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.
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 104 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.
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 102 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.
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.
R. Venczel and L. Knoke contributed equally to this work.
The online version of this article (doi: 10.1007/s15010-015-0820-8) contains supplementary material, which is available to authorized users.
The authors would like to acknowledge Cecilia Hizo-Teufel, Sylvia Stockmeier, Katja Meindl, Sabine Wolf, Jasmin Fräßdorf, A. Muminovic, AK Steinmann, Claudia Brefeld, and Simone Hanisch for excellent technical assistance. The work was partially funded by the Scientific Grant Agency of the Ministry of Education, science, research and sport of the Slovak Republic and the Slovak Academy of Sciences, Project VEGA No. 2/0108/13. This study was partially funded by EUgrant FP7-261504 EDENext and is cataloged by the EDENext Steering Committee (http:/www.edenext.eu/). The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. The work of CS was done under the frame of EurNegVec COST Action TD1303. V. Fingerle, G. Margos, and M. Derdakova are the members of ESGBOR.