title: Development of a 5' nuclease assay to detect ciprofloxacin resistant isolates of the biowarfare agent Yersinia pestis.
abstract: The detection of antibiotic resistance in agents of biowarfare is extremely critical in order to start appropriate therapy in a timely manner. We have developed a 5' nuclease assay to detect ciprofloxacin resistance (Cip(r)) in Yersinia pestis. Two groups of fluorogenic probes were developed. The first group included a probe homologous to the wild type Y. pestis gyrA sequence with two corresponding probes that were homologous with two different mis-sense mutations in codon 81 of GyrA. The second group of probes included a wild type probe and two corresponding probes that recognized mis-sense mutations in codon 83 or gyrA. These probes specifically reacted only with the homologous DNA sequences. The 5' nuclease assay was sensitive to 1 pg (approximately 1 colony forming unit) of starting template and could be used on semi-purified DNA. We tested our optimized assay against a group of 38 Cip(r) Y. pestis isolates and demonstrated that it was accurate at determining the gyrA allele encoded by these strains. The 5' nuclease assay performed on the LightCycler proved to be sensitive, rapid and accurate at identification of Cip(r) Y. pestis and thus should be useful in characterization of this organism in the event of a future act of bioterrorism.
authors.affiliation: Department of Bacterial Diseases, Division of Communicable Disease and Immunology, Walter Reed Army Institute of Research, 503 Robert Grant Ave, Silver Spring, MD 20910, USA. luther.lindler@na.amedd.army.mil
authors.forename: Luther E
authors.initials: LE
authors.lastname: Lindler
authors.forename: Wei
authors.initials: W
authors.lastname: Fan
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001483:Base Sequence; D001694:Biological Warfare; D002939:Ciprofloxacin / Q000494:pharmacology*; D015342:DNA Probes / Q000235:genetics; D024881:Drug Resistance, Bacterial; D005456:Fluorescent Dyes; D023282:Nuclease Protection Assays / Q000379:methods*; D010597:Pharmacogenetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D013379:Substrate Specificity; D015010:Yersinia pestis / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity
chemical_list: D000900:Anti-Bacterial Agents; D015342:DNA Probes; D005456:Fluorescent Dyes; D002939:Ciprofloxacin