veterinaryВетеринария сегодняVeterinary Science Today2304-196X2658-6959"Veinard"10.29326/2304-196X-2025-14-3-274-282veterinary-943Research ArticleОРИГИНАЛЬНЫЕ СТАТЬИ | БОЛЕЗНИ КРУПНОГО РОГАТОГО СКОТАORIGINAL ARTICLES | BOVINE DISEASESВалидация и применение тест-системы на основе метода ПЦР в режиме реального времени для выявления ДНК Mycoplasma disparValidation and application of qPCR test kit for detection of Mycoplasma dispar DNAhttps://orcid.org/0000-0002-1210-0303Абед АлхуссенМ.Abed AlhussenM.

Абед Алхуссен Мохаммад - канд. вет. наук, младший научный сотрудник референтной лаборатории болезней крупного рогатого скота ФГБУ «ВНИИЗЖ».

ул. Гвардейская, 6, мкр. Юрьевец, Владимир, 600901

Mohammad Abed Alhussen - Cand. Sci. (Veterinary Medicine), Junior Researcher, Reference Laboratory for Bovine Diseases, Federal Centre for Animal Health.

6 Gvardeyskaya str., Yur’evets, Vladimir 600901

alhussenmohammed85@hotmail.comhttps://orcid.org/0009-0006-0498-2904ФедороваО. Е.FedorovaO. E.

Федорова Ольга Евгеньевна - ведущий биолог референтной лаборатории болезней крупного рогатого скота ФГБУ «ВНИИЗЖ».

ул. Гвардейская, 6, мкр. Юрьевец, Владимир, 600901

Olga E. Fedorova - Leading Biologist, Reference Laboratory for Bovine Diseases, Federal Centre for Animal Health.

6 Gvardeyskaya str., Yur’evets, Vladimir 600901

fedorova@arriah.ruhttps://orcid.org/0000-0002-8311-4681КротоваА. О.KrotovaA. O.

Кротова Алена Олеговна - ведущий биолог референтной лаборатории болезней крупного рогатого скота ФГБУ «ВНИИЗЖ».

ул. Гвардейская, 6, мкр. Юрьевец, Владимир, 600901

Alena O. Krotova - Leading Biologist, Reference Laboratory for Bovine Diseases, Federal Centre for Animal Health.

6 Gvardeyskaya str., Yur’evets, Vladimir 600901

krotova@arriah.ruhttps://orcid.org/0000-0002-8326-7151БьядовскаяО. П.ByadovskayaO. P.

Бьядовская Ольга Петровна - канд. биол. наук, заведующий референтной лаборатории болезней крупного рогатого скота ФГБУ «ВНИИЗЖ».

ул. Гвардейская, 6, мкр. Юрьевец, Владимир, 600901

Olga P. Byadovskaya - Cand. Sci. (Biology), Head of Reference Laboratory for Bovine Diseases, Federal Centre for Animal Health.

6 Gvardeyskaya str., Yur’evets, Vladimir 600901

bjadovskaya@arriah.ruhttps://orcid.org/0000-0001-5982-3675СпрыгинА. В.SpryginA. V.

Спрыгин Александр Владимирович - д-р биол. наук, старший научный сотрудник референтной лаборатории болезней крупного рогатого скота ФГБУ «ВНИИЗЖ».

ул. Гвардейская, 6, мкр. Юрьевец, Владимир, 600901

Alexander V. Sprygin - Dr. Sci. (Biology), Senior Researcher, Reference Laboratory for Bovine Diseases, Federal Centre for Animal Health.

6 Gvardeyskaya str., Yur’evets, Vladimir 600901

sprygin@arriah.ruФГБУ «Федеральный центр охраны здоровья животных» (ФГБУ «ВНИИЗЖ»)РоссияFederal Centre for Animal HealthRussian Federation202517092025143274282Copyright © Абед Алхуссен М., Федорова О.Е., Кротова А.О., Бьядовская О.П., Спрыгин А.В., 20252025Абед Алхуссен М., Федорова О.Е., Кротова А.О., Бьядовская О.П., Спрыгин А.В.Abed Alhussen M., Fedorova O.E., Krotova A.O., Byadovskaya O.P., Sprygin A.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/943Введение

Введение. В настоящее время наблюдается широкое распространение и активная циркуляция Mycoplasma dispar в животноводческих хозяйствах по всему миру, в том числе и в Российской Федерации. Актуальность внедрения в ветеринарную практику тест-системы на основе полимеразной цепной реакции в режиме реального времени для выявления ДНК Mycoplasma dispar обусловлена важной ролью данного возбудителя в развитии комплекса респираторных болезней крупного рогатого скота, приводящих к значительным экономическим потерям в животноводстве, в том числе являющихся причиной падежа телят.

