Microbial DNA typing by automated repetitive-sequence-based PCR.
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.
Healy
Mimi
M
Spectral Genomics, Inc., 8080 North Stadium Dr., Suite 1200, Houston, TX 77054, USA. mhealy@spectralgenomics.com
Huong
Joe
J
Bittner
Traci
T
Lising
Maricel
M
Frye
Stacie
S
Raza
Sabeen
S
Schrock
Robert
R
Manry
Janet
J
Renwick
Alex
A
Nieto
Robert
R
Woods
Charles
C
Versalovic
James
J
Lupski
James R
JR
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
D003470:Culture Media