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

Finansieringen af ​​dette arbejde blev leveret af Natural Sciences and Engineering Research Council of Canada (NSERC) og Sentinel Bioactive Paper Network.

<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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Ingen interessekonflikter erklæret.

De fleste af de almindelige bakterielle detektionsmetoder dag er enten langsom (klassisk mikrobiel) eller teknisk krævende (antistof PCR). Derfor mener vi, at den næste generation af værktøjer til afsløring, bør tage højde mod hastighed og enkelthed. Til dette formål har vi skabt en RNA-spaltende og fluorescens-signalering DNAzyme, der kan bruges til at udvikle simple analyser til at rapportere tilstedeværelsen af ​​bakterier ved produktion af en fluorescens signal. Den udstyret DNAzyme probe, RFD-EC1, der aktiveres af CEM fremstillet under væksten af E. coli i kulturmedier. Da vores metode bruger rå ekstracellulære blandinger af en bakterie som mål for afsløring og omgår den besværlige målet udvinding og amplifikationstrinnene, kan det bruges til at oprette meget enkel, &quot;mix-og-læse&quot; type af analyser til påvisning af bakterier. Anvendelse af vores DNAzyme er ikke begrænset til fluorescens-baseret detektionsmetode. For eksempel ved anvendelse kolorimetrisk påvisning af samme DNAzyme systemet assay cen udformes ved hjælp af en tidligere beskrevet metode, der udnytter rullende cirkel amplifikation i forbindelse med et organisk farvestof. 32 Vi forudser anvendelse af DNAzymes for bakteriel detektion som en attraktiv vej til at generere nye bakterielle biosensorer med større operationel enkelhed.

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer eller model </td></tr><tr><td> Agar </td><td> BioShop Canada </td><td> AGR003 </td></tr><tr><td> Ammoniumpersulfat (APS) </td><td> BioShop Canada </td><td> AMP001 </td></tr><tr><td> Acrylamid / bis-acrylamid (40%, 29:1) </td><td> BioShop Canada </td><td> ACR004 </td></tr><tr><td> Borsyre </td><td> BioShop Canada </td><td> BOR001 </td></tr><tr><td> Bromphenolblåt </td><td> Sigma-Aldrich </td><td> B8026 </td></tr><tr><td> EDTA </td><td> EM Science </td><td> EXO539-1 </td></tr><tr><td> HCI </td><td> Sigma-Aldrich </td><td> 38281 </td></tr><tr><td> HEPES </td><td> Bioshop Canada </td><td> HEP001 </td></tr><tr><td> LB-bouillon </td><td> Sigma-Aldrich </td><td> L3022 </td></tr><tr><td> MgCl2 </td><td> EMDKemikalier </td><td> B10149-34 </td></tr><tr><td> NaCl </td><td> BioShop Canada </td><td> SOD002 </td></tr><tr><td> NaOAc </td><td> EMD Kemikalier </td><td> SXO255-1 </td></tr><tr><td> NaOH </td><td> EMD Kemikalier </td><td> SXO590-1 </td></tr><tr><td> SDS </td><td> BioShop Canada </td><td> SDS001 </td></tr><tr><td> TEMED </td><td> BioShop Canada </td><td> TEM001 </td></tr><tr><td> Tris-base </td><td> BioShop Canada </td><td> BST666 </td></tr><tr><td> Tween 20 </td><td> Sigma-Aldrich </td><td> P9416 </td></tr><tr><td> Urea </td><td> BioShop Canada </td><td> URE001 </td></tr><tr><td> Xylenecyanol FF </td><td> Sigma-Aldrich </td><td> X4126 </td></tr><tr><td> DNA-koncentrator </td><td> Thermo Scientific </td><td> Savant DNA SpeedVac 120 </td></tr><tr><td> Millex filterenhed </td><td> Millipore </td><td> SLGP033RS </td></tr><tr><td> Gel Tip til ilægning </td&gt; <td> Diamed </td><td> TEC200EX-K </td></tr><tr><td> ImageQuant software </td><td> Molecular Dynamics </td><td> Version 5,0 </td></tr><tr><td> Kimwipes </td><td> Kimberly-Clark Professional </td><td> 34705 </td></tr><tr><td> Mini hvirvelomrøreren </td><td> VWR </td><td> 58816-121 </td></tr><tr><td> Parafilm </td><td> Pechiney Plastemballage </td><td> PM996 </td></tr><tr><td> Petriskåle </td><td> Fisher Scientific </td><td> Fisherbrand 08-757-12 </td></tr><tr><td> Stripettor Plus (Pipette pistol) </td><td> Corning </td><td> 07764714 </td></tr><tr><td> Kvartskuvetter </td><td> Varian Inc </td><td> 66-100216-00 </td></tr><tr><td> Shaker / Incubator </td><td> New Brunswick Scientific </td><td> Classic Series C24 </td></tr><tr><td> Typhoon Scanner </td><td> GE Healthcare </td><td> 9200 Variabel indstilling </td></tr><tr><td> Centrifuger </td><td> Beckman Coulter </td><td> Allegra X22-R </td></tr><tr><td> UV Spektrofotometer </td><td> Thermo Scientific </td><td> GenesysUV 10 </td></tr><tr><td> Fluorescensspektrofotometer </td><td> Varian Inc </td><td> Cary Eclipse </td></tr></tbody></table>

