microbeПроблемы особо опасных инфекцийProblems of Particularly Dangerous Infections0370-10692658-719XRussian Research Anti-Plague Institute “Microbe”10.21055/0370-1069-2022-2-134-141microbe-1705Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИORIGINAL ARTICLESПодбор генетических маркеров для выявления ДНК патогенных боррелийGenetic Markers for Detecting the DNA of Pathogenic Borreliahttps://orcid.org/0000-0001-5669-1486ХаммадовН. И.KhammadovN. I.

Хаммадов Наиль Ильдарович

 420075, Республика Татарстан, Казань, Научный городок-2

Science city-2, Kazan, Republic of Tatarstan, 420075

https://orcid.org/0000-0001-5593-2399ХамидуллинаА. И.KhamidullinaA. I.

420029, Республика Татарстан, Казань, Сибирский Тракт, 35

35, Siberian Highway, Kazan, Republic of Tatarstan, 420029

kgavm_baumana@mail.ruФГБНУ «Федеральный центр токсикологической, радиационной и биологической безопасности»РоссияFederal Center for Toxicological, Radiation and Biological SafetyRussian FederationФГБОУ ВО «Казанская государственная академия ветеринарной медицины имени Н.Э. Баумана»РоссияKazan State Academy of Veterinary Medicine named after N.E. BaumanRussian Federation20221307202202134141Copyright © Хаммадов Н.И., Хамидуллина А.И., 20222022Хаммадов Н.И., Хамидуллина А.И.Khammadov N.I., Khamidullina A.I.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/1705

Цель исследования – анализ генетических маркеров возбудителей болезни Лайма, которые можно использовать для специфичной индикации их максимально большего числа штаммов и изолятов.

Материалы и методы

Материалы и методы. Нуклеотидные последовательности различных генов Borrelia garinii, B. afzelii, B. burgdorferi загружены из базы данных NCBI (Национального центра биологической информатизации). Определение встречаемости анализируемых нуклеотидных последовательностей в генетическом коде различных организмов определяли в программной утилите nBLAST. Для дизайна праймеров и зондов использовали программу Vector NTI 9.1.0 (Invitrogen Corporation, Карлсбад, США). ДНК выделяли, используя набор реагентов «МАГНО-сорб» вариант 100-200 («АмплиСенс», Москва, Россия), согласно инструкции производителя. Праймеры и зонды синтезировали в ЗАО «Евроген» (Москва, Россия). Для проведения ПЦР использовались реактивы производства ЗАО «Синтол» (Москва, Россия).

Результаты и обсуждение

Результаты и обсуждение. Для достоверной индикации патогенных боррелий методами молекулярно-генетического анализа определены специфичные локусы (гены) различных видов возбудителей: B. garinii, B. afzelii, B. burgdorferi, – которые отличались от генетических кодов других представителей рода Borrelia и от ДНК иных организмов. В результате предварительного определения аналитической значимости исследуемых локусов для дальнейшей работы выбраны следующие гены и локусы: pepX, clpA, ospA, p83/100, ospC и flaB, – из которых для практической индикации ДНК патогенных боррелий выбраны гены flaB и ospA. Индикация генетических маркеров B. burgdorferi и B. afzelii происходит при амплификации гена flaB, а B. garinii и B. afzelii – при использовании в качестве генетического маркера гена ospA.

The aim of the study was to analyze the genetic markers of Lyme disease pathogens, which can be used to specifically indicate maximum number of their strains and isolates.

Materials and methods

Materials and methods. The nucleotide sequences of various genes of Borrelia garinii, B. afzelii, B. burgdorferi were downloaded from the NCBI database (National Center for Biological Informatization). The occurrence of the analyzed nucleotide sequences in the genetic code of various organisms was determined in the nBLAST software utility. For the design of primers and probes, the Vector NTI 9.1.0 program (“Invitrogen Corporation”, Carlsbad, USA) was used. DNA was isolated using the MAGNO-sorb kit, version 100-200 (“AmpliSens”, Moscow, Russia), according to the manufacturer’s instructions. Primers and probes were synthesized at “Evrogen” company (Moscow, Russia). For PCR, reagents manufactured by “Synthol” company (Moscow, Russia) were applied.

Results and discussion

Results and discussion. In order to perform the reliable indication of pathogenic Borrelia, specific loci (genes) of B. garinii, B. afzelii, B. burgdorferi, which were significantly different from the genetic code of other representatives of the genus Borrelia and from the DNA of other organisms, have been determined by molecular-genetic methods. As a result of a preliminary determination of the analytical significance of the studied loci, the following genes and loci were selected for further work: pepX, clpA, ospA, p83/100, ospC and flaB, of which the flaB and ospA genes were selected for practical indication of pathogenic Borrelia DNA. The genetic markers of B. burgdorferi and B. afzelii are displayed during amplification of the flaB gene, while B. garinii and B. afzelii occur when the ospA gene is used as a genetic marker.

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