Abstract
Objective of the study is to analyze the genetic markers of tick-borne encephalitis virus, which can be used to specifically indicate the maximum number of virus strains and isolates.Materials and methods. Plasmid DNA and nucleic acids of the tick, the genus Ixodes and Dermacentor, were used as amplified material. Polymerase chain reaction (PCR) was conducted on C1000 amplifier with a CFX96 optical unit (BioRad). The species (strain) variety of detected organisms, using the analyzed genetic markers, was determined in the nBLAST software utility. The design of the nucleotide sequences of primers and probes was performed using “Vector NTI 9.1.0” (Invitrogen Corporation). Nucleic acids were isolated by magnetic sorption with a reagent panel MAGNO-sorb. Primers and probes were synthesized at “Evrogen” company, Moscow, Russia. The reagents for PCR were manufactured by “Syntol” company, Moscow, Russia.Results and discussion. When indicating the genome of the tick-borne encephalitis virus, the main criterion for choosing a marker sequence is the specific detection of nucleic acids of only the desired microorganism. The genomewide nucleotide sequences of various strains and isolates of tick-borne encephalitis virus were analyzed to search for a specific marker nucleotide sequence. Mutual comparison of all the above mentioned genomes made it possible to determine 7 conventionally conservative loci characterized by minimal nucleotide variability. The further work was based on the results of alignment of the nucleotide sequences of tick-borne encephalitis virus isolates, including the nucleotide sequences of heterogeneous microorganisms; primers and probes were designed to amplify each of the marker loci; the most analytically significant oligonucleotides were developed based on loci 4 and 7. Amplification with oligonucleotide primers to indicate tick-borne encephalitis virus was effective, both in a separate PCR with a positive control, and in combination with tick DNA.
Highlights
Objective of the study is to analyze the genetic markers of tick-borne encephalitis virus, which can be used to indicate the maximum number of virus strains and isolates
When indicating the genome of the tick-borne encephalitis virus, the main criterion for choosing a marker sequence is the specific detection of nucleic acids of only the desired microorganism
The genomewide nucleotide sequences of various strains and isolates of tick-borne encephalitis virus were analyzed to search for a specific marker nucleotide sequence
Summary
Поиск генетических маркеров вируса клещевого энцефалита для его специфичной индикации. Цель исследования – анализ генетических маркеров вируса клещевого энцефалита, которые можно использовать для специфичной индикации максимально большего числа штаммов и изолятов вируса. При индикации генома вируса клещевого энцефалита основным критерием выбора маркерной последовательности является специфичное выявление нуклеиновых кислот только искомого микроорганизма. Для поиска специфичной маркерной нуклеотидной последовательности произведен анализ полногеномных нуклеотидных последовательностей различных штаммов и изолятов вируса клещевого энцефалита. Опираясь в дальнейшей работе на результаты выравнивания нуклеотидных последовательностей изолятов вируса клещевого энцефалита, в том числе учитывая нуклеотидные последовательности гетерогенных микроорганизмов, выполнен дизайн праймеров и зондов для амплификации каждого из маркерных локусов, наиболее аналитически значимые олигонуклеотиды разработаны на основе локусов 4 и 7. Амплификация с олигонуклеотидными затравками для индикации вируса клещевого энцефалита была результативной как в отдельной ПЦР с положительным контролем, так и в сочетании с ДНК клещей. Federal Center of Toxicological, Radiation and Biological Safety, Kazan, Russian Federation
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