medgenМедицинская генетикаMedical Genetics2073-7998Publishing House «Genius Media» LLC10.25557/2073-7998.2022.01.34-43medgen-2021Research ArticleОРИГИНАЛЬНЫЕ ИССЛЕДОВАНИЯORIGINAL RESEARCHРазработка и характеристика метода многолокусной метилчувствительной ПЦР в режиме реального времениDevelopment and characterization of the method for multilocus methylation sensitive real-time PCRНиколаеваА. Ф.NikolaevaA. F.alex.ru97@bk.ruСигинВ. О.SiginV. O.noemail@neicon.ruКалинкинА. И.KalinkinA. I.noemail@neicon.ruТанасА. С.TanasA. S.noemail@neicon.ruСтрельниковВ. В.StrelnikovV. V.noemail@neicon.ruФГБНУ «Медико-генетический научный центр имени академика Н.П. Бочкова»РоссияResearch Centre for Medical GeneticsRussian Federation2022220420222113443Copyright © Николаева А.Ф., Сигин В.О., Калинкин А.И., Танас А.С., Стрельников В.В., 20222022Николаева А.Ф., Сигин В.О., Калинкин А.И., Танас А.С., Стрельников В.В.Nikolaeva A.F., Sigin V.O., Kalinkin A.I., Tanas A.S., Strelnikov 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://www.medgen-journal.ru/jour/article/view/2021

Введение. Метод метилчувствительной количественной ПЦР (МЧ-кПЦР) в режиме реального времени впервые описан в настоящей работе в многолокусном формате с использованием TaqMan-зондов. Многолокусный формат реакции облегчает тестирование больших выборок пациентов и способствует бережному расходованию коллекционных образцов биологического материала, а также позволяет включать в состав тест-систем необходимые контроли, обеспечивающие оценку полноты гидролиза матрицы и эффективности ПЦР. Цель: разработать тест-систему многолокусной МЧ-кПЦР для количественного анализа уровня метилирования ДНК и охарактеризовать её аналитические свойства. Методы. Материалом исследования служили смеси ДНК с заданным уровнем метилирования (препаратов ДНК, гидролизованных и негидролизованных метилчувствительной рестриктазой). Считали, что ДНК, необработанная метилчувствительной рестриктазой BstHHI, является модельной метилированной матрицей, в то время как гидролизованная ДНК - модельной неметилированной матрицей. МЧ-кПЦР проводили для двух целевых локусов - участков CpG-островков генов PRKCB и GMDS с включением положительного внутреннего контроля эффективности ПЦР и контроля полноты гидролиза геномной ДНК метилчувствительной эндонуклеазой рестрикции. Для кинетических кривых исследуемых локусов с помощью пакета qpcR языка программирования R во всех образцах получены значения Cy0, и на основе этих данных рассчитали уровни метилирования модельных препаратов с заданными уровнями метилирования. Аналитическую чувствительность метода определяли путем нахождения значений Limit of Blank (LoB, измеряемое значение при отсутствии метилирования) и Limit of Detection (LoD, наименьший измеряемый уровень метилирования). Результаты. Определены аналитические свойства метода МЧ-кПЦР: аналитическая чувствительность составила 5%; относительная погрешность составила 11,38%; коэффициент детерминации (R2) для локусов PRKCB и GMDS составил 0,98 и 0,77, соответственно. Результаты измерений по локусу GMDS позволили предположить его моноаллельное метилирование в нормальных лимфоцитах крови, что было подтверждено анализом баз данных метилома человека. Исходя из полученных результатов, аналитическая чувствительность метода МЧ-кПЦР выше таковой метода прямого бисульфитного секвенирования по Сэнгеру и не уступает методу бисульфитного пиросеквенирования.

Background. The method of multiplex methylation sensitive quantitative real-time PCR (MS-qPCR) is used for the first time in the format of a multiplex system with TaqMan probes. The previously described approaches to MSRE-qPCR are insufficient for the implementation of the technique in studies of large samples of patients, which is due to the lack of a multiplex reaction format and internal controls. We propose an MS-qPCR method with the possibility of multiplexing and the presence of internal PCR controls. Aim: to characterize the analytical properties of the developed multiplex MS-qPCR method. Methods. DNA from lymphocytes from healthy donors was used as a material for the study. We have postulated that DNA undigested with methylation sensitive restriction enzyme is a model methylated template (undigested), while hydrolyzed DNA is a model unmethylated template. To determine the analytical sensitivity, mixtures of digested and undigested DNA were used. Development of the MS-qPCR panel was carried out taking into account the design requirements developed by the team: the inclusion of a positive internal control of the PCR efficiency and a control of digestion of genomic DNA by the methylation sensitive restriction enzyme. For the kinetic curves of the studied loci in all samples, Cy0 values were obtained using the qPCR package of the R programming language, and on the basis of these data, methylation levels were calculated, and box plots were presented. The analytical sensitivity of the method was determined by finding the Limit of Blank (LoB) and Limit of Detection (LoD) values. Results. The coefficient of determination (R2) for the normally non-methylated locus is 0.98, and for the locus with normal monoallelic methylation, 0.77. Analytical sensitivity of the MS-qPCR method is 5%; the relative error was 11.38%. Conclusions. Based on the results obtained, the analytical sensitivity of the MS-qPCR method is higher than that of the direct Sanger bisulfite sequencing, and comparable to bisulfite pyrosequencing. The use of TaqMan probes allows multiplexing of several target loci in one reaction, which is a fine solution to the problem of a small amount of available biological material.

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