Multiplex PCR assay for rapid and accurate capsular typing of group B streptococci

Abstract

We developed a simple, specific, and sensitive two-multiplex-PCR assay that enabled the detection of all known group B streptococcal (GBS) capsular polysaccharides. This test is well adapted for GBS capsular polysaccharide typing in large-scale epidemiological studies.

FIG. 1.

Schematic representation of GBS cps loci. (A) Sequence analysis of two unrelated cps loci encoding the GBS serotype II capsular polysaccharide in strains CNRCCH393 and CNRCCH394. The gene nomenclatures are those used in the published sequence with GenBank accession number AY375362, thought to constitute the prototype sequence for type II cps loci (3), and the published sequence with GenBank accession number AAJO01000077 (strain 18RS21). The gray boxes indicate regions of sequence identity. Note that primers II-F and II-R match the 3′ moiety of cps2K present in all sequences, whereas primers III-F (specific for the 5′ extremity of cps2I) and III-R (specific for the 3′ extremity of cps2J) appear to be specific for the sequence with GenBank accession number AY375362. Sequencing of the strain CNRCCH393 and CNRCCH394 cps2 loci was done by chromosome walking between the 5′ and 3′ ends of cps2E and cps2K, respectively. Scales are indicated in base pairs. (B) Sequence analysis of the cps locus of NT strain CNRCCH265. Sequencing of this locus was done by chromosome walking between the 5′ and 3′ ends of cpsB and neuB, respectively. This strain should synthesize a COOH-truncated form of CpsC (CpsCΔ) but should not express any CpsM derivatives (ΔCpsM) because the appropriate translational signals have been deleted. The scale is indicated in base pairs.

FIG. 2.

Representative PCR multiplex reactions. The CPS types of the strains analyzed are indicated above their respective lanes. (A) Multiplex PCR for detection of CPS types Ia, Ib, II, III, and IV; (B) multiplex PCR for detection of CPS types V, VI, VII, and VIII; (C) GBS-specific PCR. Lanes M, molecular size standard (100-bp ladder; Invitrogen). The numbers on the right of each panel are in base pairs.