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Journal of Clinical Microbiology, September 2002, p. 3449-3454, Vol. 40, No. 9
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.9.3449-3454.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Quantitative Multiprobe PCR Assay for Simultaneous Detection and Identification to Species Level of Bacterial Pathogens

Samuel Yang,1 Shin Lin,1 Gabor D. Kelen,1 Thomas C. Quinn,1,2 James D. Dick,1 Charlotte A. Gaydos,1 and Richard E. Rothman1*

Department of Emergency Medicine, The Johns Hopkins University School of Medicine, Baltimore, and National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland

Received 29 January 2002/ Returned for modification 29 March 2002/ Accepted 16 June 2002

We describe a novel adaptation of the TaqMan PCR assay which potentially allows for highly sensitive detection of any eubacterial species with simultaneous species identification. Our system relies on a unique multiprobe design in which a single set of highly conserved sequences encoded by the 16S rRNA gene serves as the primer pair and is used in combination with both an internal highly conserved sequence, the universal probe, and an internal variable region, the species-specific probe. A pre-PCR ultrafiltration step effectively decontaminates or removes background DNA. The TaqMan system described reliabAly detected 14 common bacterial species with a detection limit of 50 fg. Further, highly sensitive and specific pathogen detection was demonstrated with a prototype species-specific probe designed to detect Staphylococcus aureus. This assay has broad potential in the clinical arena for rapid and specific diagnosis of infectious diseases.

* Corresponding author. Mailing address: Department of Emergency Medicine, Johns Hopkins School of Medicine, Baltimore, MD 21205. Phone: (410) 614-5933. Fax: (410) 502-8881. E-mail: rrothman{at}jhmi.edu.

Journal of Clinical Microbiology, September 2002, p. 3449-3454, Vol. 40, No. 9
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.9.3449-3454.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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