Routine diagnosis of Borrelia burgdorferi (sensu lato) infections using a real-time PCR assay

Objective To establish a one-tube fluorogenic real-time PCR assay for routi ne detection of Borrelia burgdorferi (sensu lato) DNA in various clinical s pecimens. Methods A fragment of the flagellin gene sequence was amplified with the Ta qMan chemistry using primers and a probe common to Borrelia burgdorferi sen su stricto, Borrelia afzelii, Borrelia garinii and Borrelia valaisiana. A r ecombinant plasmid containing the chromosomal gene coding for the flagellin protein was used as standard. Results The specificity of the assay was documented with 48 different clini cally relevant Borrelia burgdorferi strains. No cross-reaction occurred wit h unrelated bacteria, viruses and fungi. At an analytic sensitivity of 10 c opies, excellent precision within runs and between runs was observed. The p otential presence of inhibitors of the Taq DNA polymerase was monitored by spiking aliquots of each sample with a plasmid containing the target sequen ce. Among 56 cerebrospinal fluid samples taken from 54 patients with clinic al suspicion of neuroborreliosis, one (1.8%) tested positive for Borrelia b urgdorferi sensu lato DNA. Borrelia burgdorferi DNA was also detected in fi ve (17.9%) of 28 synovial fluid specimens and in one (20%) of five synovial membrane biopsies obtained from 31 patients with arthropathies. In order t o test for the absence of false-positive results, 84 samples from 83 patien ts without evidence of Lyme disease were investigated. None of these sample s showed measurable amounts of Borrelia burgdorferi DNA. Conclusion By its established features, such as speed, reliability, sensiti vity, specificity, the inclusion of carryover prevention and the monitoring of inhibitors in individual test tubes, this real-time PCR assay has prove d to be a potent tool for the detection of Borrelia burgdorferi DNA under r outine conditions in diagnostic laboratories.