title: Concurrent serotyping and genotyping of pneumococci by use of PCR and electrospray ionization mass spectrometry.
abstract: A pneumococcal serotyping/genotyping system (PSGS) was developed based upon targeted PCR, followed by electrospray ionization mass spectrometry and amplicon base composition analysis. Eight multiplex PCRs, 32 targeting serotype-determining capsular biosynthetic loci, and 8 targeting multilocus sequence typing (MLST) loci were employed for each of 229 highly diverse Streptococcus pneumoniae isolates. The most powerful aspect of the PSGS system was the identification of capsular serotypes accounting for the majority of invasive and carried pneumococcal strains. Altogether, 45 different serotypes or serogroups were correctly predicted among the 196 resolvable isolates, with only 2 unexpected negative results. All 33 isolates that represented 23 serotypes not included in the PSGS yielded negative serotyping results. A genotyping database was constructed using the base compositions of 65- to 100-bp sections of MLST alleles compiled within http://www.mlst.net. From this database, one or more MLST sequence types (STs) that comprised a PSGS genotype were identified. The end result of more PSGS genotypes (163) than conventional STs actually tested (155) was primarily due to amplification failures of 1 to 3 targets. In many instances, the PSGS genotype could provide resolution of single- and double-locus variants. This molecular serotyping/genotyping scheme is well suited to rapid characterization of large sets of pneumococcal isolates.
authors.affiliation: Ibis Biosciences, Carlsbad, California, USA. cmassire@ibisbio.com
authors.forename: Christian
authors.initials: C
authors.lastname: Massire
authors.forename: Robert E
authors.initials: RE
authors.lastname: Gertz
authors.forename: Pavel
authors.initials: P
authors.lastname: Svoboda
authors.forename: Keith
authors.initials: K
authors.lastname: Levert
authors.forename: Matthew S
authors.initials: MS
authors.lastname: Reed
authors.forename: Jan
authors.initials: J
authors.lastname: Pohl
authors.forename: Rachel
authors.initials: R
authors.lastname: Kreft
authors.forename: Feng
authors.initials: F
authors.lastname: Li
authors.forename: Neill
authors.initials: N
authors.lastname: White
authors.forename: Ray
authors.initials: R
authors.lastname: Ranken
authors.forename: Larry B
authors.initials: LB
authors.lastname: Blyn
authors.forename: David J
authors.initials: DJ
authors.lastname: Ecker
authors.forename: Rangarajan
authors.initials: R
authors.lastname: Sampath
authors.forename: Bernard
authors.initials: B
authors.lastname: Beall
mesh_terms: D015373:Bacterial Typing Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013296:Streptococcus pneumoniae / Q000145:classification* / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial