Real-time PCR-based serotyping of Streptococcus agalactiae

Abstract

Group B Streptococcus (GBS) is an encapsulated, gram-positive pathogen that is an important cause of neonatal invasive infections, including sepsis and meningitis. There are ten known GBS serotypes based on distinct capsule compositions (Ia, Ib, II-IX), and current candidate capsular polysaccharide conjugate vaccines target only a subset of these. Serotyping of GBS isolates is important for understanding local epidemiology and for monitoring for serotype replacement or capsular switching. However, serotyping generally requires either latex agglutination, multiplex PCR with analysis of band sizes, or analysis of whole genome sequences-all techniques that are either expensive or not widely available. Here we report the development of a robust real-time PCR assay for determining GBS serotypes. Using both a diverse reference set of strains encompassing all ten serotypes and a collection of clinical isolates, we demonstrate concordance between real-time PCR serotyping and latex agglutination. We propose that real-time PCR serotyping represents an attractive alternative to current serotyping methods and may allow for improved acquisition of GBS serotype data.

Figure 1. Serial dilution of GBS genomic DNA template demonstrates sensitivity of real-time PCR serotyping.

Serotyping reactions were performed on 20 validation strains using 5 ng, 500 pg, and 50 pg of template per reaction. Cycle threshold increased with decreasing template amounts but remained detectable for all strains even at the lowest concentration tested.