microbeПроблемы особо опасных инфекцийProblems of Particularly Dangerous Infections0370-10692658-719XRussian Research Anti-Plague Institute “Microbe”10.21055/0370-1069-2016-4-60-63microbe-349Research ArticleМИКРОБИОЛОГИЯMICROBIOLOGYКомплексный подход к видоспецифичной детекции вируса оспы коровComplex Approach to Species-Specific Detection of Cowpox VirusМаксютовР. А.MaksyutovR. A.

630559, Новосибирская обл., п. Кольцово

Kol’tsovo, Novosibirsk Region, 630559

maksyutov_ra@vector.nsc.ruФБУН «Государственный научный центр вирусологии и биотехнологии «Вектор». п. КольцовоРоссияState Scientific Center of Virology and Biotechnology “Vector”, Kol’tsovoRussian Federation201620122016046063Copyright © Максютов Р.А., 20162016Максютов Р.А.Maksyutov R.A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://journal.microbe.ru/jour/article/view/349

Цель. Разработка и апробация метода видоспецифичной детекции вируса оспы коров (ВОК). Материалы и методы. В работе использованы олигонуклеотидные праймеры и флуоресцентно- меченные зонды для родоспецифичной детекции ортопоксвирусов (ОПВ) и видоспецифичной детекции вирусов оспы коров и эктромелии (ВЭ). Пары флуоресцентных красителей и соответствующих тушителей флуоресценции вводили в зонды в соответствии с их специфичностью: ОПВ-специфичнй зонд содержал пару FAM/BHQ1, ВОК-специфичный зонд – JOE/BHQ1, ВЭ-специфичный зонд – Cy5/ BHQ3. Для определения чувствительности и специфичности метода видоспецифичной детекции вируса оспы коров исследованы образцы 68 различных штаммов ортопоксвирусов. Результаты и выводы. Реализован комплексный подход к видоспецифичной детекции вируса оспы коров за счет использования мультиплексного варианта ПЦР в реальном времени для одновременной мультилокусной детекции на основе трех независимых генов-мишеней вируса оспы коров, родоспецифичной детекции для исключения ложноотрицательных результатов и дополнительных олигонуклеотидных праймеров и зонда, обеспечивающих специфичную детекцию вируса эктромелии, с целью исключения ложноположительных результатов. 

Objective of the study is to develop and validate the method for species-specific detection of cowpox virus (CPV). Materials and methods. Utilized were oligonucleotide primers and fluorescence-labeled probes for genus-specific detection of orthopoxviruses (OPV) and species-specific detection of cowpox and ectromelia viruses (EV). Pairs of fluorescent dyes and corresponding fluorescence extinguishers were embodied into probes in accordance with their specificity: OPV-specific probe contained FAM/BHQ1 pair, CPVspecific probe – JOE/BHQ1, and EV-specific – Cy5/BHQ3. For evaluation of sensitivity and specificity of the method for species-specific detection of CPV, investigated were samples of 68 different strains of orthopoxviruses. Results and conclusions. Implemented was complex approach to species-specific detection of CPV using multiplex real-time PCR variant for simultaneous multilocus detection on the basis of three independent target genes of CPV, to genus-specific detection for the exclusion of false-negative results, and additional oligonucleotide primers and probes, providing for specific detection of EV, aimed at the exclusion of false-positive results. 

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The authors declare that there are no conflicts of interest present.