Optimization of a Quantitative Real-Time RT-PCR Technique for Evaluation of Concentration of Genomic +RNA of Tick-Borne Encephalitis Virus

Abstract

Background. The specific detection of genomic/template +RNA and replicative –RNA of tick-born encephalitis virus (TBEV) is necessary to study the mechanisms of viral replication in the cells of reservoir and accidental hosts. However, the current approaches of quantitative reverse transcription – polymerase chain reaction (qRT-PCR) are rather focused on the detection of total viral RNA load in the sample. Thus, the significant optimization is necessary both for RT-PCR and for RNA copy number standard preparation.Aims. To develop the set of standard samples of synthetic +RNA of TBEV and to optimize qRT-PCR for quantification of genomic +RNA of the virus.Materials and methods. Fragment of the genomic +RNA of TBEV was synthesized using pTZ57R-T\A plasmid vector with embedded T7 promoter and T7 RNA polymerase. The DNA contamination was removed using RNase-free DNase I treatment followed by additional RNA purification step. Reverse transcription was performed using specific antisense primer 11154R 5`- AGCGGGTGTTTTTCCG-3` and qPCR detection was used according to the modified procedure of M. Schwaiger and P. Cassinotti (2003).Results. As a result of the amplification of standard samples, the concentration of positive polarity ТBEV RNA, carried out in five independent repetitions on different days, the correlation coefficient R2 between the quantification cycle and the concentration of the standard sample was 0.99, and the efficiency of PCR was 100 %. The coefficient of variation in assessing the inter-test accuracy of determination averaged 2.8 %.Conclusions. Optimized qRT-PCR procedure and set of +RNA standards allow to determine the concentration of genomic +RNA of TBEV in routine laboratory practice.