title: Microbial DNA typing by automated repetitive-sequence-based PCR.
abstract: Repetitive sequence-based PCR (rep-PCR) has been recognized as an effective method for bacterial strain typing. Recently, rep-PCR has been commercially adapted to an automated format known as the DiversiLab system to provide a reliable PCR-based typing system for clinical laboratories. We describe the adaptations made to automate rep-PCR and explore the performance and reproducibility of the system as a molecular genotyping tool for bacterial strain typing. The modifications for automation included changes in rep-PCR chemistry and thermal cycling parameters, incorporation of microfluidics-based DNA amplicon fractionation and detection, and Internet-based computer-assisted analysis, reporting, and data storage. The performance and reproducibility of the automated rep-PCR were examined by performing DNA typing and replicate testing with multiple laboratories, personnel, instruments, DNA template concentrations, and culture conditions prior to DNA isolation. Finally, we demonstrated the use of automated rep-PCR for clinical laboratory applications by using isolates from an outbreak of Neisseria meningitidis infections. N. meningitidis outbreak-related strains were distinguished from other isolates. The DiversiLab system is a highly integrated, convenient, and rapid testing platform that may allow clinical laboratories to realize the potential of microbial DNA typing.
authors.affiliation: Spectral Genomics, Inc., 8080 North Stadium Dr., Suite 1200, Houston, TX 77054, USA. mhealy@spectralgenomics.com
authors.forename: Mimi
authors.initials: M
authors.lastname: Healy
authors.forename: Joe
authors.initials: J
authors.lastname: Huong
authors.forename: Traci
authors.initials: T
authors.lastname: Bittner
authors.forename: Maricel
authors.initials: M
authors.lastname: Lising
authors.forename: Stacie
authors.initials: S
authors.lastname: Frye
authors.forename: Sabeen
authors.initials: S
authors.lastname: Raza
authors.forename: Robert
authors.initials: R
authors.lastname: Schrock
authors.forename: Janet
authors.initials: J
authors.lastname: Manry
authors.forename: Alex
authors.initials: A
authors.lastname: Renwick
authors.forename: Robert
authors.initials: R
authors.lastname: Nieto
authors.forename: Charles
authors.initials: C
authors.lastname: Woods
authors.forename: James
authors.initials: J
authors.lastname: Versalovic
authors.forename: James R
authors.initials: JR
authors.lastname: Lupski
mesh_terms: D001331:Automation; D015373:Bacterial Typing Techniques; D003470:Culture Media; D006801:Humans; D008589:Meningococcal Infections / Q000453:epidemiology / Q000382:microbiology; D017720:Molecular Epidemiology; D009345:Neisseria meningitidis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D015203:Reproducibility of Results; D012984:Software
chemical_list: D003470:Culture Media