A Multiplex TaqMan qPCR Assay for Rapid Detection of Streptococcus suis Serotypes 1, 2, 7, and 9 with Cross-Reactivity to Serotypes 14 and 1/2.

Streptococcus suis (S. suis) is a zoonotic pathogen that causes severe diseases including meningitis, septic shock, and endocarditis in both pigs and humans. In swine, S. suis is one of the most important bacterial pathogens causing invasive diseases such as meningitis, septicemia, arthritis, and sudden death, with serotypes 1, 2, 7, and 9 being among the most frequently recovered from clinical cases in affected herds across Southeast Asia. We developed a multiplex TaqMan qPCR assay targeting four serotype-specific genes (cps1I, cps2I, cps7L, and cps9J) for detection of major S. suis serotypes in porcine samples. The assay achieved a detection limit of 10 copies/μL for all four target serogroups, amplification efficiencies of 89.64% to 100.42% (R2 ≥ 0.997), and no cross-reactivity with any of the negative controls, including SS4, APP, HPS, Salmonella, E. coli, and S. aureus. Intra-assay and inter-assay repeatability were below 3%. Testing 98 clinical porcine samples from Guangxi, China, yielded positivity rates of 26.53% for the serotype 1/14 group, 19.39% for the serotype 2/1/2 group, 17.34% for serotype 7, and 29.59% for serotype 9, respectively, with mixed-target detection in 30.61% of samples. This assay enables rapid, reliable detection of major zoonotic S. suis serotypes in porcine samples, though it detects serotypes 1 and 14 as a group, and serotypes 2 and 1/2 as a group, due to capsular locus sequence similarity-a limitation acceptable for surveillance given the high pathogenicity within each group. This tool supports One Health-based veterinary surveillance for zoonotic disease control.