Количественное определение возбудителей гемотрансмиссивных инфекций методом полимеразной цепной реакции на олигонуклеотидном микрочипе

Заключение

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Разработанный метод количественного определения возбудителей гемотрансмиссивных инфекций на биологическом микрочипе является чувствительным, специфичным инструментом многопараметрического количественного анализа, позволяющим получать воспроизводимые результаты. Его дальнейшее развитие до стадии диагностической тест-системы и последующее её внедрение в клиническую лабораторную диагностику будет способствовать снижению риска передачи возбудителей гемотрансмиссивных инфекций и позволит в кратчайшие сроки определять вирусную нагрузку с целью оценки тяжести протекания заболевания и эффективности применения противовирусной терапии. Предложенный метод также может быть применён для выполнения широкого спектра исследований, связанных с многосторонней идентификацией и количественным определением других генетических мишеней.

Разработан метод мультиплексной PCR на олигонуклеотидном микрочипе с регистрацией результатов амплификации в режиме реального времени с одним флуоресцентным красителем.

Изучено влияние компонентов реакционной смеси, температурно-временных режимов, концентраций иммобилизованных праймеров и размера гелевых ячеек на эффективность протекания qPCR на микрочипе и кинетику накопления флуоресцентных сигналов.

Создан биологический микрочип для одновременного выявления нуклеиновых кислот вирусов гепатитов В, С и иммунодефицита человека 1-го типа методом мультиплексной qPCR.

Разработанный метод применён для одновременного количественного анализа вирусных нуклеиновых кислот в образцах плазмы крови человека. Чувствительность и специфичность системы составили 100% (на выборке из 132 образцов), пределы количественного определения (с вероятностью обнаружения 95%) составили 14, 10 и 15 геном-эквивалентов для HIV-1, HBV и HCV, соответственно.

Благодарности.

Выражаю свою глубокую благодарность моему научному руководителю. Владимиру Михайловичу Михайловичу. Я искренне признателен, Дмитрию Грядунову, Максиму Донникову, Сергею Лапе, Ольге Антоновой и Наталье Захаровой за помощь в работе, поддержку, ценные замечания при обсуждении результатов и рукописи диссертации, Сергею Панькову, Эдуарду Яковлевичу Крейндлину — за подготовку микрочипов для проведения экспериментов, Сергею Суржикову и Ирине Гречишниковой за синтез олигонуклеотидов, Александру Сергеевичу Заседателеву за помощь и поддержку.

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