Understanding epidemiology of the tick-borne pathogens requires the accurate 
identification of the vector ticks. Morphological analysis of ticks is difficult 
and often leads to misidentification. Molecular techniques offer an alternative 
approach of tick identification. To date, no practical and reliable molecular 
assays for discrimination of Euro-Asian ticks are available. Our aim was to 
develop such an assay for discrimination between four Euro-Asian tick species of 
high medical importance such as Ixodes ricinus, Ixodes persulcatus, Ixodes 
hexagonus, and Dermacentor reticulatus. As a basis, we have chosen conventional 
species-specific polymerase chain reaction (PCR), a technique providing a good 
combination of simplicity and reliability. The DNA information available on 
ticks was searched for orthologous loci containing stretches of sequence 
dissimilarity sufficient for designing species-specific primers. ITS2 locus 
(second internal transcribed region of the rRNA gene cluster) was found to be 
the most favorable for primer design. Finally, for each of the three Ixodes 
species a PCR was developed amplifying only for the targeted species. One PCR 
amplified the entire ITS2 locus of the four species and allowed discrimination 
of D. reticulatus from the Ixodes species on the basis of the size difference of 
the respective PCR products. This PCR system was successfully tested for 
discrimination of the ticks at different maturation stages (larva, nymph, and 
adult) in engorged and unfed conditions, and therefore it may be useful for 
large-scale epidemiological studies. Differentiation between the closely related 
I. ricinus and I. persulcatus, the two species most often occurring in the 
tick-borne diseases in Eurasia, is of special importance.