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

TB Morrison, Y Ma, JH Weis… - Journal of clinical …, 1999 - Am Soc Microbiol
TB Morrison, Y Ma, JH Weis, JJ Weis
Journal of clinical microbiology, 1999Am Soc Microbiol
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 …
Abstract
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 ofB. burgdorferi quantities to the mouse nidogengene 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.
American Society for Microbiology