detection of bacteria using fluorogenic dnazymes

Udbrud knyttet til fødevarebåren og hospitalserhvervede patogener højde for millioner af dødsfald og indlæggelser samt kolossale økonomiske tab hvert eneste år. Forebyggelse af sådanne udbrud og minimering af konsekvenserne af en igangværende epidemi sted en stadig stigende efterspørgsel efter de analysemetoder, der nøjagtigt kan identificere synderen patogener på den tidligste fase. Selv om der er en bred vifte af effektive metoder til påvisning af patogener, kan ingen af ​​dem opfylder alle de følgende fem førende krav indeholdt i en ideel påvisningsmetode: høj specificitet (detektere kun bakterie af interesse), høj følsomhed (i stand til at opdage så lavt som en enkelt levende bakteriecelle), kort tid-til-resultater (minutter til timer), stor operationelle enkelhed (ikke behov for langvarige prøveudtagningsprocedurer og brug af specialudstyr), og omkostningseffektivitet. For eksempel er traditionelle mikrobiologiske metoder højspecifik men kræver en lang tid (days til uger) at opnå et endeligt resultat. 1 PCR-og antistof-baserede teknikker giver kortere ventetider (timer og dage), men de kræver anvendelse af dyre reagenser og / eller sofistikeret udstyr. 2-4 Der er derfor stadig stor efterspørgsel på videnskabelig forskning i retning af udviklingen af ​​innovative bakterielle påvisningsmetoder, der giver forbedrede egenskaber i en eller flere af de ovennævnte krav. Vores laboratorium er interesseret i at undersøge potentialet i DNAzymes som en ny klasse af molekylære prober for biosensorer applikationer, herunder påvisning af bakterier. 5 DNAzymes (også kendt som deoxyribozymes eller DNA-enzymer), er syntetiske enkeltstrengede DNA-molekyler med evnen til at katalysere kemiske reaktioner 6-8 Disse molekyler kan isoleres fra et bredt tilfældig sekvens DNA pulje (som indeholder så mange som 10 16 individuelle sekvenser) ved en proces, der kaldes &quot;in vitro selektion&quot; or &quot;SELEX&quot; (systematisk evolution af ligander ved eksponentiel berigelse). 9-16 Disse specielle DNA-molekyler er blevet bredt undersøgt i de seneste år som molekylære værktøjer til biosensorer anvendelser 6-8 Vores laboratorium har etablerede in vitro-udvælgelse for isolering af RNA-spaltende fluorescerende DNAzymes (RFDs. Figur 1) og undersøgt anvendelsen af RFDs som analytiske værktøjer 17-29 RFDs katalyserer spaltning af et DNA-RNA-kimær substratet ved en enkelt ribonukleotid. junction (R), som er flankeret af en fluorofor (F) og en quencher (Q). Den tætte nærhed af F-og Q gør det uspaltede substrat minimal fluorescens. Imidlertid spaltningshændelse fører til adskillelse af F-og Q, som er ledsaget af signifikant forøgelse af fluorescensintensitet. For nylig har vi udviklet en fremgangsmåde til isolering af RFDs til bakteriel detektion. 5 Disse særlige RFDs blev isoleret til &quot;lyser &quot;i nærværelse af den rå ekstracellulære blanding (CEM) efterladt af en specifik type af bakterier i deres miljø eller i medierne de dyrkes (fig. 1). Anvendelse af rå blanding omgår omstændelig proces til oprensning og identificere en passende mål fra mikrobe af interesse for biosensor udvikling (hvilket kan tage måneder eller år at fuldføre). Brugen af ​​ekstracellulære mål betyder, at analysering proceduren er enkel, fordi der ikke er behov for foranstaltninger til at indhente intracellulære mål. Ved hjælp af ovennævnte fremgangsmåde, har vi opnået en RFD der spalter dens substrat (FS1. Figur 2A) kun i nærvær af CEM fremstillet af E. coli (CEM-EF). 5 Denne E. coli-sensing RFD, kaldet RFD-EC1 (fig. 2A), blev fundet at være strengt reagerer på CEM-EF men nonresponsive til CEMS fra en lang række andre bakterier (fig. 3). Her Present de centrale eksperimentelle procedurer for etablering E. coli påvisningsanalyser med RFD-EC1 og repræsentative resultater.

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

Detection of Bacteria Using Fluorogenic DNAzymes

Vi har for nylig rapporteret om en ny tilgang til at generere fluorogene DNAzyme sonder, der kan anvendes til at oprette en enkel, &quot;mix-and-læser&quot; fluorescenstest for påvisning af bakterier. Disse særlige DNA-prober katalyserer spaltningen af ​​en chromophor-modificeret DNA-RNA-kimær substrat i nærvær af rå ekstracellulært blanding (CEM) fremstillet ved en specifik bakterie, hvorved omsætte bakteriel detektion i fluorescenssignal generation. I denne rapport vil vi beskrive de vigtigste eksperimentelle procedurer, hvor et bestemt DNAzyme sonde betegnes &quot;RFD-EC1&quot; er anvendt til påvisning af modellen bakterie,<em&gt; Escherichia coli (E. coli)</em&gt;.

Påvisning af bakterier under anvendelse Fluorogene DNAzymes

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

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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

Biomimetiske Materialer, der skal karakterisere Bakterier-vært interaktioner

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

Fødevarebårne patogen Screening ved hjælp af Magneto-fluorescerende Nanosensor: Hurtig påvisning af E. Coli 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

Ultrasensitive påvisning af biomarkører ved hjælp af en molekylær prægning baseret kapacitiv Biosensor

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.

En høj overførselshastighed Platform for Screening af 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

Kørsler til uorganiske Polyphosphate i bakterier

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 ...