Simple, accurate, serotype-specific PCR assay to differentiate Streptococcus pneumoniae serotypes 6A, 6B, and 6C

Abstract

In this study, we developed a simple, reliable, serotype-specific PCR method to differentiate Streptococcus pneumoniae serotypes 6A, 6B, and 6C. It was more efficient and practical than the assays currently being used to identify serotypes 6A, 6B, and 6C. Of 120 selected serogroup 6 isolates from subjects with invasive (n = 101) and noninvasive (n = 19) pneumococcal disease, most of which were collected after 2003 in New South Wales, 45 had been identified as 6A and 75 had been identified as 6B by the Quellung reaction. PCR analysis confirmed the results for serotype 6B isolates and identified two different subtypes. Fourteen of 45 isolates that had been identified as serotype 6A actually belonged to serotype 6C.

FIG. 1.

Design of primers to differentiate serotypes 6A/6C and 6B. The figure shows part of the alignment of the CR931638, CR931639 and AF246897, and EF538714 sequences by the ClustalW tool provided in BioManager (Sydney Bioinformatics [http://www.angis.org.au/]). The gray highlights show primer regions. The white letters at the 3′ ends of the primers represent the key SNP, G584A, in serotypes 6A/6C and 6B. Two sense primers are positioned with their 3′ ends at the heterogeneity site. Primer pairs wciP584gS/wciP-r and wciP584aS/wciP-r are expected to amplify sequences of serotypes 6A/6C and 6B, respectively.

FIG. 2.

Algorithm for identification of serogroup 6 serotypes. Sequences of primers wciP584gS/wciP-r, wciP584aS/wciP-r, wciN_betaS1/wciN_betaA2, and wciN_betaS2/wciN_betaA1 are listed in Table 1.