Pedersen et al.
Development of a real-time PCR method for the detection of European and Siberian subtypes of tick-borne encephalitis virus
Microbiol Res. 2023;14(4):1545-1558. doi:10.3390/microbiolres14040106
TBE virus subtypes can be found in various regions within Eurasia. The European subtype mainly circulates in Europe (in Ixodes ricinus), and the Far Eastern subtype in Far Eastern and Siberia (in Ixodes persulcatus). The Siberian subtype is mainly distributed in Siberia (Ixodes persulcatus as host), but it seems to move westwards, as it was also identified in the Baltic countries, Finland and Sweden, where it was recently shown to spread out (see, e.g., Snapshot week 42/2023).
The Siberian subtype has a higher virulence compared to the European subtype, and therefore raises the concern of further expansion into regions where this subtype has so far not been detected. To monitor the situation, a real-time PCR has been developed which sensitively detects both the European and the Siberian subtype with a refined primer set.
In this study, a new approach of primer design improvement based on the melting temperature (Tm) determination of fully matched and partially matched duplexes using synthetic target sequences was explored. The efficiency of the real-time PCR varied with the different subtypes of TBE virus, though it was above 80% for both subtype strains tested; the amplification efficiency ranged from 83% to 94%, depending on the strain tested. The designed primers do not cross-react with louping ill virus, the most closely related flavivirus. The limit of detection was 10–100 DNA copies per reaction.
The developed real-time PCR should prove a useful tool in the surveillance of the spread of the virulent strains of both the Siberian and the Far Eastern subtypes in Europe.