Establishment of a minor groove binder-probe based quantitative real time PCR to detect Borrelia burgdorferi sensu lato and differentiation of Borrelia spielmanii by ospA-specific conventional PCR

Abstract

<p>Abstract</p> <p>Background</p> <p><it>Borrelia burgdorferi </it>sensu lato (sl), the causative agent of Lyme borreliosis, is transmitted by ticks of the genus <it>Ixodes </it>as vector. For identification of <it>Borrelia </it>infections in ticks a TaqMan™ minor groove binder (MGB) probe-based quantitative real time PCR (qPCR) was established targeting the 5S-23S intergenic spacer. Extension to a duplex qPCR included an <it>Ixodes </it>spp. positive control to verify successful DNA isolation. Besides qPCR, an <it>osp</it>A-specific conventional PCR for species-specific identification of <it>B. spielmanii </it>was established. Afterwards 1000 <it>I. ricinus </it>flagged in the city of Hanover, Germany, were investigated for <it>B. burgdorferi </it>sl infections followed by species identification. Furthermore, <it>I. hexagonus </it>ticks were investigated to proof applicability of the PCRs.</p> <p>Results</p> <p>Quantitative real time PCR (qPCR) identifying <it>B. burgdorferi </it>sl in ticks was able to detect 1-10 copies per reaction. <it>B. spielmanii osp</it>A-specific conventional PCR was also highly specific and showed no cross reactions with the other tested <it>Borrelia </it>species. From 1000 hanoveranian ticks 24.3% were positive compared to only 7.4% positives by dark-field microscopy. Related to tick stage 1.7% larvae, 18.1% nymphs, and 34.6% adults were positive. The most frequent species was <it>B. garinii</it>, followed by <it>B. afzelii</it>, <it>B. spielmanii</it>, <it>B. valaisiana </it>and <it>B. burgdorferi </it>sensu stricto (ss). 70.6% of <it>I. ricinus </it>were mono-infected, whereas 28.0% and 1.4% were infected with two and three <it>Borrelia </it>species, respectively. From 232 <it>I. hexagonus </it>collected from hedgehogs in different sites of Germany, qPCR detected 5.7% to be infected with <it>B. burgdorferi </it>sl, which were identified as <it>B. afzelii</it>, <it>B. garinii </it>and <it>B. spielmanii</it>.</p> <p>Conclusions</p> <p>The evaluated qPCR to detect <it>B. burgdorferi </it>sl in <it>Ixodes </it>spp. is highly specific and sensitive. As a duplex qPCR including detection of <it>Ixodes </it>spp. DNA it is the first DNA based technique incorporating a control for successful DNA isolation from the vector tick. Establishment of a <it>B. spielmanii </it>specific conventional PCR filled the gap in PCR identification of principal European <it>Borrelia </it>genospecies. Practical application showed that all European pathogenic <it>Borrelia </it>spp. were present in <it>I. ricinus </it>flagged in recreational areas of the city of Hanover and confirmed <it>I. hexagonus </it>as reservoir for pathogenic <it>Borrelia </it>spp.</p