Очищенная вода для ПЦР анализаторов
Сейчас, в разгар пандемии ДНК-амплификаторы используются особенно интенсивно в большинстве профильных медучреждениях. Именно эти приборы используются для выполнения ПЦР-тестов на наличие вируса. Одним из главных моментов в использовании анализаторов является качество воды, которая используется для изготовления реагентов и в обработке сосудов.
Примеси, мешающие или препятствующие процессу ПЦР
В первую очередь это – наличие нуклеаз.
ПЦР-анализаторы основаны на применении ферментов ДНК-полимеразы для синтеза двухцепочечных молекул ДНК из одноцепочечных матриц с целю многократного копирования необходимого гена. Несмотря на то, что обычно в этом анализе используются термостабильные ДНК-полимеразы, эффективность реакции, которая ускоряется ферментами зависят от физических условий (температура, кислотность). Кроме того, зависит и от состава реакционной смеси. Наличие нуклеаз внутри реакционного сосуда - ферментов, расщепляющих фосфодиэфирные связи между субединицами нуклеиновых кислот приведут к большому нарушению процесса пилемеразной цепной реакции, т.к. генетический материал будет разделяться в условиях реакции. Именно поэтому необходимо гарантировать, что все реагенты, которые применяют в ПЦР не имеют нуклеаз.
Наличие бактерий
Наличие ДНК бактерий может приводить к неверным результатам, а также к амплификации нецелевых последовательностей. Помимо этого, большинство бактерий выделяют нуклеазы и иные молекулы, мешающие полимеризации ДНК.
Термостабильные ДНК-полимеразы требуют наличия кофактора магния (Mg2+) для лучшего связывания субстрата. Именно поэтому, концентрация ионов Mg2+ имеет основное значение для оптимизации активности полимеразы.
Ферменты полимеразы тоже чувствительны к другим обычным двухвалентным катионам (Cu2+, Fe2+, Ni2+ и т.д.), которые могут мешать координации кофакторов и нарушают связывание субстрата в активном центре. Также, наличие остаточных ионов тяжелых металлов целиком подавляет активность ферментов.
Система получения ультрачистой воды для ПЦР анализаторов.
ПЦР требует использования воды избавленной от нуклеаз, бактерий, микроорганизмов, органических соединений и микроэлементов, для создания всех реагентов и буферов.
Это может быть получено только при использовании сверхчистой воды Тип I со следующими характеристиками:
удельное сопротивление 18,2 МОм·см,
низкое значение общего органического углерода (ТОС), менее 20 ppb
уровень бактерий ниже 1 КОЕ/мл
уровень эндотоксинов ниже 0,01 ЕС/мл.
Оптимальной системой для получения ультрачистой воды с подобными характеристиками является система DUO (Avidity Science, Великобритания).
Источник: https://findlab.ru/news/ochishchennaja-voda-dlja-pcr-analizatorov/
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