Analytical and diagnostic performances of a triplex real-time polymerase chain reaction assay for quantification of Streptococcus suis species, serotypes 2 and 1/2 and serotype 9 in swine biological samples

The epidemiological control of Streptococcus ( S .) suis infections require detection and quantification of the serotypes most frequently implicated in pathological cases (serotypes 2 and 9), and even more so of all serotypes of the bacterial specie directly in pig organs including tonsils. This study developed a new multiplex qPCR assay for the simultaneous quantification of these three targets: S . suis species (all the 29 serotypes), S. suis serotype 2 (and 1/2 indistinguishable by qPCR) and S. suis serotype 9. This qPCR assay based on the recN and cps genes was tested with 84 S. suis strains. The specificity was verified with a collection of 43 strains of bacterial species other than S. suis . The estimated detection thresholds of the recN , cps2J and cps9H targets were 5 (±1), 6 (±2) and 5 (±3) ge/assay, respectively. The amplification efficiencies and correlation coefficients were 99.1% (±3.1%) and 0.999 (±0.002), 97.4% (±2.9%) and 0.999 (±0.002), 100% (±3.2%) and 0.998 (±0.002), respectively. The diagnostic specificity, measured with 74 samples, was 100%. The diagnostic sensitivity, evaluated from 126 samples, was 98%. These performances enabled its use to assess tonsillar carriage in a farm with streptococcal infections . S. suis was detected in all tonsils (100%) whereas S. suis serotypes 2 and 1/2 and S. suis serotype 9 were detected in 38% and 29% of 180 samples, with mean amounts at 4.80 (±0.78), 3.26 (±0.69) and 3.27 (±0.60) log(10) ge/ml, respectively. This triplex qPCR assay validated has significant potential for applications in monitoring and controlling this zoonotic pathogen. • New triplex qPCR test for quantification of S. suis, serotypes 2 - 1/2 and serotype 9 • The detection thresholds are 5 (±1), 6 (±2) and 5 (±3) genome equivalent by assay • The diagnostic specificity is 100% and the diagnostic sensitivity is 98% • This test was applied to assess tonsillar carriage in a farm with infections • It will help understand the S. suis epidemiology and guide veterinarian decisions