R Venczel1,2, L Knoke3, M Pavlovic4, E Dzaferovic5, T Vaculova6, C Silaghi7,8, E Overzier9,10, R Konrad11, S Kolenčík12, M Derdakova13, A Sing14, G A Schaub15, G Margos16,17, V Fingerle18,19. 1. German National Reference Centre for Borrelia, Veterinärstr. 2, 85764, Oberschleissheim, Germany. rita.venczel92@gmail.com. 2. Bavarian Health and Food Safety Authority, Veterinärstr. 2, 85764, Oberschleissheim, Germany. rita.venczel92@gmail.com. 3. Zoology/Parasitology, Ruhr University Bochum, Universitätsstr. 150, 44780, Bochum, Germany. Lisa.Knoke@ruhr-uni-bochum.de. 4. Bavarian Health and Food Safety Authority, Veterinärstr. 2, 85764, Oberschleissheim, Germany. Melanie.Pavlovic@lgl.bayern.de. 5. German National Reference Centre for Borrelia, Veterinärstr. 2, 85764, Oberschleissheim, Germany. eldina.dzaferovic@student.hswt.de. 6. Institute of Zoology, Slovak Academy of Sciences, Dúbravská cesta 9, 84506, Bratislava, Slovakia. tana.vaculova@gmail.com. 7. Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universität München, Munich, Leopoldstr. 5, 80802, Munich, Germany. cornelia.silaghi@uzh.ch. 8. Swiss National Reference Center for Vector Entomology, Institute of Parasitology, Vetsuisse Faculty, University of Zurich, Winterthurerstr. 266A, 8057, Zurich, Switzerland. cornelia.silaghi@uzh.ch. 9. Comparative Tropical Medicine and Parasitology, Ludwig-Maximilians-Universität München, Munich, Leopoldstr. 5, 80802, Munich, Germany. Evelyn.Overzier@micro.vetmed.uni-muenchen.de. 10. Lehrstuhl für Bakteriologie und Mykologie, Veterinärwissenschaftliches Department, Tierärztliche Fakultät der LMU, Veterinärstr. 13, 80539, Munich, Germany. Evelyn.Overzier@micro.vetmed.uni-muenchen.de. 11. Bavarian Health and Food Safety Authority, Veterinärstr. 2, 85764, Oberschleissheim, Germany. regina.konrad@lgl.bayern.de. 12. University of Veterinary and Pharmaceutical Sciences, Palackého 1/3, 612 42, Brno, Czech Republic. stanislav.kolencik@gmail.com. 13. Institute of Zoology, Slovak Academy of Sciences, Dúbravská cesta 9, 84506, Bratislava, Slovakia. marketa.derdakova@gmail.com. 14. Bavarian Health and Food Safety Authority, Veterinärstr. 2, 85764, Oberschleissheim, Germany. andreas.sing@lgl.bayern.de. 15. Zoology/Parasitology, Ruhr University Bochum, Universitätsstr. 150, 44780, Bochum, Germany. Guenter.schaub@rub.de. 16. German National Reference Centre for Borrelia, Veterinärstr. 2, 85764, Oberschleissheim, Germany. gabriele.margos@lgl.bayern.de. 17. Bavarian Health and Food Safety Authority, Veterinärstr. 2, 85764, Oberschleissheim, Germany. gabriele.margos@lgl.bayern.de. 18. German National Reference Centre for Borrelia, Veterinärstr. 2, 85764, Oberschleissheim, Germany. volker.fingerle@lgl.bayern.de. 19. Bavarian Health and Food Safety Authority, Veterinärstr. 2, 85764, Oberschleissheim, Germany. volker.fingerle@lgl.bayern.de.
Abstract
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.
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.
Entities:
Keywords:
Borrelia burgdorferi sensu lato; Borrelia miyamotoi; Real-time PCR
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