Detection of ciprofloxacin-resistant Yersinia pestis by fluorogenic PCR using the lightcycler

We have developed a fluorescence resonance energy transfer (FRET)-based ass ay to detect ciprofloxacin resistant (Cp-r) mutants of the biothreat agent Yersinia pestis. We selected spontaneous mutants of the attenuated Y. pesti s KIM 5 strain that were resistant to a ciprofloxacin (CIP) concentration o f at least 1 mug/ml. DNA sequencing of gyrA encoded by 65 of these mutants revealed that all isolates contained one of four different point mutations within the quinolone resistance-determining region of gyrA. We developed a FRET-based assay that detected all of these mutations by using a single pai r of fluorescent probes with sequences complementary to the wild-type Y. pe stis gyrA sequence. Melting peak analysis revealed that the probe-PCR produ ct hybrid was less stable when amplification occurred from any of the four mutant templates. This instability resulted in the PCR product obtained fro m the Cpr Y. pestis strains displaying a 4 to 11 degreesC shift in probe me lting temperature. Following optimization of the reaction conditions, we we re able to detect approximately 10 pg of purified wild-type template DNA or the presence of approximately 4 CFU of wild-type Y. pestis KIM 5 or Cpr mu tants in crude lysates. Taken together, our results demonstrate the utility of FRET-based assays for detection of Cpr mutants of Y. pestis. This metho d is both sensitive and rapid.