9-2005) for <i>S. suis</i> serotype 2, and the PCR as recommended by the Chinese agricultural industry standard (NY/T 564-2016) for <i>P. multocida</i> by detecting the same clinical samples. Both methods are reasonably consistent, while the triplex qPCR assay was more sensitive. In summary, triplex qPCR serves not only as a rapid and accurate detection and early prevention method for these pathogens but also constitutes a robust tool for microbial quality control in specific pathogen-free pigs.