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J. Clin. Microbiol. doi:10.1128/JCM.00226-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Multiplexed identification of blood borne bacterial pathogens using a novel 16s rDNA PCR/LDR/Capillary Electrophoresis assay

Department of Microbiology, Department of Pathology and Laboratory Medicine, and Division of Infectious Disease and International Medicine, Weill Medical College of Cornell University, New York, NY and; Department of Pathology, Stony Brook University Medical Center, Stony Brook, NY

^* To whom correspondence should be addressed. Email: barany{at}med.cornell.edu.

	Abstract

We have developed a novel high throughput PCR/Ligase Detection reaction/Capillary electrophoresis assay for the multiplexed identification of twenty blood borne pathogens (Staphylococcus epidermidis, Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Enterococcus faecium, Listeria monocytogenes, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Acinetobacter baumannii, Neisseria meningitidis, and Bacteroides fragilis) including four biothreat agents (Bacillus anthracis, Yersinia pestis, Francisella tularensis and Brucella abortus). The method relies on the amplification of two regions within the bacterial 16s ribosomal DNA using universal PCR primers and querying the identity of specific SNPs within the amplified regions in a subsequent ligase detection reaction (LDR) reaction. The ligation products vary in color and size and are separated by capillary electrophoresis (CE). Each organism generates a specific pattern of ligation products which can be used to distinguish the pathogens using an automated software program we developed for that purpose. The assay has been verified on 315 clinical isolates and demonstrated a detection sensitivity of 98%. Additionally, 484 seeded blood cultures were tested with a detection sensitivity of 97.7%. The ability to identify geographically variant strains of the organisms was determined by testing 132 isolates obtained from across the United States. In summary, the PCR/LDR/CE assay can successfully identify, in a multiplexed fashion, a panel of twenty blood borne pathogens with high sensitivity and specificity.

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