Quantitative detection of Borrelia burgdorferi with a microtiter-based competitive polymerase chain reaction assay.
BACKGROUND
The current understanding of the inflammation associated with Lyme disease directly involves infection caused by Borrelia burgdorferi within specific target tissues, accompanied by a significant host immunologic component driving the inflammatory process. The measurement of spirochetal tissue 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 chain reaction assay based on amplification of the B. burgdorferi flagellin gene. The assay makes use of a competitive internal standard and a commercially available enzyme-linked immunosorbent assay detection kit.


CONCLUSIONS
The assay clearly discriminated between infected and 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. Normalization 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, which will be extremely useful for the study of Lyme disease pathogenesis in the murine model.
Germer
J
J
Division of Experimental Pathology, Mayo Clinic, Rochester, Minnesota, USA.
Ryckmann
B
B
Moro
M
M
Hofmeister
E
E
Barthold
S W
SW
Bockenstedt
L
L
Persing
D H
DH
D000818:Animals; D001667:Binding, Competitive; D015748:Borrelia burgdorferi Group / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004198:Disease Susceptibility; D005408:Flagellin / Q000235:genetics; D018884:Ixodes / Q000382:microbiology; D008193:Lyme Disease / Q000175:diagnosis / Q000382:microbiology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008809:Mice, Inbred C3H; D008832:Microchemistry; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D005408:Flagellin