Sensitive Detection of Borrelia burgdorferi Sensu Lato DNA and Differentiation of Borrelia Species by LightCycler PCR

Abstract

In order to differentiate species within the Borrelia burgdorferi sensu lato complex, LightCyler PCR and melting-curve analysis of the amplicons of two genes with intraspecies variability, the p66 gene and the recA gene, were performed. It was demonstrated that nested LightCycler PCR amplification of p66 is more sensitive in the detection of borrelia DNA than amplification of the recA gene. B. burgdorferi sensu stricto could be differentiated from Borrelia garinii and Borrelia afzelii by melting-curve analysis of the p66 gene amplicon. B. garinii could be differentiated from B. afzelii and B. burgdorferi sensu stricto by melting-curve analysis of the recA gene amplicon. Therefore, the PCRs complement each other in subtyping different Borrelia species, and combined LightCycler PCR and melting-curve analysis of both target genes is a rapid method to distinguish the three species of B. burgdorferi sensu lato.Lyme disease is the most prevalent tick-borne disease of the Northern Hemisphere (3). Its etiologic agent, Borrelia burgdorferi sensu lato, has been divided into different species. B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii are the common human pathogenic species (4, 21). The infection leads to a variety of clinical symptoms involving the skin, nervous system, heart, and joints (19).Erythema migrans (EM) and acrodermatitis chronica atrophicans (ACA) represent common cutaneous manifestations of an infection by B. burgdorferi sensu lato (1, 13, 21), whereas the role of this spirochete in the pathogenesis of morphea and lichen sclerosus et atrophicus is controversial (13,15,23,25).Since PCR has proved to be a sensitive and fast method for the diagnosis of microrganisms which are difficult to culture, the technique has been applied to the detection of B. burgdorferi sensu lato DNA in infected ticks (6) as well as in human specimens, such as cerebrospinal fluid (4) or synovial fluid (5, 20), urine (2, 16), and skin (9, 24). Established PCR protocols amplify different segments of borrelial chromosomal genes, such as the flagellin gene (10, 15), the one-copy 16S rRNA gene (7), the 23S rRNA gene (17), the p66 gene segment encoding a 66-kDa protein (14), the recA gene (8), and the plasmid-encoded ospA gene (9,20).Common PCR with a conventional thermocycler and subsequent separation of the amplicon by agarose gel electrophoresis allows the detection of B. burgdorferi sensu lato DNA. Subtyping of Borrelia species DNA is not possible, since the intraspecies sequence polymorphisms of PCR amplicons are only a few base pairs long.Several postamplification methods were employed to identify Borrelia species commonly associated with Lyme borreliosis, e.g., oligonucleotide typing with PCR fragments (5), randomly amplified polymorphic DNA fingerprinting analysis (22), pulsed-field gel electrophoresis (4), single-strand conformation polymorphism (18), and subtype-specific PCR targeting the 16S rRNA gene (6). These techniques are usually time-consuming, and some of them require high technical standards and ...

Quantitative Detection of Borrelia burgdorferi in 2-Millimeter Skin Samples of Erythema Migrans Lesions: Correlation of Results with Clinical and Laboratory Findings

“…The higher sensitivity of qPCR over that of nested PCR may be attributed to the fact that sample amplification and quantitative analysis were monitored in real time by a fluorometric assay with the double-stranded DNA-specific dye SYBR Green I. Amplicon detection was performed at the end of each cycle, not at the end point of amplification, as was done for nested PCR. The apparent discrepancy between the two PCR amplification methods was most likely due to inherent differences in amplicon detection technology and not to target amplification differences, as noted previously (11,24). Both PCR amplification procedures targeted single-copy chromosomal genes with comparable specificities and sensitivities.…”

“…The method used was an adaptation of one previously reported by Morrison et al (12) for use with mouse tissues. While previous studies have used the LightCycler instrument to type B. burgdorferi sensu lato (11,17,18), to our knowledge the present study is the first one in which qPCR has been used to enumerate the B. burgdorferi organisms in specimens from Lyme disease patients and the first to attempt to correlate spirochete numbers with clinical findings and other laboratory results. In addition to qPCR, conventional nested PCR and culture were also performed with the same skin biopsy specimens.…”

Distribution of Clinically RelevantBorreliaGenospecies in Ticks Assessed by a Novel, Single-Run, Real-Time PCR

“…Recently, two LightCycler PCR-based assays for the differentiation of Borrelia species were described (15,19). The second method, an amendment of the first, could distinguish among all three Borrelia species known to be pathogenic for humans.…”

“…Furthermore, calculation of the melting point of the DNA-probe adduct enables identification of the PCR product. This method can be exploited to distinguish sequence deviations, e.g., polymorphisms of different bacterial strains.Recently, two LightCycler PCR-based assays for the differentiation of Borrelia species were described (15,19). The second method, an amendment of the first, could distinguish among all three Borrelia species known to be pathogenic for humans.…”

Real-Time PCR for Simultaneous Detection and Quantification of Borrelia burgdorferi in Field-Collected Ixodes scapularis Ticks from the Northeastern United States

“…The technique has recently been employed to detect the presence of B. burgdorferi DNA in I. ricinus ticks from Switzerland (15), to quantify the spirochete loads in experimentally infected animal tissues (23,24,35), and to differentiate the three B. burgdorferi sensu lato species that are pathogenic to humans in Europe (22,28,30). By targeting the B. burgdorferi-specific recA gene, we have successfully developed a real-time PCR protocol to quantify the spirochetes in infected animal tissues and in skin biopsy specimens of patients with erythema migrans (19,36,37).…”