title: Use of the Agilent 2100 bioanalyzer for rapid and reproducible molecular typing of Streptococcus pneumoniae.
abstract: Restriction fragment length polymorphism (RFLP) analysis is an economic and fast technique for molecular typing but has the drawback of difficulties in accurately sizing DNA fragments and comparing banding patterns on agarose gels. We aimed to improve RFLP for typing of the important human pathogen Streptococcus pneumoniae and to compare the results with the commonly used typing techniques of pulsed-field gel electrophoresis and multilocus sequence typing. We designed primers to amplify a noncoding region adjacent to the pneumolysin gene. The PCR product was digested separately with six restriction endonucleases, and the DNA fragments were analyzed using an Agilent 2100 bioanalyzer for accurate sizing. The combined RFLP results for all enzymes allowed us to assign each of the 47 clinical isolates of S. pneumoniae tested to one of 33 RFLP types. RFLP analyzed using the bioanalyzer allowed discrimination between strains similar to that obtained by the more commonly used techniques of pulsed-field gel electrophoresis, which discriminated between 34 types, and multilocus sequence typing, which discriminated between 35 types, but more quickly and with less expense. RFLP of a noncoding region using the Agilent 2100 bioanalyzer could be a useful addition to the molecular typing techniques in current use for S. pneumoniae, especially as a first screen of a local population.
authors.affiliation: Institute for Infectious Diseases, University of Bern, Friedbühlstrasse 51, CH-3010 Bern, Switzerland.
authors.forename: Lucy J
authors.initials: LJ
authors.lastname: Hathaway
authors.forename: Silvio
authors.initials: S
authors.lastname: Brugger
authors.forename: Alina
authors.initials: A
authors.lastname: Martynova
authors.forename: Suzanne
authors.initials: S
authors.lastname: Aebi
authors.forename: Kathrin
authors.initials: K
authors.lastname: Mühlemann
mesh_terms: D001322:Autoanalysis / Q000295:instrumentation / Q000379:methods; D001426:Bacterial Proteins / Q000235:genetics; D015373:Bacterial Typing Techniques / Q000295:instrumentation* / Q000379:methods*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D016521:Electrophoresis, Gel, Pulsed-Field; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D013296:Streptococcus pneumoniae / Q000145:classification* / Q000235:genetics; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; C027908:endodeoxyribonuclease DdeI; D015252:Deoxyribonucleases, Type II Site-Specific