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Journal of Clinical Microbiology, January 2005, p. 199-207, Vol. 43, No. 1
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.1.199-207.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Microbial DNA Typing by Automated Repetitive-Sequence-Based PCR

Mimi Healy,1* Joe Huong,1 Traci Bittner,1 Maricel Lising,1 Stacie Frye,1 Sabeen Raza,1 Robert Schrock,1 Janet Manry,1 Alex Renwick,1 Robert Nieto,1 Charles Woods,2 James Versalovic,3,4,5 and James R. Lupski6,7,8

Spectral Genomics, Inc.,1 Departments of Pathology,3 Molecular Virology and Microbiology,4 Molecular and Human Genetics,6 Pediatrics, Baylor College of Medicine,7 Departments of Pathology,5 Pediatrics, Texas Children's Hospital, Houston, Texas,8 Department of Pediatrics, Wake Forest University School of Medicine, Winston-Salem, North Carolina2

Received 16 April 2004/ Returned for modification 30 July 2004/ Accepted 10 September 2004

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.

* Corresponding author. Mailing address: Spectral Genomics, Inc., 8080 North Stadium Dr., Suite 1200, Houston, TX 77054. Phone: (713) 467-8500. Fax: (713) 467-7766. E-mail: mhealy{at}spectralgenomics.com.

Journal of Clinical Microbiology, January 2005, p. 199-207, Vol. 43, No. 1
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.1.199-207.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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