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Multiplexed identification of blood-borne bacterial pathogens by use of a novel 16S rRNA gene PCR-ligase detection reaction-capillary electrophoresis assay.

TitleMultiplexed identification of blood-borne bacterial pathogens by use of a novel 16S rRNA gene PCR-ligase detection reaction-capillary electrophoresis assay.
Publication TypeJournal Article
Year of Publication2007
AuthorsPingle MR, Granger K, Feinberg P, Shatsky R, Sterling B, Rundell M, Spitzer E, Larone D, Golightly LM, Barany F
JournalJ Clin Microbiol
Volume45
Issue6
Pagination1927-35
Date Published2007 Jun
ISSN0095-1137
KeywordsBacteria, Bioterrorism, Blood-Borne Pathogens, Electrophoresis, Capillary, Genes, rRNA, Humans, Ligase Chain Reaction, Polymerase Chain Reaction, RNA, Ribosomal, 16S, Sensitivity and Specificity
Abstract

We have developed a novel high-throughput PCR-ligase detection reaction-capillary electrophoresis (PCR-LDR-CE) assay for the multiplexed identification of 20 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, Bacteroides fragilis, Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella abortus), the last four of which are biothreat agents. The method relies on the amplification of two regions within the bacterial 16S rRNA gene, using universal PCR primers and querying the identity of specific single-nucleotide polymorphisms within the amplified regions in a subsequent LDR. The ligation products vary in color and size and are separated by 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 20 blood-borne pathogens with high sensitivity and specificity.

DOI10.1128/JCM.00226-07
Alternate JournalJ. Clin. Microbiol.
PubMed ID17428930
PubMed Central IDPMC1933048
Grant ListAI062579 / AI / NIAID NIH HHS / United States

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