Abstract
Objective. Construction of positive control samples based on recombinant retroviral particles and their application in RT-PCR diagnostic assays for RNA detection of agents of dangerous and particularly dangerous viral infections.Materials and methods. Molecular biological, genetic engineering, and immunological methods were used: polymerase chain reaction, restriction, ligation, cloning, transformation, transfection, flow cytometry.Results and discussion. Technology of positive control samples producing based on recombinant virions has been developed and tested. It includes construction of retroviral vector with cloned diagnostic sequence of the viral genome; obtaining a packaging cell line producing chimeric retroviral particles; determination of recombinant virions titer by flow cytometry and polymerase chain reaction; application of the obtained preparation as a control sample for PCR diagnostics of infectious agents. Positive controls based on retroviral vectors as carriers of genomic RNA fragments of pathogenic viruses were used in the development of PCR diagnostic kits for dangerous and particularly dangerous viral infections. Their application increased the kits quality and made it possible to exclude the work with concentrated hazardous infectious agents (Lassa virus, tick-borne encephalitis virus, lymphocytic choriomeningitis virus, Puumala virus).
Highlights
Construction of positive control samples based on recombinant retroviral particles and their application in RT-PCR diagnostic assays for RNA detection of agents of dangerous and dangerous viral infections
Technology of positive control samples producing based on recombinant virions has been developed and tested
Positive controls based on retroviral vectors as carriers of genomic RNA fragments of pathogenic viruses were used in the development of PCR diagnostic kits for dangerous and dangerous viral infections
Summary
Рекомбинантные ретровирусные частицы: технология получения и использование в качестве положительных контрольных образцов для ПЦР-диагностики опасных вирусных инфекций. Цель работы – разработка технологии получения положительных контрольных образцов на основе рекомбинантных ретровирусных частиц, а также ее применение при создании наборов реагентов для выявления РНК возбудителей опасных и особо опасных вирусных инфекций методом полимеразной цепной реакции с обратной транскрипцией. Разработана и апробирована технология получения положительных контрольных образцов на основе рекомбинантных вирионов, включающая в себя создание генноинженерной конструкции на базе ретровирусного вектора с клонированной в него диагностической последовательностью вирусного генома; получение «пакующей» клеточной линии, продуцирующей химерные ретровирусные частицы; определение титра рекомбинантных вирионов методом проточной цитометрии и полимеразной цепной реакции; использование полученного на их основе препарата в качестве положительного контрольного образца при ПЦР-диагностике инфекционного агента. Republican Research and Practical Center of Epidemiology and Microbiology, Minsk, Republic of Belarus
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