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Journal of Clinical Microbiology, October 2001, p. 3649-3655, Vol. 39, No. 10
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.10.3649-3655.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Detection of Ciprofloxacin-Resistant Yersinia pestis by Fluorogenic PCR Using the LightCycler

Luther E. Lindler,* Wei Fan, and Nazma Jahan

Department of Bacterial Diseases, Division of Communicable Diseases and Immunology, Walter Reed Army Institute of Research, Silver Spring, Maryland 20910

Received 7 May 2001/Returned for modification 9 July 2001/Accepted 26 July 2001

We have developed a fluorescence resonance energy transfer (FRET)-based assay to detect ciprofloxacin resistant (Cpr) mutants of the biothreat agent Yersinia pestis. We selected spontaneous mutants of the attenuated Y. pestis KIM 5 strain that were resistant to a ciprofloxacin (CIP) concentration of at least 1 µg/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 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 to 11°C 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 CFU of wild-type Y. pestis KIM 5 or Cpr mutants in crude lysates. Taken together, our results demonstrate the utility of FRET-based assays for detection of Cpr mutants of Y. pestis. This method is both sensitive and rapid.

* Corresponding author. Mailing address: Department of Bacterial Diseases, WRAIR, 503 Robert Grant Ave., Silver Spring, MD 20910. Phone: (301)-319-9388. Fax: (301) 319-9123. E-mail: Luther.Lindler{at}na.amedd.army.mil.

Journal of Clinical Microbiology, October 2001, p. 3649-3655, Vol. 39, No. 10
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.10.3649-3655.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.