Detection of 89K PAI gene from Streptococcus suis serotype 2 by using duplex fluorescence quantitative PCR combined with Taqman-MGB fluorescence probe.

To develop a duplex fluorescence quantitative PCR to detect pathogenicity island 89K DNA (89K PAI) in Streptococcus suis serotype 2 (SS2) from Chinese virulent strain, two pairs of primer and two TaqMan-MGB hybridization probes were designed for identifying 16S rRNA and 89K PAI gene simultaneously. PCR reaction conditions were optimized. 21 SS2, 18 other Streptococcus, 9 Staphylococcus and 5 other bacteria strains were tested. The specificity, sensitivity and reproducibility of the assay were evaluated. It was demonstrated that all of the SS2 strains were 16S rRNA-positive. and 71.45% (15/21) of SS2 strains were 89K PAI gene-positive. The sensitivity was 6 copies/reaction or 12cfu/mL for detection of the 16Sr RNA gene and 27copies/reaction or 58cfu/mL for detection of 89K PAI. The coefficients of variation(CV) value were 0.30%-2.11%. It took only 2 hours from DNA ABStraction to gene detection. Forty-nine unknown samples were test in this assay in which 2 of them were 16S rRNA-positive, and one was 89K DNA positive. It indicates that 89K PAI gene of SS2 can be detected by using duplex fluorescence quantitative PCR established here, which is specific, rapid and high-sensitive. In addition, SS2, non SS2 and 89K PAI gene of SS2 can be discriminated. This method provided the possibility of early detection for Streptococcus suis infection, and quantitative pathogenic identification for disease epidemic or outbreak.