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

وقدم التمويل لهذا العمل من جانب العلوم الطبيعية والهندسة مجلس البحوث كندا (NSERC) وشبكة الحارس ورقة النشطة بيولوجيا.

<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. Bioelectron. 22, 1205-1217 (2007).</li><li>Velusamy, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+foodborne+pathogen+detection:+In+the+perspective+of+biosensors.">An overview of foodborne pathogen detection: In the perspective of biosensors.</a> Biotechnol. Adv. 28, 232-254 (2010).</li><li>Ali, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorogenic+DNAzyme+Probes+as+Bacterial+Indicators.">Fluorogenic DNAzyme Probes as Bacterial Indicators.</a> Angew. Chem. Int. Ed. 50, 3751-3754 (2011).</li><li>Navani, N. 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=Nucleic+acid+aptamers+and+enzymes+as+sensors.">Nucleic acid aptamers and enzymes as sensors.</a> Curr. Opin. Chem. Biol. 10, 272-281 (2006).</li><li>Liu, J., Cao, Z., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Nucleic+Acid+Sensors.">Functional Nucleic Acid Sensors.</a> Chem. Rev. 109, 1948-1998 (2009).</li><li>Li, Y., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Functional Nucleic Acids for Analytical Applications. , (2009).</li><li>Tuerk, C., Gold, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+evolution+of+ligands+by+exponential+enrichment:+RNA+ligands+to+bacteriophage+T4+DNA+polymerase.">Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.</a> Science. 249, 505-510 (1990).</li><li>Ellington, A. D., Szostak, J. 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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. 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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. 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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. 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الإعلان عن أي تضارب في المصالح.

معظم طرق الكشف البكتيرية شيوعا اليوم اما ان تكون بطيئة (الجراثيم الكلاسيكية) أو مطالبة من الناحية الفنية (الضد، PCR). وبالتالي، فإننا نعتقد أن الجيل القادم من أدوات الكشف وينبغي أن تلبي نحو السرعة والبساطة. تحقيقا لهذه الغاية، لقد أنشأنا الرنا الانفطار ومضان، مما يشير DNAzyme التي يمكن استخدامها لتطوير فحوصات بسيطة للإبلاغ عن وجود البكتيريا من خلال توليد إشارة مضان. يتم تنشيط الميزات DNAzyme التحقيق، تكتل القوى الديمقراطية، EC1، التي أنتجت خلال CEM نمو E. القولونية في وسائل الإعلام ثقافة. منذ أسلوبنا يستخدم خليط من النفط الخام خارج الخلية جرثومة كهدف الكشف واستخراج يتجاوز الهدف شاقة والخطوات التضخيم، أنها يمكن أن تستخدم لإعداد بسيط جدا &quot;، ومزيج، وقراءة&quot; نوع من فحوصات للكشف عن البكتيريا. ولا يقتصر استخدام DNAzyme جهدنا لمضان طريقة كشف القائم. على سبيل المثال، كشف اللونية باستخدام نظام DNAzyme نفسه فحص جتصميم لاستخدام أسلوب ذكرت في وقت سابق ان يستغل المتداول التضخيم دائرة بالتعاون مع لصبغ العضوية (32). ونحن نتوقع استخدام DNAzymes للكشف عن البكتريا كوسيلة جذابة لتوليد أجهزة الاستشعار بكتيرية جديدة مع بساطة تشغيلية أكبر.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> عدد دليل أو نموذج </td></tr><tr><td> أجار </td><td> BioShop كندا </td><td> AGR003 </td></tr><tr><td> بيرسلفات الأمونيوم (APS) </td><td> BioShop كندا </td><td> AMP001 </td></tr><tr><td> الأكريلاميد / مكرر الأكريلاميد (40٪، 29:1) </td><td> BioShop كندا </td><td> ACR004 </td></tr><tr><td> حمض البوريك </td><td> BioShop كندا </td><td> BOR001 </td></tr><tr><td> زرقة البروموفينول </td><td> سيغما الدريخ </td><td> B8026 </td></tr><tr><td> EDTA </td><td> EM العلم </td><td> EXO539-1 </td></tr><tr><td> حمض الهيدروكلوريك </td><td> سيغما الدريخ </td><td> 38281 </td></tr><tr><td> HEPES </td><td> Bioshop كندا </td><td> HEP001 </td></tr><tr><td> LB مرق </td><td> سيغما الدريخ </td><td> L3022 </td></tr><tr><td> MgCl 2 </td><td> EMDالمواد الكيميائية </td><td> B10149-34 </td></tr><tr><td> كلوريد الصوديوم </td><td> BioShop كندا </td><td> SOD002 </td></tr><tr><td> NaOAc </td><td> EMD كيماويات </td><td> SXO255-1 </td></tr><tr><td> هيدروكسيد الصوديوم </td><td> EMD كيماويات </td><td> SXO590-1 </td></tr><tr><td> SDS </td><td> BioShop كندا </td><td> SDS001 </td></tr><tr><td> TEMED </td><td> BioShop كندا </td><td> TEM001 </td></tr><tr><td> تريس القاعدة </td><td> BioShop كندا </td><td> BST666 </td></tr><tr><td> توين 20 </td><td> سيغما الدريخ </td><td> P9416 </td></tr><tr><td> اليوريا </td><td> BioShop كندا </td><td> URE001 </td></tr><tr><td> Xylenecyanol FF </td><td> سيغما الدريخ </td><td> X4126 </td></tr><tr><td> الحمض النووي المكثف </td><td> الحرارية العلمية </td><td> رجل علم الحمض النووي SpeedVac 120 </td></tr><tr><td> Millex مرشح وحدة </td><td> ميليبور </td><td> SLGP033RS </td></tr><tr><td> نصائح هلام التحميل </tد&gt; <td> Diamed </td><td> TEC200EX-K </td></tr><tr><td> ImageQuant البرمجيات </td><td> جزيئية حيوية </td><td> الإصدار 5.0 </td></tr><tr><td> Kimwipes </td><td> كيمبرلي كلارك الفنية </td><td> 34705 </td></tr><tr><td> مصغرة Vortexer </td><td> VWR </td><td> 58816-121 </td></tr><tr><td> Parafilm </td><td> بيشيني تغليف بلاستيكية </td><td> PM996 </td></tr><tr><td> أطباق بتري </td><td> فيشر العلمية </td><td> Fisherbrand 08-757-12 </td></tr><tr><td> Stripettor زائد (ماصة بندقية) </td><td> كورنينج </td><td> 07764714 </td></tr><tr><td> الكوارتز cuvettes </td><td> فاريان المؤتمر الوطني العراقي </td><td> 66-100216-00 </td></tr><tr><td> شاكر / حاضنة </td><td> نيو برونزويك العلمي </td><td> الكلاسيكي سلسلة C24 </td></tr><tr><td> الاعصار سكانر </td><td> جنرال إلكتريك للرعاية الصحية </td><td> 9200 وضع متغير </td></tr><tr><td> نبذ </td><td> بيكمان كولتر </td><td> أليجرا X22-R </td></tr><tr><td> الأشعة فوق البنفسجية الطيف </td><td> الحرارية العلمية </td><td> GenesysUV 10 </td></tr><tr><td> مضان الطيف </td><td> فاريان المؤتمر الوطني العراقي </td><td> كاري الكسوف </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

