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Journal of Clinical Microbiology, April 1999, p. 987-992, Vol. 37, No. 4
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Rapid and Sensitive Quantification of Borrelia burgdorferi-Infected Mouse Tissues by Continuous Fluorescent Monitoring of PCR

Tom B. Morrison, Ying Ma, John H. Weis, and Janis J. Weis*

Department of Pathology, Division of Cell Biology and Immunology, University of Utah School of Medicine, Salt Lake City, Utah 84132

Received 14 May 1998/Returned for modification 28 July 1998/Accepted 21 December 1998

The quantity of Borrelia burgdorferi organisms in tissue samples is an important determinant for infection studies in the mouse model of Lyme disease. This report presents the development of a rapid and sensitive external-standard-based PCR assay for the absolute quantification of B. burgdorferi in mouse tissue samples. The assay uses a double-stranded DNA dye to continuously monitor product formation and in less than an hour was able to quantify samples ranging up to 6 log units in concentration. The PCR efficiencies of the sample and the standard were matched by using a standard composed of purified B. burgdorferi chromosome mixed with tissue-matched mouse genome lacking bacterial DNA. Normalization of B. burgdorferi quantities to the mouse nidogen gene allowed comparison of B. burgdorferi numbers in samples isolated from different tissues and strains. PCR analysis of the chromosomal gene recA in cultured B. burgdorferi was consistent with a single recA per bacterium. The parameters defined in this assay should be applicable to quantification of other organisms, even infectious agents for which no ready source of DNA standard is available. In summary, this report presents a rapid external-standard-based PCR method for the quantification of B. burgdorferi in mouse DNA samples.

* Corresponding author. Mailing address: Department of Pathology, University of Utah Medical School, Salt Lake City, UT 84132. Phone: (801) 581-8386. Fax: (801) 581-4517. E-mail: janis.weis{at}path.med.utah.edu.

Journal of Clinical Microbiology, April 1999, p. 987-992, Vol. 37, No. 4
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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