Name: detection of bacteria using fluorogenic dnazymes
Uploaded: 2012-05-28T13:00:00
Duration: 13 min 20 s
Description: Watch this Scientific Journal Video about Detection of Bacteria Using Fluorogenic DNAzymes at JoVE.com

Финансирование этой работы была предоставлена ​​естественным наукам и инженерным исследованиям Совета Канады (NSERC) и биологически активных Страж сети бумаги.

<ol>
	<li>Zourob, M., Elwary, S., Truner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems. , (2008).</li><li>Call, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+and+Opportunities+for+Pathogen+Detection+Using+DNA+Microarrays.">Challenges and Opportunities for Pathogen Detection Using DNA Microarrays.</a> Crit. Rev. Microbiol. 31, 91-99 (2005).</li><li>Lazcka, O., Campo, D. e. l., J, F., Muñoz, F. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen+detection:+A+perspective+of+traditional+methods+and+biosensors.">Pathogen detection: A perspective of traditional methods and biosensors.</a> Biosens. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+as+a+versatile+chemical+component+for+catalysis,+encoding,+and+stereocontrol.">DNA as a versatile chemical component for catalysis, encoding, and stereocontrol.</a> Angew. Chem. Int. Ed. 49, 7180-7201 (2010).</li><li>Mei, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+RNA-cleaving+DNA+enzyme+that+synchronizes+catalysis+with+fluorescence+signaling.">An efficient RNA-cleaving DNA enzyme that synchronizes catalysis with fluorescence signaling.</a> J. Am. Chem. Soc. 125, 412-420 (2003).</li><li>Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assemblage+of+signaling+DNA+enzymes+with+intriguing+metal-ion+specificities+and+pH+dependences.">Assemblage of signaling DNA enzymes with intriguing metal-ion specificities and pH dependences.</a> J. Am. Chem. Soc. 125, 7539-7545 (2003).</li><li>Kandadai, S. A., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+catalytically+efficient+acidic+RNA-cleaving+deoxyribozyme.">Characterization of a catalytically efficient acidic RNA-cleaving deoxyribozyme.</a> Nucleic Acids Res. 33, 7164-7175 (2005).</li><li>Rupcich, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quenching+of+fluorophore-labeled+DNA+oligonucleotides+by+divalent+metal+ions:+implications+for+selection,+design,+and+applications+of+signaling+aptamers+and+signaling+deoxyribozymes.">Quenching of fluorophore-labeled DNA oligonucleotides by divalent metal ions: implications for selection, design, and applications of signaling aptamers and signaling deoxyribozymes.</a> J. Am. Chem. Soc. 128, 780-790 (2005).</li><li>Shen, Y., Brennan, J. D., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+the+secondary+structure+and+identifying+functionally+essential+nucleotides+of+pH6DZ1,+a+fluorescence-signaling+and+RNA-cleaving+deoxyribozyme.">Characterizing the secondary structure and identifying functionally essential nucleotides of pH6DZ1, a fluorescence-signaling and RNA-cleaving deoxyribozyme.</a> Biochemistry. 44, 12066-12076 (2005).</li><li>Chiuman, W., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revitalization+of+six+abandoned+catalytic+DNA+species+reveals+a+common+three-way+junction+framework+and+diverse+catalytic+cores.">Revitalization of six abandoned catalytic DNA species reveals a common three-way junction framework and diverse catalytic cores.</a> J. Mol. Biol. 357, 748-754 (2006).</li><li>Chiuman, W., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+high-branching+deoxyribozymes+from+a+catalytic+DNA+with+a+three-way+junction.">Evolution of high-branching deoxyribozymes from a catalytic DNA with a three-way junction.</a> Chem. Biol. 13, 1061-1069 (2006).</li><li>Shen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalysis+and+rational+engineering+of+trans-acting+pH6DZ1,+an+RNA-cleaving+and+fluorescence-signaling+deoxyribozyme+with+a+four-way+junction+structure.">Catalysis and rational engineering of trans-acting pH6DZ1, an RNA-cleaving and fluorescence-signaling deoxyribozyme with a four-way junction structure.</a> Chem BioChem. 7, 1343-1348 (2006).</li><li>Ali, M. M., Kandadai, S. A., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+pH3DZ1+-+An+RNA-cleaving+deoxyribozyme+with+optimal+activity+at+pH+3.">Characterization of pH3DZ1 - An RNA-cleaving deoxyribozyme with optimal activity at pH 3.</a> Can. J. Chem. 85, 261-273 (2007).</li><li>Chiuman, W., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+signaling+platforms+built+from+a+small+catalytic+DNA+and+doubly+labeled+fluorogenic+substrates.">Efficient signaling platforms built from a small catalytic DNA and doubly labeled fluorogenic substrates.</a> Nucleic Acids Res. 35, 401-405 (2007).</li><li>Chiuman, W., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+fluorescent+sensors+engineered+with+catalytic+DNA+MgZ+based+on+a+non-classic+allosteric+design.">Simple fluorescent sensors engineered with catalytic DNA MgZ based on a non-classic allosteric design.</a> PLoS ONE. 2, e1224 (2007).</li><li>Shen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entrapment+of+fluorescence+signaling+DNA+enzymes+in+sol+gel-derived+materials+for+metal+ion+sensing.">Entrapment of fluorescence signaling DNA enzymes in sol gel-derived materials for metal ion sensing.</a> Anal. Chem. 79, 3494-3503 (2007).</li><li>Kandadai, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+an+RNA-cleaving+deoxyribozyme+with+optimal+activity+at+pH+5.">Characterization of an RNA-cleaving deoxyribozyme with optimal activity at pH 5.</a> Biochemistry. 48, 7383-7391 (2009).</li><li>Zhao, W., Brook, M. A., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodic+assembly+of+nanospecies+on+repetitive+DNA+sequences+generated+on+gold+nanoparticles+by+rolling+circle+amplification.">Periodic assembly of nanospecies on repetitive DNA sequences generated on gold nanoparticles by rolling circle amplification.</a> Methods Mol. Biol. 474, 79-90 (2008).</li><li>Navani, N. K., Mok, W. K., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+selection+of+protein-binding+DNA+aptamers+as+ligands+for+biosensing+applications.">In vitro selection of protein-binding DNA aptamers as ligands for biosensing applications.</a> Methods Mol. Biol. 504, 399-415 (2009).</li><li>Ali, M. M., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+sensing+by+using+allosteric-DNAzyme-coupled+rolling+circle+amplification+and+a+peptide+nucleic+acid-organic+dye+probe.">Colorimetric sensing by using allosteric-DNAzyme-coupled rolling circle amplification and a peptide nucleic acid-organic dye probe.</a> Angew. Chem. Int. Ed. 48, 3512-3515 (2009).</li></ol>

