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

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 – po...

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Autores principales:	N. A. Liapunovа, M. A. Khasnatinov, G. A. Danchinova
Formato:	article
Lenguaje:	RU
Publicado:	Scientific Сentre for Family Health and Human Reproduction Problems 2019
Materias:	flavivirus tick-borne encephalitis virus rna quantitative rt-pcr in real time Science Q
Acceso en línea:	https://doaj.org/article/04a37aa28601493e827853f840f201c0
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spelling	oai:doaj.org-article:04a37aa28601493e827853f840f201c02021-11-23T06:14:43ZOptimization of a Quantitative Real-Time RT-PCR Technique for Evaluation of Concentration of Genomic +RNA of Tick-Borne Encephalitis Virus2541-94202587-959610.29413/ABS.2019-4.5.18https://doaj.org/article/04a37aa28601493e827853f840f201c02019-11-01T00:00:00Zhttps://www.actabiomedica.ru/jour/article/view/2179https://doaj.org/toc/2541-9420https://doaj.org/toc/2587-9596Background. 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.N. A. LiapunovаM. A. KhasnatinovG. A. DanchinovaScientific Сentre for Family Health and Human Reproduction Problemsarticleflavivirustick-borne encephalitis virusrnaquantitative rt-pcr in real timeScienceQRUActa Biomedica Scientifica, Vol 4, Iss 5, Pp 116-121 (2019)
institution	DOAJ
collection	DOAJ
language	RU
topic	flavivirus tick-borne encephalitis virus rna quantitative rt-pcr in real time Science Q
spellingShingle	flavivirus tick-borne encephalitis virus rna quantitative rt-pcr in real time Science Q N. A. Liapunovа M. A. Khasnatinov G. A. Danchinova Optimization of a Quantitative Real-Time RT-PCR Technique for Evaluation of Concentration of Genomic +RNA of Tick-Borne Encephalitis Virus
description	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.
format	article
author	N. A. Liapunovа M. A. Khasnatinov G. A. Danchinova
author_facet	N. A. Liapunovа M. A. Khasnatinov G. A. Danchinova
author_sort	N. A. Liapunovа
title	Optimization of a Quantitative Real-Time RT-PCR Technique for Evaluation of Concentration of Genomic +RNA of Tick-Borne Encephalitis Virus
title_short	Optimization of a Quantitative Real-Time RT-PCR Technique for Evaluation of Concentration of Genomic +RNA of Tick-Borne Encephalitis Virus
title_full	Optimization of a Quantitative Real-Time RT-PCR Technique for Evaluation of Concentration of Genomic +RNA of Tick-Borne Encephalitis Virus
title_fullStr	Optimization of a Quantitative Real-Time RT-PCR Technique for Evaluation of Concentration of Genomic +RNA of Tick-Borne Encephalitis Virus
title_full_unstemmed	Optimization of a Quantitative Real-Time RT-PCR Technique for Evaluation of Concentration of Genomic +RNA of Tick-Borne Encephalitis Virus
title_sort	optimization of a quantitative real-time rt-pcr technique for evaluation of concentration of genomic +rna of tick-borne encephalitis virus
publisher	Scientific Сentre for Family Health and Human Reproduction Problems
publishDate	2019
url	https://doaj.org/article/04a37aa28601493e827853f840f201c0
work_keys_str_mv	AT naliapunova optimizationofaquantitativerealtimertpcrtechniqueforevaluationofconcentrationofgenomicrnaoftickborneencephalitisvirus AT makhasnatinov optimizationofaquantitativerealtimertpcrtechniqueforevaluationofconcentrationofgenomicrnaoftickborneencephalitisvirus AT gadanchinova optimizationofaquantitativerealtimertpcrtechniqueforevaluationofconcentrationofgenomicrnaoftickborneencephalitisvirus
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Optimization of a Quantitative Real-Time RT-PCR Technique for Evaluation of Concentration of Genomic +RNA of Tick-Borne Encephalitis Virus

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