veterinaryВетеринария сегодняVeterinary Science Today2304-196X2658-6959"Veinard"10.29326/2304-196X-2019-4-31-3-7veterinary-436Research ArticleВЕТЕРИНАРНАЯ МИКРОБИОЛОГИЯVETERINARY MICROBIOLOGYВЫЯВЛЕНИЕ БАКТЕРИЙ CAMPYLOBACTER SPP. С ИСПОЛЬЗОВАНИЕМ ПОЛИМЕРАЗНОЙ ЦЕПНОЙ РЕАКЦИИ В РЕАЛЬНОМ ВРЕМЕНИDETECTION OF CAMPYLOBACTER SPP. WITH REAL-TIME POLYMERASE CHAIN REACTIONhttps://orcid.org/0000-0003-0714-3912СкитовичГ. С.SkitovichG. S.

Старший научный сотрудник, кандидат биологических наук

Владимир 

Senior Researcher, Candidate of Science (Biology)

Vladimir

skitovich@arriah.ruСероваК. В.SerovaK. V.

Ведущий ветеринарный врач

Владимир 

Leading Veterinarian

Vladimir

serova@arriah.ruhttps://orcid.org/0000-0001-7510-1269ШадроваН. Б.ShadrovaN. B.

Заведующий лабораторией, кандидат биологических наук

Владимир

Head of Laboratory, Candidate of Science (Biology)

Vladimir

shadrova@arriah.ruhttps://orcid.org/0000-0003-3143-7339ПрунтоваО. В.PruntovaO. V.

Главный эксперт, доктор биологических наук, профессор

Владимир

Chief Expert, Doctor of Science (Biology), Professor

Vladimir

pruntova@arriah.ruФГБУ «ВНИИЗЖ»РоссияFGBI “ARRIAH”Russian Federation2019261220190437Copyright © Скитович Г.С., Серова К.В., Шадрова Н.Б., Прунтова О.В., 20192019Скитович Г.С., Серова К.В., Шадрова Н.Б., Прунтова О.В.Skitovich G.S., Serova K.V., Shadrova N.B., Pruntova O.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://veterinary.arriah.ru/jour/article/view/436

Бактерии рода Campylobacter – одни из основных зоонозных патогенов, вызывающих заболевания человека и животных. Кампилобактерии являются микроаэрофилами, поэтому для роста им требуется низкая концентрация кислорода (3–5%) и высокая концентрация диоксида углерода (3–10%). В качестве источника энергии они используют не углеводы, а аминокислоты. Классические бактериологические методы выявления бактерий рода Campylobacter не всегда являются успешными из-за сложности обеспечения оптимальных условий для их роста. В связи с этим разработка и внедрение в практику молекулярных методов обнаружения и идентификации Campylobacter является актуальной задачей. Была оптимизирована методика качественного обнаружения генома бактерий Campylobacter spp. посредством полимеразной цепной реакции в режиме реального времени с использованием термоциклера CFX-96. Мишенью амплификации был выбран высокоспецифичный участок гена 16S pРНК, позволяющий определить шесть видов кампилобактерий: C. jejuni, C. coli, C. lari, C. upsaliensis, C. helveticus и C. hyointestinalis. Подобрана оптимальная концентрация ионов магния (2,5 мМ) и температура отжига праймеров (58 °С). Было протестировано 18 референтных штаммов различных бактерий. Положительный результат был показан только со штаммами, относящимися к роду Campylobacter. Чувствительность метода составила 40 молекул-мишеней. С использованием данной методики исследовали 76 проб сырья животного происхождения. Геном Campylobacter spp. был обнаружен в 18 образцах. Полученные результаты показывают, что оптимизированный вариант полимеразной цепной реакции в режиме реального времени, основанный на амплификации гена 16S pРНК, представляет собой специфичный, чувствительный, быстрый, воспроизводимый и точный метод для качественного обнаружения Campylobacter spp. в пробах сырья животного происхождения.

Bacteria of Campylobacter genus are ones of the main zoonotic pathogens causing human and animal diseases. Campylobacter organisms are microaerophiles and, therefore, require low oxygen concentration (3–5%) and high carbon dioxide concentration (3–10%) for their growth. They use amino acids rather than carbons as a source of energy. Classical bacteriological methods for Campylobacter spp. detection are not always successful due to diffi culties in creating optimal conditions for their growth. Therewith, development and implementation of molecular methods for Campylobacter detection and identifi cation are of current importance. Assay for qualitative Campylobacter spp. detection with real-time polymerase chain reaction using CFX-96 thermocycler was optimized. Highly specifi c segment of 16S rRNA gene allowing identifi cation of 6 Campylobacter species: C. jejuni, C. coli, C. lari, C. upsaliensis, C. helveticus и C. hyointestinalis was selected as an amplifi cation target. Optimal magnesium ion concentration (2.5 мМ) and primer annealing temperature (58 °С) were determined. Eighteen reference strains of various bacteria were tested. Only tests of Campylobacter genus strains gave positive results. The method sensitivity was 40 target molecules. The said method was used for testing 76 samples of raw materials of animal origin. Campylobacter spp. genome was detected in 18 samples. Obtained results showed that the optimized variant of real-time polymerase chain reaction based on 16S rRNA gene amplifi cation was a specifi c, sensitive, rapid, reproducible and accurate method for qualitative detection of Campylobacter spp. in samples of raw animal materials.

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