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Background  Rickettsioses are caused by pathogenic species of the genus  Rickettsia  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  Rickettsia  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  Ixodes  ticks, which are  R. helvetica ,  R. monacensis ,  R. massiliae , and  R. felis . Tick material from a quantitative real-time PCR (qPCR) based study on  Rickettsia -infections in  I. ricinus  allowed direct comparison of both sequencing techniques, Sanger and real-time pyrosequencing.    Methods  A sequence stretch of rickettsial citrate synthase ( gltA ) gene was identified to contain divergent single nucleotide polymorphism (SNP) sites suitable for  Rickettsia  species differentiation. Positive control plasmids inserting the respective target sequence of each  Rickettsia  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  Ixodes  tick samples already detected  Rickettsia -positive in previous qPCR experiments.    Results  Establishment of real-time pyrosequencing using positive control plasmids resulted in accurate detection of all SNPs in all included  Rickettsia  species. The method was then applied to 263  Rickettsia -positive  Ixodes ricinus  samples, of which 153 (58.2%) could be identified for their species (151  R. helvetica  and 2  R. monacensis ) 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  gltA  copy numbers in the reaction setup. Whereas less than 10 1  copies in the initial amplification reaction resulted in identification of 15.1% of the samples only, the percentage increased to 54.2% at 10 1 -10 2  copies, to 95.6% at >10 2 -10 3  copies and reached 100% samples identified for their  Rickettsia  species if more than 10 3  copies were present in the template.    Conclusions  The established real-time pyrosequencing approach represents a reliable method for detection and differentiation of  Rickettsia  spp. present in  I. ricinus  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.

bmc infectious diseases

SNP-based real-time pyrosequencing as a sensitive and specific tool for identification and differentiation of Rickettsia species in Ixodes ricinus ticks