وذكرت ونحن في الآونة الأخيرة نهجا جديدا لتوليد تحقيقات DNAzyme fluorogenic التي يمكن تطبيقها لاقامة بسيطة، &quot;مزيج والقراءة&quot; فلوري فحص للكشف عن البكتيريا. هذه التحقيقات DNA خاص تحفيز الانقسام من الحمض النووي الريبي الركيزة حامل اللون معدلة وخيالية في وجود مزيج النفط الخام خارج الخلية (CEM) التي تنتجها بكتيريا معينة، وترجمة وبالتالي اكتشاف بكتيريا في توليد إشارة مضان. في هذا التقرير سنقوم بشرح الإجراءات التجريبية الرئيسية التي يعمل تحقيقا DNAzyme محددة تدل على &quot;تكتل القوى الديمقراطية، EC1&quot; للكشف عن نموذج للبكتيريا،<em&gt; القولونية (اي كولي)</em&gt;.

الكشف عن البكتيريا عن طريق DNAzymes Fluorogenic

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

Research

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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

مزيج من الشريط اللاصق على أساس أخذ العينات والإسفار في الموقع تهجين للكشف السريع عن السالمونيلا على المنتجات الطازجة

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

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

كشف متعددة من البكتيريا الموجودة في مجمع العينات السريرية والبيئية باستخدام قليل النوكليوتيد ، بالإضافة نيون المجهرية

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

التالية في الوقت الحقيقي تأثير العوامل الالتهاب الرئوي الحاد الفوعة في نموذج الفأر الالتهاب الرئوي عن طريق البكتيريا للإضاءة الحيوية

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

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

التعرف السريع على البكتيريا سالبة الجرام من ثقافة الدم في مرق عن طريق MALDI-TOF الطيف الكتلي

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

الكشف الموضعي من البكتيريا داخل أنسجة البارافين جزءا لا يتجزأ من استخدام المسمى الديجوكسين DNA التحقيق استهداف 16S الريباسي

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

المواد الجزيئية الحيوية لوصف التفاعلات البكتيريا في استضافة

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

مسببات الأمراض المنقولة عن طريق الأغذية الفحص باستخدام المغناطيسية-فلوري نانوسينسور: الكشف السريع عن O157:H7 كولاي

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

الكشف عن أولتراسينسيتيفي للمؤشرات الحيوية باستخدام طابعة جزيئية أساس Biosensor سعوية

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.

منصة الفائق لفحص السالمونيلا spp./دوسنتاريا 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

المعايرة وميلامين متعدد الفوسفات غير العضوية في البكتيريا

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 ...