title: A novel duplex real-time PCR permits simultaneous detection and differentiation of Borrelia miyamotoi and Borrelia burgdorferi sensu lato.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: German National Reference Centre for Borrelia, Veterinärstr. 2, 85764, Oberschleissheim, Germany. rita.venczel92@gmail.com.|Bavarian Health and Food Safety Authority, Veterinärstr. 2, 85764, Oberschleissheim, Germany. rita.venczel92@gmail.com.
authors.forename: R
authors.initials: R
authors.lastname: Venczel
authors.affiliation: Zoology/Parasitology, Ruhr University Bochum, Universitätsstr. 150, 44780, Bochum, Germany. Lisa.Knoke@ruhr-uni-bochum.de.
authors.forename: L
authors.initials: L
authors.lastname: Knoke
authors.affiliation: Bavarian Health and Food Safety Authority, Veterinärstr. 2, 85764, Oberschleissheim, Germany. Melanie.Pavlovic@lgl.bayern.de.
authors.forename: M
authors.initials: M
authors.lastname: Pavlovic
authors.affiliation: German National Reference Centre for Borrelia, Veterinärstr. 2, 85764, Oberschleissheim, Germany. eldina.dzaferovic@student.hswt.de.
authors.forename: E
authors.initials: E
authors.lastname: Dzaferovic
authors.affiliation: Institute of Zoology, Slovak Academy of Sciences, Dúbravská cesta 9, 84506, Bratislava, Slovakia. tana.vaculova@gmail.com.
authors.forename: T
authors.initials: T
authors.lastname: Vaculova
authors.affiliation: Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universität München, Munich, Leopoldstr. 5, 80802, Munich, Germany. cornelia.silaghi@uzh.ch.|Swiss National Reference Center for Vector Entomology, Institute of Parasitology, Vetsuisse Faculty, University of Zurich, Winterthurerstr. 266A, 8057, Zurich, Switzerland. cornelia.silaghi@uzh.ch.
authors.forename: C
authors.initials: C
authors.lastname: Silaghi
authors.affiliation: Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universität München, Munich, Leopoldstr. 5, 80802, Munich, Germany. Evelyn.Overzier@micro.vetmed.uni-muenchen.de.|Lehrstuhl für Bakteriologie und Mykologie, Veterinärwissenschaftliches Department, Tierärztliche Fakultät der LMU, Veterinärstr. 13, 80539, Munich, Germany. Evelyn.Overzier@micro.vetmed.uni-muenchen.de.
authors.forename: E
authors.initials: E
authors.lastname: Overzier
authors.affiliation: Bavarian Health and Food Safety Authority, Veterinärstr. 2, 85764, Oberschleissheim, Germany. regina.konrad@lgl.bayern.de.
authors.forename: R
authors.initials: R
authors.lastname: Konrad
authors.affiliation: University of Veterinary and Pharmaceutical Sciences, Palackého 1/3, 612 42, Brno, Czech Republic. stanislav.kolencik@gmail.com.
authors.forename: S
authors.initials: S
authors.lastname: Kolenčík
authors.affiliation: Institute of Zoology, Slovak Academy of Sciences, Dúbravská cesta 9, 84506, Bratislava, Slovakia. marketa.derdakova@gmail.com.
authors.forename: M
authors.initials: M
authors.lastname: Derdakova
authors.affiliation: Bavarian Health and Food Safety Authority, Veterinärstr. 2, 85764, Oberschleissheim, Germany. andreas.sing@lgl.bayern.de.
authors.forename: A
authors.initials: A
authors.lastname: Sing
authors.affiliation: Zoology/Parasitology, Ruhr University Bochum, Universitätsstr. 150, 44780, Bochum, Germany. Guenter.schaub@rub.de.
authors.forename: G A
authors.initials: GA
authors.lastname: Schaub
authors.affiliation: German National Reference Centre for Borrelia, Veterinärstr. 2, 85764, Oberschleissheim, Germany. gabriele.margos@lgl.bayern.de.|Bavarian Health and Food Safety Authority, Veterinärstr. 2, 85764, Oberschleissheim, Germany. gabriele.margos@lgl.bayern.de.
authors.forename: G
authors.initials: G
authors.lastname: Margos
authors.affiliation: German National Reference Centre for Borrelia, Veterinärstr. 2, 85764, Oberschleissheim, Germany. volker.fingerle@lgl.bayern.de.|Bavarian Health and Food Safety Authority, Veterinärstr. 2, 85764, Oberschleissheim, Germany. volker.fingerle@lgl.bayern.de.
authors.forename: V
authors.initials: V
authors.lastname: Fingerle
mesh_terms: D000818:Animals; D001898:Borrelia / Q000145:classification* / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D005408:Flagellin / Q000235:genetics; D005858:Germany; D006801:Humans; D018884:Ixodes / Q000382:microbiology*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D018154:Slovakia
chemical_list: D017931:DNA Primers; D015345:Oligonucleotide Probes; D005408:Flagellin
keywords: Borrelia burgdorferi sensu lato; Borrelia miyamotoi; Real-time PCR