| Summary: | <p>Abstract</p> <p>Background</p> <p>Rickettsioses are caused by pathogenic species of the genus <it>Rickettsia</it> and play an important role as emerging diseases. The bacteria are transmitted to mammal hosts including humans by arthropod vectors. Since detection, especially in tick vectors, is usually based on PCR with genus-specific primers to include different occurring <it>Rickettsia</it> species, subsequent species identification is mainly achieved by Sanger sequencing. In the present study a real-time pyrosequencing approach was established with the objective to differentiate between species occurring in German <it>Ixodes</it> ticks, which are <it>R. helvetica</it>, <it>R. monacensis</it>, <it>R. massiliae</it>, and <it>R. felis</it>. Tick material from a quantitative real-time PCR (qPCR) based study on <it>Rickettsia</it>-infections in <it>I. ricinus</it> allowed direct comparison of both sequencing techniques, Sanger and real-time pyrosequencing.</p> <p>Methods</p> <p>A sequence stretch of rickettsial citrate synthase (<it>gltA</it>) gene was identified to contain divergent single nucleotide polymorphism (SNP) sites suitable for <it>Rickettsia</it> species differentiation. Positive control plasmids inserting the respective target sequence of each <it>Rickettsia</it> species of interest were constructed for initial establishment of the real-time pyrosequencing approach using Qiagen’s PSQ 96MA Pyrosequencing System operating in a 96-well format. The approach included an initial amplification reaction followed by the actual pyrosequencing, which is traceable by pyrograms in real-time. Afterwards, real-time pyrosequencing was applied to 263 <it>Ixodes</it> tick samples already detected <it>Rickettsia</it>-positive in previous qPCR experiments.</p> <p>Results</p> <p>Establishment of real-time pyrosequencing using positive control plasmids resulted in accurate detection of all SNPs in all included <it>Rickettsia</it> species. The method was then applied to 263 <it>Rickettsia</it>-positive <it>Ixodes ricinus</it> samples, of which 153 (58.2%) could be identified for their species (151 <it>R. helvetica</it> and 2 <it>R. monacensis</it>) by previous custom Sanger sequencing. Real-time pyrosequencing identified all Sanger-determined ticks as well as 35 previously undifferentiated ticks resulting in a total number of 188 (71.5%) identified samples. Pyrosequencing sensitivity was found to be strongly dependent on <it>gltA</it> copy numbers in the reaction setup. Whereas less than 10<sup>1</sup> copies in the initial amplification reaction resulted in identification of 15.1% of the samples only, the percentage increased to 54.2% at 10<sup>1</sup>-10<sup>2</sup> copies, to 95.6% at >10<sup>2</sup>-10<sup>3</sup> copies and reached 100% samples identified for their <it>Rickettsia</it> species if more than 10<sup>3</sup> copies were present in the template.</p> <p>Conclusions</p> <p>The established real-time pyrosequencing approach represents a reliable method for detection and differentiation of <it>Rickettsia</it> spp. present in <it>I. ricinus</it> diagnostic material and prevalence studies. Furthermore, the method proved to be faster, more cost-effective as well as more sensitive than custom Sanger sequencing with simultaneous high specificity.</p>
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