Microbial DNA Typing by Automated Repetitive-Sequence-Based PCR

Author:

Healy Mimi1,Huong Joe1,Bittner Traci1,Lising Maricel1,Frye Stacie1,Raza Sabeen1,Schrock Robert1,Manry Janet1,Renwick Alex1,Nieto Robert1,Woods Charles2,Versalovic James343,Lupski James R.567

Affiliation:

1. Spectral Genomics, Inc.

2. Department of Pediatrics, Wake Forest University School of Medicine, Winston-Salem, North Carolina

3. Departments of Pathology

4. Molecular Virology and Microbiology

5. Molecular and Human Genetics

6. Pediatrics, Baylor College of Medicine

7. Pediatrics, Texas Children's Hospital, Houston, Texas

Abstract

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.

Publisher

American Society for Microbiology

Subject

Microbiology (medical)

Reference54 articles.

1. Borgdorff, M. W., A. Kolk, D. van Soolingen, J. W. van der Meer, and T. H. Ottenhoff. 2003. Research into new methods for diagnosing, treating and preventing tuberculosis. Ned. Tijdschr. Geneeskd.147:1838-1841.

2. Evaluation of a High-Throughput Repetitive-Sequence-Based PCR System for DNA Fingerprinting of Mycobacterium tuberculosis and Mycobacterium avium Complex Strains

3. Application of Real-Time Quantitative PCR to Molecular Analysis of Candida albicans Strains Exhibiting Reduced Susceptibility to Azoles

4. PCR Analyses of tRNA Intergenic Spacer, 16S-23S Internal Transcribed Spacer, and Randomly Amplified Polymorphic DNA Reveal Inter- and Intraspecific Relationships of Enterobacter cloacae Strains

5. Cousins, D. V., R. A. Skuce, R. R. Kazwala, J. D. van Embden, et al. 1998. Towards a standardized approach to DNA fingerprinting of Mycobacterium bovis.Int. J. Tuberc. Lung Dis.2:471-478.

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