microbeПроблемы особо опасных инфекцийProblems of Particularly Dangerous Infections0370-10692658-719XRussian Research Anti-Plague Institute “Microbe”10.21055/0370-1069-2018-4-39-47microbe-1086Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИORIGINAL ARTICLESРазработка и испытание метода выявления РНК вируса Ласса на основе полимеразной цепной реакции с обратной транскрипцией в режиме реального времениDevelopment and Testing of the Method for the Detection of Lassa virus RNA, Based on real-Time Polymerase Chain reaction with reverse TranscriptionДедковВ. Г.DedkovV. G.

Дедков Владимир Георгиевич.

197101, Санкт-Петербург, ул. Мира, д. 14. 

 Vladimir G. Dedkov.

14, Mira St., Saint-Petersburg, 197101.

vgdedkov@yandex.ruСафоноваМ. В.SafonovaM. V.

4, Musorgskogo St., Moscow, 127490.

protivochym@nln.ruНайденоваЕ. В.NaidenovaE. V.

46, Universitetskaya St., Saratov, 410005.

rusrapi@microbe.ruMagassoubaN. F.MagassoubaN. F.

Conakry.

АйгининА. А.AygininA. A.

3a, Novogireevskaya St., Moscow, 111123.

crie@pcr.ruSoropoguiB.SoropoguiB.

Conakry.

KouroumaF.KouroumaF.

Conakry.

CamaraA. B.CamaraA. B.

Conakry.

CamaraJ.CamaraJ.

Conakry.

КрицкийА. А.KritskyA. A.

46, Universitetskaya St., Saratov, 410005.

rusrapi@microbe.ruЩелкановМ. Ю.ShchelkanovM. Yu.МалеевВ. В.MaleevV. V.

3a, Novogireevskaya St., Moscow, 111123.

crie@pcr.ruСанкт-Петербургский научно-исследовательский институт эпидемиологии и микробиологии им. ПастераРоссияSaint-Petersburg Pasteur Research Institute of Epidemiology and MicrobiologyRussian FederationПротивочумный центрРоссияPlague Control CenterRussian FederationРоссийский научно-исследовательский противочумный институт «Микроб»РоссияRussian Research Anti-Plague Institute "Microbe"Russian FederationУниверситет им. Гамаль Абдель НасераГвинеяGamal Abdel Naser UniversityGuineaЦентральный научно-исследовательский институт эпидемиологииРоссияCentral Research Institute of EpidemiologyRussian FederationДальневосточный Федеральный университетРоссияFar East Federal UniversityRussian Federation201829122018043947Copyright © Дедков В.Г., Сафонова М.В., Найденова Е.В., Magassouba N.F., Айгинин А.А., Soropogui B., Kourouma F., Camara A.B., Camara J., Крицкий А.А., Щелканов М.Ю., Малеев В.В., 20182018Дедков В.Г., Сафонова М.В., Найденова Е.В., Magassouba N.F., Айгинин А.А., Soropogui B., Kourouma F., Camara A.B., Camara J., Крицкий А.А., Щелканов М.Ю., Малеев В.В.Dedkov V.G., Safonova M.V., Naidenova E.V., Magassouba N.F., Ayginin A.A., Soropogui B., Kourouma F., Camara A.B., Camara J., Kritsky A.A., Shchelkanov M.Y., Maleev V.V.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/1086

Цель. Разработка метода выявления и количественного анализа (ОТ-ПЦР в реальном времени) для выявления генетических маркеров вируса Ласса - LASV-Fl. Материалы и методы. Для работы взяты все доступные в базе данных GenBank (https://www.ncbi.nlm.nih.gov/genbank/) последовательности вируса Ласса, которые были выровнены для идентификации консервативных сайтов с использованием программного пакета BioEdit 7.2.5 (IbisBiosciences, США). для апробации разработанного ПЦР-набора была использована контрольная панель РНК вируса Ласса и псевдовирусных частиц, 27 вирусных штаммов, относящихся к различным семействам, а также 37 образцов сывороток крови от пациентов с лихорадочными заболеваниями, отобранных в лечебных учреждениях Республики Гвинея в 2016-2018 гг., и 55 проб суспензий органов от многососковых мышей. Результаты и обсуждение. Аналитическая чувствительность метода варьировала от 103 до 105 копий/мл и имела 96,4 % диагностическую чувствительность, тогда как аналитическая и диагностическая специфичность составляли 100 %. показано, что разработанная методика может быть успешно применена на практике для обнаружения вируса Ласса в Гвинейской Республике, с использованием различных типов материала от мелких млекопитающих, включая цельную кровь и суспензии органов M. natalensis,а также образцы сывороток крови людей, собранных через 3-7 дней после начала заболевания. Сделано также предположение, что данный метод может быть использован для штаммов вируса Ласса, распространенных не только в Гвинее, но и на других эндемичных территориях, но данный факт необходимо подтвердить в дальнейших исследованиях.

Abstract. Objective of the study was the development of a method for the detection and quantitative analysis (realtime RT-PCR) to identify genetic markers of Lassa virus - LASV-Fl. Materials and methods. We utilized all the available in the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) Lassa virus sequences that have been aligned to identify conservative sites applying the BioEdit 7.2.5 software package (IbisBiosciences, USA). To test the developed PCR kit, the control panel of Lassa virus RNA and pseudo-viral particles, 27 viral strains belonging to different fami­lies, as well as 37 serum samples from patients with feverish diseases selected in medical institutions of the Republic of Guinea in 2016-2018 and 55 samples of organ suspensions from multi-spiked mice were used. Results and discussion. The analytical sensitivity of the method varied from 103 copies/ml to 105 copies/ml and had 96.4 % diagnostic sensitivity, while the analytical and diagnostic specificity was 100 %. It is shown that the developed technique can be successfully introduced into practice for the detection of Lassa virus in the Republic of Guinea, using various types of material from small mammals, including whole blood and organ suspensions of M. natalensis, as well as samples of human blood sera collected 3-7 days after the onset of the disease. It is also suggested that this method can be used for strains of Lassa virus, common not only in Guinea but also in other endemic areas, but this fact must be confirmed in further studies.

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