Abstract
Developed was the DNA-biochip to identify subtypes of influenza A virus, pathogenic for humans. Microchip was capable of detecting H1, H3, H5-subtypes of hemagglutinin (including H1-subtype of pandemic A/H1N1(2009) influenza virus ) and neuraminidase subtypes N1,N2 of influenza virus. This microchip was successfully tested on the strains of A/H5N1 highly pathogenic avian influenza virus, A/H1N1(2009) pandemic influenza virus, A/H1N1 and A/H3N2 seasonal influenza viruses.
Highlights
Developed was the DNA-biochip to identify subtypes of influenza A virus, pathogenic for humans
Microchip was capable of detecting H1, H3, H5-subtypes of hemagglutinin (including H1-subtype of pandemic A/H1N1(2009) influenza virus ) and neuraminidase subtypes N1,N2 of influenza virus
This microchip was successfully tested on the strains of A/H5N1 highly pathogenic avian influenza virus, A/H1N1(2009) pandemic influenza virus, A/H1N1 and A/H3N2 seasonal influenza viruses
Summary
В работе использовались штаммы вируса гриппа типа А: А/Aichi/2/ 68(Н3N2), A/chicken/Kurgan/05/2005(H5N1), A/Nov o sibirsk/1/09(H1N1), A/Ekaterinburg/01/2009(H1N1)v. Экстракция РНК и синтез кДНК вируса гриппа типа А. Комплиментарная ДНК синтезировалась на матрице суммарной РНК с использованием синтетического праймера 5'-GACTAATACGACTCACTAT AGGGAGCAAAAGCAGG-3', универсального для всех субтипов вируса гриппа. AGG-3' (20 мкМ), 9 мкл РНК и 2 мкл dNTP (10 мМ) нагревали 10 мин при температуре 65 °C, охлаждали во льду, добавляли 4 мкл 5x кДНК буфера, 1 мкл 0,1 M DTT, 1 мкл РНКзина (40 а.е.), 1 мкл воды, 1 мкл обратной транскриптазы ThermoScript («Invitrogen», США). Высушенный образец ампликона растворяли в 10 мкл воды, добавляли 10 мкл 2-кратного гибридизационного буфера (1-кратный гибридизационный буфер: 6x SSC, 5x раствор Денхардта, 0,1 % Tween 20) и прогревали перед нанесением на слайд 2 мин при температуре 97 °C, а затем охлаждали во льду.
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