Name: ampliphox colorimetric detection on a dna microarray for influenza
Uploaded: 2011-06-09T12:00:00
Description: Watch this Scientific Journal Video about ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza at JoVE.com

InDevR признает NIH / NIAID U01AI070276 и R43AI077112 для финансирования этой работы.

1. Примеры усиления использованием RT-PCR <ol><li> Выделите вирусную РНК из клинического материала или вирусной изолировать использованием вирусов Qiagen MinElute Спиновые Kit в сочетании с автоматизированной QIAcube нуклеиновые кислоты платформы добычи. Экстракция выполняется на 200 мкл образца с кон...

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	<li>Kumar, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Widely+Used+Diagnostics+"DNA-Microarray"-A+Review.">The Widely Used Diagnostics "DNA-Microarray"-A Review.</a> Amer J Inf Dis. 5, 207-218 (2009).</li><li>Miller, M. B., Tang, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Concepts+of+Microarrays+and+Potential+Applications+in+Clinical+Microbiology.">Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology.</a> Clin Microbiol Rev. 22, 611-633 (2009).</li><li>Mikhailovich, V., Gryadunov, D., Kolchinsky, A., Makarov, A. 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Обнаружение ampliPHOX колориметрических технология, представленная здесь, быстрый, недорогой альтернативой один цвет флуоресценции обнаружения для нижних приложений плотность микрочипов. Схематически показано на рисунке 1, принцип обнаружения основан на использовани?...

<table border="1"><tbody><tr><th> Реагент / оборудование </th><th> Производитель </th><th> Каталог # </th><th> Комментарии </th></tr><tr><td> Qiagen MinElute Вирус Спиновые Kit </td><td> Qiagen </td><td> 57704 </td><td> один 60 мкл элюирования </td></tr><tr><td> QIAcube </td><td> Qiagen </td><td> 9001292 </td><td> необязательный </td></tr><tr><td> ABI 9800 Быстрый термоциклер </td><td> Applied Biosystems </td><td> 4441166 </td><td></td></tr><tr><td> Qiagen OneStep RT-PCR комплект </td><td> Qiagen </td><td> 210210 </td><td> Комплект дНТФ не используется </td></tr><tr><td> 2x Зрительные буфера </td><td> InDevR ООО </td><td> MI-5007 </td><td></td></tr><tr><td> Биотинилированные Mix дНТФ </td><td> InDevR ООО </td><td> МИ-5009 </td><td></td></tr><tr><td> Lambda экзонуклеазная </td><td> Эпицентр Биотехнологии </td><td> LE032K </td><td> 2500 U, 10U/μl </td></tr><tr><td> FluChip грунтовку перемешать </td><td> InDevR </td><td> N / A </td><td> пока не доступны для продажи </td></tr><tr><td> Орбитальный Шейкер </td><td> Madell технологии </td><td> ZD-9556- </td><td></td></tr><tr><td> Вымойте Бункеры </td><td> InDevR ООО </td><td> MI-4002 </td><td></td></tr><tr><td> Вымойте стойки </td><td> InDevR ООО </td><td> MI-4003 </td><td></td></tr><tr><td> 2x буфера Гибридизация </td><td> InDevR ООО </td><td> MI-5004 </td><td></td></tr><tr><td> Калибровка Фишки </td><td> InDevR ООО </td><td> AP-5006 </td><td></td></tr><tr><td> Вымойте Буферы н.э. </td><td> InDevR ООО </td><td> MI-5005 </td><td></td></tr><tr><td> ampliRED </td><td> InDevR ООО </td><td> AP-5004 </td><td></td></tr><tr><td> ampliTAG </td><td> InDevR ООО </td><td> AP-5001 </td><td></td></tr><tr><td> 2x ampliTAG буфера </td><td> InDevR ООО </td><td> AP-5002 </td><td></td></tr><tr><td> ampliPHY, ampliPHY усилитель </td><td> InDevR ООО </td><td> AP-5003 </td><td></td></tr></tbody></table>

ampliphox colorimetric detection on a dna microarray for influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

Watch this Scientific Journal Video about ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza at JoVE.com

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

ampliPHOX колориметрических технологии обнаружения представлена ​​как недорогая альтернатива флуоресценции обнаружения микрочипов. На основе фотополимеризации, ampliPHOX производит твердые пятна полимера видна невооруженным глазом в течение нескольких минут. Результаты затем отображаемого и автоматически интерпретировать с простой, но мощный пакет программного обеспечения.

ampliPHOX Колориметрические обнаружения на ДНК-микрочипов по гриппу

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

Research

JoVE Journal

Immunology and Infection

Generation of Recombinant Influenza Virus from Plasmid DNA

Генерация рекомбинантного вируса гриппа от ДНК плазмиды

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Сочетание Клей-ленты на основе выборки и флуоресценции На месте Гибридизация для быстрого обнаружения Сальмонеллы На Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

Использование изображений Биолюминесцентный по расследованию синергизм между Пневмококк И вируса гриппа А в младенческой Мыши

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

Использование Пан-Вирусные Microarray анализ (Virochip), чтобы экран клинических образцов для вирусных патогенов

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

High-throughput Detection Method for Influenza Virus

Высокая пропускная способность Метод обнаружения для вируса гриппа

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Носовые салфетки для гриппа обнаружения вируса и изоляции от свиного

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Миниатюризированное Glycan Microarray Анализ для оценки авидности и специфичность вируса гриппа А агглютинины

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

In Vitro разборке вируса гриппа капсиды по градиентное центрифугирование

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Быстрое Молекулярная Обнаружение и дифференциация вирусов гриппа А и В

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Метод тампон Отбор проб для обнаружения человека норовирус на поверхностях

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...