PURPOSE: For simultaneous detection of Borrelia miyamotoi (relapsing fever spirochete) and Borrelia burgdorferi sensu lato, we have developed a duplex real-time PCR targeting the flagellin gene (flaB; p41), a locus frequently used in routine diagnostic PCR for B. burgdorferi s.l. detection. METHODS: Primers and probes were designed using multiple alignments of flaB sequences of B. miyamotoi and B. burgdorferi s.l. species. The sensitivity and specificity of primers and probes were determined using serial dilutions (ranging from 10(4) to 10(-1)) of B. miyamotoi and B. burgdorferi s.l. DNA and of several species of relapsing fever spirochetes. Conventional PCR on recG and glpQ and sequencing of p41 PCR products were used to confirm the species assignment. RESULTS: The detection limit of both singleplex and duplex PCR was 10 genome equivalents except for B. spielmanii and two B. garinii genotypes which showed a detection limit of 10(2) genome equivalents. There was no cross reactivity of the B. miyamotoi primers/probes with B. burgdorferi s.l. DNA, while the B. burgdorferi s.l. primer/probe generated a signal with B. hermsii DNA. Out of 2341 Ixodes ricinus ticks from Germany and Slovakia that were screened simultaneously for the presence of B. miyamotoi and B. burgdorferi s.l., 52 were positive for B. miyamotoi and 276 for B. burgdorferi s.l., denoting an average prevalence of 2.2% for B. miyamotoi and 11.8% for B. burgdorferi s.l., and B. miyamotoi DNA was also detectable by PCR using artificial clinical samples. CONCLUSION: The duplex real-time PCR developed here represents a method that permits simultaneous detection and differentiation of B. burgdorferi s.l. and B. miyamotoi in environmental and potentially clinical samples.

Bavarian Health and Food Safety Authority Veterinärstr 2 85764 Oberschleissheim Germany

Comparative Tropical Medicine and Parasitology Ludwig Maximilians Universität München Munich Leopoldstr 5 80802 Munich Germany Lehrstuhl für Bakteriologie und Mykologie Veterinärwissenschaftliches Department Tierärztliche Fakultät der LMU Veterinärstr 13 80539 Munich Germany

Comparative Tropical Medicine and Parasitology Ludwig Maximilians Universität München Munich Leopoldstr 5 80802 Munich Germany Swiss National Reference Center for Vector Entomology Institute of Parasitology Vetsuisse Faculty University of Zurich Winterthurerstr 266A 8057 Zurich Switzerland

German National Reference Centre for Borrelia Veterinärstr 2 85764 Oberschleissheim Germany

German National Reference Centre for Borrelia Veterinärstr 2 85764 Oberschleissheim Germany Bavarian Health and Food Safety Authority Veterinärstr 2 85764 Oberschleissheim Germany

Institute of Zoology Slovak Academy of Sciences Dúbravská cesta 9 84506 Bratislava Slovakia

University of Veterinary and Pharmaceutical Sciences Palackého 1 3 612 42 Brno Czech Republic

Zoology Parasitology Ruhr University Bochum Universitätsstr 150 44780 Bochum Germany

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