title: Sequetyping: serotyping Streptococcus pneumoniae by a single PCR sequencing strategy.
abstract: The introduction of pneumococcal conjugate vaccines necessitates continued monitoring of circulating strains to assess vaccine efficacy and replacement serotypes. Conventional serological methods are costly, labor-intensive, and prone to misidentification, while current DNA-based methods have limited serotype coverage requiring multiple PCR primers. In this study, a computer algorithm was developed to interrogate the capsulation locus (cps) of vaccine serotypes to locate primer pairs in conserved regions that border variable regions and could differentiate between serotypes. In silico analysis of cps from 92 serotypes indicated that a primer pair spanning the regulatory gene cpsB could putatively amplify 84 serotypes and differentiate 46. This primer set was specific to Streptococcus pneumoniae, with no amplification observed for other species, including S. mitis, S. oralis, and S. pseudopneumoniae. One hundred thirty-eight pneumococcal strains covering 48 serotypes were tested. Of 23 vaccine serotypes included in the study, most (19/22, 86%) were identified correctly at least to the serogroup level, including all of the 13-valent conjugate vaccine and other replacement serotypes. Reproducibility was demonstrated by the correct sequetyping of different strains of a serotype. This novel sequence-based method employing a single PCR primer pair is cost-effective and simple. Furthermore, it has the potential to identify new serotypes that may evolve in the future.
authors.affiliation: Centre for Clinical Microbiology, University College London Medical School, Royal Free Campus, London, United Kingdom.
authors.forename: Marcus H
authors.initials: MH
authors.lastname: Leung
authors.forename: Kevin
authors.initials: K
authors.lastname: Bryson
authors.forename: Kathrin
authors.initials: K
authors.lastname: Freystatter
authors.forename: Bruno
authors.initials: B
authors.lastname: Pichon
authors.forename: Giles
authors.initials: G
authors.lastname: Edwards
authors.forename: Bambos M
authors.initials: BM
authors.lastname: Charalambous
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Gillespie
mesh_terms: D019295:Computational Biology; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D058889:Molecular Typing / Q000379:methods*; D011008:Pneumococcal Infections / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012703:Serotyping / Q000379:methods; D013296:Streptococcus pneumoniae / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial