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Journal of Clinical Microbiology, October 2001, p. 3649-3655, Vol. 39, No. 10
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.
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
*
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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