Detection of Borrelia burgdorferi using the polymerase chain reaction

Abstract

Segments of the ospA gene of Borrelia burgdorferi were amplified by the polymerase chain reaction (PCR). Oligonucleotide primers used in the reaction flank a 309-base-pair segment within the ospA gene. After 35 cycles of amplification, the product could be detected by agarose gel electrophoresis or dot hybridization with a 32P-labeled probe. This segment was amplified in all strains of B. burgdorferi tested, but it was not detected in other bacterial species. An additional primer pair which has a broad specificity for conserved 16S rRNA sequences that are present in eubacteria amplified a 215-base-pair fragment in all organisms tested. The sensitivity of PCR for the detection of B. burgdorferi in clinical samples was evaluated by seeding blood and urine specimens with B. burgdorferi and subjecting them to amplification. We were able to detect 10 organisms per ml of blood or urine, using PCR with dot hybridization detection. DNA extraction is not required for sample preparation. Blood and urine specimens were obtained from canines with clinical and serologic evidence of Lyme disease and subjected to PCR analysis. Of 17 clinical specimens from 15 animals, one blood specimen showed reactivity in the PCR.