Detection of species-specific DNA sequences of Borrelia burgdorferi in infected humans, animal reservoirs, and ixodid tick vectors

Abstract

Segments of the ospA gene that encode hydrophobic regions of the outer membrane protein, OspA, of Borrelia burgdorferi strain B31 were synthesized for use as oligonucleotide primers in the polymerase chain reaction (PCR). These oligonucleotide primers flank a 309-base-pair segment within the ospA gene. Optimal amplification conditions were achieved in a reaction mixture containing 0.2 uM of each oligonucleotide primer and 2 mMMgCl{dollar}\sb2.{dollar} Dimethyl sulfoxide at a concentration of 10% or higher was found to inhibit amplification and gelatin had no effect at concentrations below 100 ug/ml, and slight inhibition was seen at concentrations higher than 100 ug/ml. After 30 cycles of amplification under optimal conditions, the target fragment could be detected by agarose gel electrophoresis or dot hybridization with a {dollar}\sp{lcub}32{rcub}{dollar}P- or digoxigenin-labeled probe. This segment was amplified in all strains of B. burgdorferi, but it was not detected in other bacterial species. 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. Ten organisms per ml of blood or urine could be detected using PCR with dot hybridization detection. In a blinded study of Lyme disease patients, the OspA PCR was positive in 31% of patients who were early in disease and who had not received oral antibiotic therapy. No patient who had received antibiotics was positive in the PCR. 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 canines, one blood specimen showed reactivity in the PCR. Two of 32 cerebrospinal fluid specimens from suspected neuroborreliosis patients showed reactivity in the PCR. B. burgdorferi could be detected optimally in tissue only after DNA extraction. Nine of ten mice from a highly endemic Lyme disease area in Wisconsin showed reactivity in the PCR when DNA extracted from heart, kidney, or bladder was used as the target. Two of five punch biopsy tissue samples from skin lesions from suspected Lyme disease patients showed reactivity in the PCR. Of all tissues studied, one yielded a positive spirochete stain and all were negative by immunoperoxidase staining with a polyclonal antibody to B. burgdorferi. The conclusion of this study is that PCR can detect and identify B. burgdorferi in clinical samples from Lyme disease with greater sensitivity than any other currently available method and that this tool can be used to detect the spirochete in tick and animal reservoirs.