A quadruplex real-time fluorescent quantitative PCR detection method for identification and serotyping of Pasteurella multocida

Abstract

Pasteurella multocida is a zoonotic pathogen with the ability to infect a diverse range of hosts, including humans through contact with pets. Due to its diverse capsular serogroups, rapid and accurate identification, as well as serotyping, are essential for the effective prevention and control of infections caused by this bacterium. This study developed a quadruplex quantitative PCR (qPCR) method for the identification and serotyping of P. multocida, specifically differentiating capsular serogroups A, D, and F in cats and other hosts. Primers and probes targeted the kmt1 gene (species-specific), hyaD-hyaC intergenic region (serogroup A), dcbF (serogroup D), and fcbD (serogroup F). The developed method demonstrated a detection limit of 1.7 copies/μL for the identification of P. multocida and 2.03, 11.24, and 14.52 copies/μL for the serotyping of capsular serogroups A, D, and F, respectively. The assay did not display cross-reactivity with other pathogens or different serogroups of P. multocida. Both the intra-assay and inter-assay coefficients of variation are below 1 %, highlighting the excellent reproducibility and specificity of the method. Applying this method, we examined 121 respiratory swabs from domestic cats, and 96 were tested positive for P. multocida. Among these positive samples, 66 were determined as capsular serogroup A, 1 as capsular serogroup D, and 24 as capsular serogroup F. The typing results exhibited high concordance with those obtained through bacterial isolation from the positive samples. The method developed in this study offers a rapid, accurate, and high-throughput approach for the identification and serotyping of P. multocida capsular serogroups A, D, and F. It serves as a powerful tool for advancing research into the pathogenic mechanisms of P. multocida and for implementing public health control measures.

Keywords: Capsular serogroups; Cats; Pasteurella multocida; Quadruplex fluorescent quantitative PCR.