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

Abstract

Objective: To establish a one-tube fluorogenic real-time PCR assay for routine detection of Borrelia burgdorferi (sensu lato) DNA in various clinical specimens.

Methods: A fragment of the flagellin gene sequence was amplified with the TaqMan chemistry using primers and a probe common to Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii and Borrelia valaisiana. A recombinant 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 clinically relevant Borrelia burgdorferi strains. No cross-reaction occurred with unrelated bacteria, viruses and fungi. At an analytic sensitivity of 10 copies, excellent precision within runs and between runs was observed. The potential presence of inhibitors of the Taq DNA polymerase was monitored by spiking aliquots of each sample with a plasmid containing the target sequence. Among 56 cerebrospinal fluid samples taken from 54 patients with clinical suspicion of neuroborreliosis, one (1.8%) tested positive for Borrelia burgdorferi sensu lato DNA. Borrelia burgdorferi DNA was also detected in five (17.9%) of 28 synovial fluid specimens and in one (20%) of five synovial membrane biopsies obtained from 31 patients with arthropathies. In order to test for the absence of false-positive results, 84 samples from 83 patients without evidence of Lyme disease were investigated. None of these samples showed measurable amounts of Borrelia burgdorferi DNA.

Conclusion: By its established features, such as speed, reliability, sensitivity, specificity, the inclusion of carryover prevention and the monitoring of inhibitors in individual test tubes, this real-time PCR assay has proved to be a potent tool for the detection of Borrelia burgdorferi DNA under routine conditions in diagnostic laboratories.