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Journal of Clinical Microbiology, June 1999, p. 1958-1963, Vol. 37, No. 6
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Quantitative Detection of Borrelia burgdorferi by Real-Time PCR

Andreas Pahl,1 Uta Kühlbrandt,2 Kay Brune,1 Martin Röllinghoff,2 and André Gessner2,*

Institute of Pharmacology and Toxicology1 and Institute of Clinical Microbiology, Immunology and Hygiene,2 University of Erlangen-Nürnberg, D-91054 Erlangen, Germany

Received 7 December 1998/Returned for modification 21 January 1999/Accepted 17 March 1999

Currently, no easy and reliable methods allowing for the quantification of Borrelia burgdorferi in tissues of infected humans or animals are available. Due to the lack of suitable assays to detect B. burgdorferi CFU and the qualitative nature of the currently performed PCR assays, we decided to exploit the recently developed real-time PCR. This technology measures the release of fluorescent oligonucleotides during the PCR. Flagellin of B. burgdorferi was chosen as the target sequence. A linear quantitative detection range of 5 logs with a calculated detection limit of one to three spirochetes per assay reaction mixture was observed. The fact that no signals were obtained with closely related organisms such as Borrelia hermsii argues for a high specificity of this newly developed method. A similar method was developed to quantify mouse actin genomic sequences to allow for the standardization of spirochete load. The specificity and sensitivity of the B. burgdorferi and the actin real-time PCR were not altered when samples were spiked with mouse cells or spirochetes, respectively. To evaluate the applicability of the real-time PCR, we used the mouse model of Lyme disease. The fate of B. burgdorferi was monitored in different tissues from inbred mice and from mice treated with antibiotics. Susceptible C3H/HeJ mice had markedly higher burdens of bacterial DNA than resistant BALB/c mice, and penicillin G treatment significantly reduced the numbers of spirochetes. Since these results show a close correlation between clinical symptoms and bacterial burden of tissues, we are currently analyzing human biopsy specimens to evaluate the real-time PCR in a diagnostic setting.

* Corresponding author. Mailing address: Institute of Clinical Microbiology, Immunology, and Hygiene, University of Erlangen-Nürnberg, Wasserturmstr. 3, 91054 Erlangen, Germany. Phone: 49-9131-8522580. Fax: 49-9131-851001. E-mail: gessner{at}mikrobio.med.uni-erlangen.de.

Journal of Clinical Microbiology, June 1999, p. 1958-1963, Vol. 37, No. 6
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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