Development of a Real-Time PCR Method for the Detection of European and Siberian Subtypes of Tick-Borne Encephalitis Virus

Author:

Pedersen Benedikte N.12ORCID,Jenkins Andrew1ORCID,Paulsen Katrine M.2,Basset Coraline2,Andreassen Åshild K.12ORCID

Affiliation:

1. Department of Natural Science and Environmental Health, University of South-Eastern Norway, NO-3800 Bø, Norway

2. Department of Virology, Division for Infection Control and Environmental Health, Norwegian Institute of Public Health, NO-0456 Oslo, Norway

Abstract

The tick-borne encephalitis virus (TBEV) is transmitted to humans through tick bites. In recent years, the appearance of the Siberian subtype of TBEV in Ixodes ricinus in Finland, together with deaths from the normally mild European subtype in the same country, have raised concerns about a possible spread of virulent variants of TBEV in Western Europe. Thus, there is a need to monitor the spread of strains, particularly of the European and Siberian subtypes. In this study, we develop a new real-time PCR method targeting Siberian and European subtypes of TBEV. The primers amplify a 176 bp fragment of the E gene, which is suitable for subsequent strain identification by Sanger sequencing. This study pioneers a new approach to primer design where the melting temperature (Tm) of primers annealed to representative mismatched target sequences is empirically determined and used to guide improvements in primer sequence. This allowed the range of TBEV strains detected to be extended to cover most European and Siberian strains tested, in addition to a strain of the Far-Eastern subtype. The limit of detection was 10–100 DNA copies per reaction and amplification efficiency varied between 83% and 94%, depending on the TBEV strain. Experimental determination of primer Tm proved to be a fruitful approach and will be a useful tool for future primer design and diagnostics.

Funder

EU Interreg IV A program

Publisher

MDPI AG

Subject

Microbiology (medical),Molecular Biology,Microbiology

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