Establishment of a multiplex real-time fluorescent PCR method for simultaneous detection of Streptococcus suis and Pasteurella multocida

Abstract:

[Background] Streptococcus suis (SS) and Pasteurella multocida (Pm) are zoonotic pathogens that can cause severe diseases in humans, which are easily confused with swine fever and swine erysipelas in clinical diagnosis because of mixed infections. [Objective] To establish a multiplex real-time fluorescent quantitative PCR assay for simultaneous detection of SS and Pm, so as to realize early diagnosis and treatment. [Methods] Two pairs of specific primers and TaqMan were designed based on the gdh gene of SS and the plpE gene of Pm. The universal primers and probe were designed based on the sequence of bacterial 16S rRNA gene. After optimization of the reaction conditions, a multiplex real-time fluorescence quantitative PCR method was established for the simultaneous detection of SS and Pm.[Results] This method can specifically detect SS and Pm, and the results was entirely consistent with the sequencing results after isolation of bacteria. The lower limits of detection for recombinant plasmid standards were 4.53×10² copies/μL and 3.97×10² copies/μL, respectively. The results of repeated experiments showed that the intra- and inter-group coefficients of variation were less than 3%. [Conclusion] The method established in this study was simple, accurate, and reliable. It can be used for the simultaneous detection of SS and Pm and provides an effective detection tool for the prevention and treatment of the diseases caused by SS and Pm.