Нет конфликта интересов объявлены.

Большинство общих методов обнаружения бактериальной сегодня либо медленно (классический микробных) или технически сложных (антитела, ПЦР). Таким образом, мы считаем, что новое поколение средств обнаружения должно удовлетворить на скорость и простоту. С этой целью мы создали РНК-раскалывания и флуоресценции сигнализации DNAzyme которые могут быть использованы для разработки простых тестов, чтобы сообщить о наличии бактерий через поколение сигнала флуоресценции. Рекомендуемые DNAzyme зонд, RFD-EC1, активируется CEM производятся в процессе роста E. палочки в культуре средств массовой информации. Так как наш метод использует сырье внеклеточной смеси бактерий в качестве целевого обнаружения и обходит трудоемкая добыча цели и усиление шагов, он может быть использован для создания очень простая, &quot;сочетание и читать&quot; типа тестов для выявления бактериальных. Использование наших DNAzyme не ограничивается флуоресценции на основе метода обнаружения. Например, колориметрический обнаружения с использованием той же системы DNAzyme анализ сбыть разработаны с использованием ранее метод, который использует прокатки усиления круга в сочетании с органическим красителем 32. Мы предвидим использования DNAzymes для обнаружения бактериальных как привлекательный путь к созданию новых бактериальных биосенсоров с большей простотой эксплуатации.