Цель исследования

Цель исследования. Определение основных валидационных характеристик и внедрение в ветеринарную практику разработанной тест-системы для выявления ДНК Mycoplasma dispar методом полимеразной цепной реакции в режиме реального времени.

Материалы и методы

Материалы и методы. Референтный штамм Mycoplasma dispar (ATCC № 27140) культивировали на питательной среде 1699 Revised Mycoplasma Medium, рекомендованной American Type Culture Collection. ДНК выделяли с использованием коммерческого набора, постановка ПЦР в режиме реального времени осуществлялась по заранее подобранным параметрам. Были определены основные валидационные характеристики тест-системы: аналитическая чувствительность, аналитическая специфичность, эффективность амплификации, повторяемость и воспроизводимость. Показана возможность практического применения тест-системы на основе ПЦР в режиме реального времени для выявления ДНК Mycoplasma dispar.

Результаты

Результаты. Аналитическая чувствительность (предел обнаружения) тест-системы по выявлению ДНК Mycoplasma dispar составила 10 копий ДНК/мкл (или 100 копий ДНК/реакцию), специфичность – 100% (детектирует только ДНК Mycoplasma dispar), эффективность амплификации – 99,01%, среднее значение коэффициента вариации при оценке повторяемости – 0,91%, воспроизводимости – 0,66–1,26% в рамках 5 повторений и 0,91% в рамках 15 повторений. Апробацию тест-системы проводили на 228 пробах биоматериала, отобранных от крупного рогатого скота из 13 регионов Российской Федерации, при этом ДНК Mycoplasma dispar была обнаружена в 39,47% исследуемых проб.

Заключение

Заключение. Разработанная тест-система для выявления ДНК Mycoplasma dispar продемонстрировала высокие валидационные показатели и может быть использована в диагностике микоплазмоза крупного рогатого скота.

Introduction

Introduction. Currently, Mycoplasma dispar is widely spread and circulating in livestock farms around the world, including in the Russian Federation. The implementation of a real-time polymerase chain reaction test kit for detecting Mycoplasma dispar DNA in veterinary practice is highly relevant, as this pathogen can cause respiratory diseases in cattle and contribute to calf mortality, leading to significant economic losses in livestock production.

Objective

Objective. To introduce a newly developed real-time polymerase chain reaction test kit Mycoplasma dispar DNA detection kit into veterinary practice and determine its major validation parameters.

Materials and methods

Materials and methods. Mycoplasma dispar reference strain (ATCC No. 27140) was cultured in 1699 Revised Mycoplasma Medium recommended by the American Type Culture Collection. DNA was extracted using a commercial kit, real-time polymerase chain reaction was performed using pre-selected parameters. The major validation parameters of the test kit were determined: analytical sensitivity, analytical specificity, amplification efficiency, repeatability and reproducibility. Applicability of real-time polymerase chain reaction test kit for detection of Mycoplasma dispar DNA was demonstrated.

Results

Results. The Mycoplasma dispar DNA detection test kit demonstrated an analytical sensitivity (detection limit) of 10 copies/μL (100 copies/reaction), 100% specificity (exclusive to Mycoplasma dispar DNA), 99.01% amplification efficiency, and an average repeatability coefficient of variation of 0.91%. Reproducibility coefficient of variation ranged from 0.66% to 1.26% across 5 replicates and was 0.91% across 15 replicates. The test kit was validated using 228 biological samples from cattle from 13 regions of the Russian Federation, while Mycoplasma dispar DNA was detected in 39.47% of the samples tested.

Conclusion

Conclusion. The developed Mycoplasma dispar DNA test kit has demonstrated high validation performance and is suitable for diagnosing bovine mycoplasmosis.

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