Voprosy virusologii · 2026
[Test systems for the laboratory detection and differentiation of Powassan virus from tick-borne encephalitis virus (Flaviviridae: Orthoflavivirus) using RT-PCR and RT-qPCR].
Аннотация на английскомСтатьяНазвание и аннотация: Перевод в очереди
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Fedina M P, Ivannikov R I, Karganova G G, Litov A G
Аннотация
INTRODUCTION: Powassan virus (POWV) is a tick-borne orthoflavivirus capable of causing neurological diseases. In the Russian Far East, the area affected by POWV overlaps with that of another neurotropic orthoflavivirus, tick-borne encephalitis virus (TBEV). Currently, there are no differentiated test systems for specific detection of POWV in the presence of TBEV. The aim of this work is to create differentiating test systems for the detection of POWV in a mixture with TBEV and other orthoflaviviruses.
MATERIALS AND METHODS: We constructed a genome alignment of eight POWV and five TBEV strains to select differentiating primers using MEGA X. To assess the specificity of the assay, we used POWV, several TBEV strains, and other orthoflaviviruses. PCR and qPCR were performed with the selected oligonucleotides, and the specificity of amplification was verified by electrophoresis in agarose gel and Sanger sequencing. cDNA was obtained from virus-containing material by isolating RNA and performing a reverse transcription reaction.
RESULTS: The selected oligonucleotides for the qPCR-based differentiation system ensured specific detection of POWV without detecting signals from other orthoflaviviruses. The assay's limit of detection was 102 copies/sample. Analysis of mixed samples containing POWV and TBEV demonstrated no distortion of the measurement results. Furthermore, differentiating oligonucleotides were selected and tested to amplify extended regions of the POWV and TBEV genomes for subsequent sequencing and phylogenetic analysis.
CONCLUSION: A laboratory technique based on PCR has been developed that allows to detect the POWV in samples containing other orthoflaviviruses.
MATERIALS AND METHODS: We constructed a genome alignment of eight POWV and five TBEV strains to select differentiating primers using MEGA X. To assess the specificity of the assay, we used POWV, several TBEV strains, and other orthoflaviviruses. PCR and qPCR were performed with the selected oligonucleotides, and the specificity of amplification was verified by electrophoresis in agarose gel and Sanger sequencing. cDNA was obtained from virus-containing material by isolating RNA and performing a reverse transcription reaction.
RESULTS: The selected oligonucleotides for the qPCR-based differentiation system ensured specific detection of POWV without detecting signals from other orthoflaviviruses. The assay's limit of detection was 102 copies/sample. Analysis of mixed samples containing POWV and TBEV demonstrated no distortion of the measurement results. Furthermore, differentiating oligonucleotides were selected and tested to amplify extended regions of the POWV and TBEV genomes for subsequent sequencing and phylogenetic analysis.
CONCLUSION: A laboratory technique based on PCR has been developed that allows to detect the POWV in samples containing other orthoflaviviruses.