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер по каталогу или модели </td></tr><tr><td> Агар </td><td> BioShop Канаде </td><td> AGR003 </td></tr><tr><td> Персульфат аммония (APS) </td><td> BioShop Канаде </td><td> AMP001 </td></tr><tr><td> Акриламид / бис-акриламид (40%, 29:1) </td><td> BioShop Канаде </td><td> ACR004 </td></tr><tr><td> Борная кислота </td><td> BioShop Канаде </td><td> BOR001 </td></tr><tr><td> Bromophenol синий </td><td> Sigma-Aldrich </td><td> B8026 </td></tr><tr><td> ЭДТА </td><td> EM науки </td><td> EXO539-1 </td></tr><tr><td> HCl </td><td> Sigma-Aldrich </td><td> 38281 </td></tr><tr><td> HEPES </td><td> Bioshop Канаде </td><td> HEP001 </td></tr><tr><td> LB бульоне </td><td> Sigma-Aldrich </td><td> L3022 </td></tr><tr><td> MgCl 2 </td><td> EMDХимические вещества </td><td> B10149-34 </td></tr><tr><td> NaCl </td><td> BioShop Канаде </td><td> SOD002 </td></tr><tr><td> NaOAc </td><td> EMD химическими веществами </td><td> SXO255-1 </td></tr><tr><td> NaOH </td><td> EMD химическими веществами </td><td> SXO590-1 </td></tr><tr><td> SDS </td><td> BioShop Канаде </td><td> SDS001 </td></tr><tr><td> TEMED </td><td> BioShop Канаде </td><td> TEM001 </td></tr><tr><td> Трис-основание </td><td> BioShop Канаде </td><td> BST666 </td></tr><tr><td> Твин-20 </td><td> Sigma-Aldrich </td><td> P9416 </td></tr><tr><td> Мочевина </td><td> BioShop Канаде </td><td> URE001 </td></tr><tr><td> Xylenecyanol FF </td><td> Sigma-Aldrich </td><td> X4126 </td></tr><tr><td> ДНК концентратор </td><td> Thermo Scientific </td><td> Savant ДНК SpeedVac 120 </td></tr><tr><td> Millex фильтра </td><td> Millipore </td><td> SLGP033RS </td></tr><tr><td> Советы гель загрузкой </tг&gt; <td> Диамед </td><td> TEC200EX-K </td></tr><tr><td> ImageQuant программного обеспечения </td><td> Молекулярная динамика </td><td> Версия 5.0 </td></tr><tr><td> Kimwipes </td><td> Kimberly-Clark Professional </td><td> 34705 </td></tr><tr><td> Мини Vortexer </td><td> VWR </td><td> 58816-121 </td></tr><tr><td> Парафильмом </td><td> Pechiney пластиковой упаковки </td><td> PM996 </td></tr><tr><td> Чашки Петри </td><td> Fisher Scientific </td><td> Fisherbrand 08-757-12 </td></tr><tr><td> Stripettor Plus (Пипетка пушка) </td><td> Гранулирование </td><td> 07764714 </td></tr><tr><td> Кварцевые кюветы </td><td> Varian Inc </td><td> 66-100216-00 </td></tr><tr><td> Шейкер / инкубатор </td><td> New Brunswick Scientific </td><td> Классическая серия C24 </td></tr><tr><td> Тайфун сканер </td><td> GE Healthcare </td><td> 9200 Переменный режим </td></tr><tr><td> Центрифугировать </td><td> Beckman Coulter </td><td> Allegra X22-R </td></tr><tr><td> УФ-спектрофотометр </td><td> Thermo Scientific </td><td> GenesysUV 10 </td></tr><tr><td> Флуоресцентный спектрофотометр </td><td> Varian Inc </td><td> Кэри затмение </td></tr></tbody></table>

detection of bacteria using fluorogenic dnazymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

Watch this Scientific Journal Video about Detection of Bacteria Using Fluorogenic DNAzymes at JoVE.com

Detection of Bacteria Using Fluorogenic DNAzymes

Недавно мы сообщали о новом подходе для создания флуорогенных зондов DNAzyme которые могут быть применены для создания простых, &quot;сочетание и читать&quot; флуоресцентный тест для обнаружения бактериальной. Эти специальные ДНК-зонды катализируют расщепление хромофора модифицированных ДНК-РНК химерных субстрата в присутствии внеклеточных сырой смеси (CEM) производства конкретных бактерий, тем самым переводя бактериальных обнаружения флуоресценции в поколение сигнала. В этом докладе мы опишем основной экспериментальной процедуры, когда конкретный зонд DNAzyme обозначается &quot;RFD-EC1&quot; используется для определения модели бактерии,<em&gt; Кишечной палочки (E.coli)</em&gt;.

Обнаружение бактерий с помощью флуорогенных DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Research

JoVE Journal

Immunology and Infection

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

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Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

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Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

Быстрое выявление грамотрицательных бактерий из крови культуральной жидкости Использование MALDI-TOF масс-спектрометрии

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

В Ситу обнаружения бактерий внутри парафин тканей с использованием дигоксина меченных ДНК зонд таргетинга 16S рРНК

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Биомиметические Материалы для характеристики Бактерии-хозяин взаимодействий

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Пищевых патогенов скрининга с помощью магнитно люминесцентные нано: Быстрое обнаружение кишечной палочки O157: H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

Сверхчувствительная обнаружения биомаркеров с помощью молекулярного импринтинга основе емкостной биосенсор

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Высок объём платформы для проверки Salmonella spp. /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Assaying for Inorganic Polyphosphate in Bacteria

Опробование для неорганических полифосфат бактерий

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many ...