A nested RT-PCR for the detection of tick-borne encephalitis virus (TBEV) in ticks in natural foci

We have developed a sensitive nested reverse-transcriptase polymerase chain reaction assay (n RT-PCR) for the detection of the tick-borne encephalitis virus (TBEV) RNA, especially in ticks. The primer pairs were selected from the 5'-terminal noncoding region, a highly conserved part of the virus. Th e specificity was tested by computer homology searches of sequences as well as by the sequencing of the first and second amplificate, by Southern blot hybridization with a DIG-labelled oligonucleotide probe, and by restrictio n enzyme analysis. The method has proved to be very sensitive. The detectio n limit is about 20 fg of TBEV RNA per PCR run (25 mu l), or a single posit ive tick, i.e. (adult or nymph). The method can be used for comparative studies of the epidemiological situa tion, as well as for the screening of natural foci for the presence and cir culation of TBEV or for the detection of TBEV-genome-sequences in clinical materials.