Development of a two-step multiplex PCR assay for typing of capsular polysaccharide synthesis gene clusters of Streptococcus suis

Abstract

We developed a practical and easy two-step multiplex PCR assay to aid in serotyping of Streptococcus suis. The assay accurately typed almost all of the serotype reference strains and field isolates of various serotypes and also identified the genotypes of capsular polysaccharide synthesis gene clusters of some serologically nontypeable strains.

FIG 1

Scheme of cps typing using a two-step multiplex PCR assay and the PCR products amplified from 35 S. suis serotype reference strains. The first PCR (grouping PCR) classified the tested strains into 7 groups (cps groups I to VII). Numbers and sizes of grouping PCR products amplified from strains of each cps group and the electrophoresis image are shown in the upper panel. Approximately 1.5-kbp bands that appeared in all grouping PCR assays are the internal control products (16S rRNA gene products). Only the internal control products were amplified from cps group VII strains. The strains classified by grouping PCR were then subjected to the second PCR (typing PCR) using primers specific to the respective cps groups in order to identify the cps type of each strain. Number and size of typing PCR products amplified from all S. suis serotype reference strains and the electrophoresis images are shown in the bottom panels. Approximately 1.5-kbp bands that appeared in all typing PCR assays are the internal control products (16S rRNA gene products). The serotypes of reference strains are indicated below the lanes. All PCR products were electrophoresed on a 2% agarose gel (100 V, 40 min), stained with ethidium bromide, and photographed under UV light. Lanes M show DNA molecular weight markers (in thousands [k]) (100-bp DNA ladder; Bioneer, Daejeon, South Korea).