Quantitative detection of Borrelia burgdorferi with a microtiter-based competitive polymerase chain reaction assay

Background: The current understanding of the inflammation associated with L yme disease directly involves infection caused by Borrelia burgdorferi with in specific target tissues, accompanied by a significant host immunologic c omponent driving the inflammatory process. The measurement of spirochetal t issue burden may thus be useful for studying animal models of Lyme disease pathogenesis. Widely available methods based on the culture of spirochetes from tissues do not provide quantitative information. Methods: We developed and evaluated a quantitative-competitive polymerase c hain reaction assay based on amplification of the B. burgdorferi flagellin gene. The assay makes use of a competitive internal standard and a commerci ally available enzyme-linked immunosorbent assay detection kit. Results and Conclusion: The assay clearly discriminated between infected an d uninfected mouse tissues, and an accurate quantitation range of 500 to 20 ,000 spirochetes per milligram of tissue was obtained. C3H mice were shown to harbor greater amounts of spirochetal genomic DNA than BALB/c mice. Norm alization of samples by tissue weight and genomic DNA content both provided acceptable results. These data indicate this assay can be used to provide reliable and meaningful measurements of spirochetal infectious burden, whic h will be extremely useful for the study of Lyme disease pathogenesis in th e murine model.