mesЭкстремальная биомедицинаExtreme Medicine3033-89643033-8972Centre for Strategic Planning of the Federal Medical and Biological Agency10.47183/mes.2022.003mes-159Research ArticleМЕТОДMETHODРазработка ПЦР-теста для выявления генетических вариантов альфа, бета, гамма, дельта вируса SARS-COVDevelopment of PCR test for detection of the SARS-CoV-2 genetic variants Alpha, Beta, Gamma, DeltaШипулинГ. А.ShipulinG. A.

г. Москва.

Moscow.

СавочкинаЮ. А.SavochkinaYu. A.

г. Москва.

Moscow.

ШуряеваА. К.ShuryaevaA. K.

Анна Константиновна Шуряева,

д. 10, стр. 1, ул. Погодинская, г. Москва; 119121.

Anna K. Shuryaeva,

10, bldg 1, Pogodinskaya str., Moscow, 119121.

ashuryaeva@cspmz.ruШивлягинаЕ. Е.ShivlyaginaE. E.

г. Москва.

Moscow.

НосоваА. О.NosovaA. O.

г. Москва.

Moscow.

ДавыдоваЕ. Е.DavydovaE. E.

г. Москва.

Moscow.

ЛупаревА. Р.LuparevA. R.

г. Москва.

Moscow.

МаловаТ. В.MalovaT. V.

г. Москва.

Moscow.

ЮдинС. М.YudinS. M.

г. Москва.

Moscow.

Центр стратегического планирования и управления медико-биологическими рисками здоровью Федерального медико-биологического агентстваРоссияCentre for Strategic Planning and Management of Biomedical Health Risks of Federal Medical Biological AgencyRussian Federation202227102024241512Copyright © Шипулин Г.А., Савочкина Ю.А., Шуряева А.К., Шивлягина Е.Е., Носова А.О., Давыдова Е.Е., Лупарев А.Р., Малова Т.В., Юдин С.М., 20242024Шипулин Г.А., Савочкина Ю.А., Шуряева А.К., Шивлягина Е.Е., Носова А.О., Давыдова Е.Е., Лупарев А.Р., Малова Т.В., Юдин С.М.Shipulin G.A., Savochkina Y.A., Shuryaeva A.K., Shivlyagina E.E., Nosova A.O., Davydova E.E., Luparev A.R., Malova T.V., Yudin S.M.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.extrememedicine.ru/jour/article/view/159

Возникновение новых вариантов коронавируса SARS-CoV-2, обладающих повышенной трансмиссивностью и снижающих эффективность его нейтрализации выработанными ранее антителами, представляет угрозу для здоровья населения во всем мире. Метод полимеразной цепной реакции с обратной транскрипцией (ОТ-ПЦР) с флуоресцентными зондами, позволяющими детектировать единичные нуклеотидные замены, подходит для скрининга содержащих РНК SARS-CoV-2 образцов на наличие уже известных мутаций в S-гене, которые имеют функциональное значение, и выявление которых позволяет определять и дифференцировать геноварианты, имеющие наибольшее эпидемиологическое значение. Целью настоящей работы было разработать набор реагентов и методику для оперативного мониторинга распространения приоритетно значимых вариантов SARS-CoV-2. На основе выравнивания полногеномных последовательностей SARS-CoV-2, опубликованных в базе данных GISAID, были выбраны праймеры и LNAмодифицированные зонды для выявления мутаций в S-гене, характерных для генетических линий альфа, бета/гамма и дельта особой эпидемиологической значимости (VOC). Разработан и зарегистрирован набор реагентов в формате ОТ-ПЦР в реальном времени для выявления наиболее значимых мутаций в S-гене SARS-CoV-2, продемонстрированы его высокие аналитические и диагностические характеристики. Показано соответствие результатов выявления линий VOC и их основных мутаций разработанным набором с данными полногеномного секвенирования для 1500 образцов РНК SARSCoV-2. Применение набора реагентов в сочетании с последующим секвенированием РНК SARS-CoV-2 в рамках проведения эпидемиологического мониторинга позволило оперативно установить факт появления на территории России геноварианта дельта и в дальнейшем отследить динамику изменения его распространенности в Московском регионе в период с апреля по сентябрь 2021 г.

The emergence of novel SARS-CoV-2 genetic variants with increased transmissivity and reduced antibody neutralization efficiency is a threat to global public health. Reverse transcription polymerase chain reaction (RT-PCR) with the use of fluorescent probes, which make it possible to detect the single nucleotide substitutions, is a technique suitable for screening the SARS-CoV-2 RNA-containing samples for the already known functionally significant mutations in the S-gene, identification of which allows to define and differentiate the most epidemiologically significant genetic variants. The study was aimed to develop an assay for the large-scale monitoring of the spread of the SARS-CoV-2 top-priority variants. Based on the whole-genome alignment of the SARS-CoV-2 sequences, deposited in the GISAID database, primers and LNA-modified probes were selected to detect mutations in the S gene, typical for the Alpha, Beta/Gamma and Delta variants of concern (VOC). The developed reagent kit for detection of the key mutations in the SARS-CoV-2 S gene by the real time RT-PCR has good analytical and diagnostic characteristics and was authorized as a medical device (reagent) for in vitro use. The results of detecting the VOC and the key mutations with the use of the developed reagent kit were consistent with the data of the whole genome sequencing of 1,500 SARS-CoV-2 RNA samples. The developed reagent kit and the subsequent SARS-CoV-2 RNA sequencing assay used to perform the epidemiological monitoring of SARS-CoV-2 variants made it possible to promptly report the emergence of the Delta genetic variant in Russia, and to trace the dynamic changes in the prevalence of Delta in Moscow Region in April–September 2021. 

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