Detection of Ciprofloxacin-Resistant Yersinia pestis by Fluorogenic PCR Using the LightCycler
Abstract
mutants of the biothreat agent Yersinia pesfis. We selected spontaneous mutants of the attenuated Y. pestis KIM 5 strain that were resistant to at least 1 mgiml Cp. DNA sequencing of gyrA encoded by sixty-five 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 assay that detected all of these mutations using a single pair of fluorescent probes with sequences complementary to the wild type Y. pestis gyrA sequence. Melting peak analysis revealed that the probe-PCR product hybrid was less stable when amplification occurred from any of the four mutant templates. This instability resulted in the PCR product obtained from the Cpr Y. pestis strains displaying a 4-I lOC shift in probe melting temperature. Following optimization of the reaction conditions we were able to detect approximately 10 pg of purified wild type template DNA or the presence of approximately 4 CIll of Y. pestis KIM 5 wild type or Cpr mutants in cmde lysates. Taken together, our results demonstrate the utility of FRET-based assays to detect Cpr mutants of Y. pestis that is both sensitive and rapid.
Document Details
- Document Type
- Technical Report
- Publication Date
- Oct 01, 2001
- Accession Number
- ADA395206
Entities
People
- Luther E. Lindler
- Nazma Jahan
- Wei Fan
Organizations
- Walter Reed Army Institute of Research