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

Funding for this work was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Sentinel Bioactive Paper Network.

<ol>
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No conflicts of interest declared.

Most of the common bacterial detection methods today are either slow (classic microbial) or technically demanding (antibody, PCR). Thus, we believe that the next generation of detection tools should cater toward speed and simplicity. To this end, we have created an RNA-cleaving and fluorescence-signaling DNAzyme that can be used to develop simple assays to report the presence of bacteria through the generation of a fluorescence signal. The featured DNAzyme probe, RFD-EC1, is activated by the CEM produced during the growth of E. coli in culture media. Since our method uses crude extracellular mixtures of a bacterium as the target of detection and bypasses the laborious target extraction and amplification steps, it can be used to set up very simple, "mix-and-read" type of assays for bacterial detection. The use of our DNAzyme is not restricted to fluorescence based detection method. For example, colorimetric detection using the same DNAzyme system assay can be designed using a previously reported method that exploits rolling circle amplification in conjunction with an organic dye.32 We foresee the use of DNAzymes for bacterial detection as an attractive avenue to generate new bacterial biosensors with greater operational simplicity.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number or model</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>BioShop Canada</td>
			<td>AGR003</td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>BioShop Canada</td>
			<td>AMP001</td>
		</tr>
		<tr>
			<td>Acrylamide/Bis-acrylamide (40%, 29:1)</td>
			<td>BioShop Canada</td>
			<td>ACR004</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>BioShop Canada</td>
			<td>BOR001</td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>B8026</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>EM Science</td>
			<td>EXO539-1</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sigma-Aldrich</td>
			<td>38281</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Bioshop Canada</td>
			<td>HEP001</td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>EMD Chemicals</td>
			<td>B10149-34</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>BioShop Canada</td>
			<td>SOD002</td>
		</tr>
		<tr>
			<td>NaOAc</td>
			<td>EMD Chemicals</td>
			<td>SXO255-1</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>EMD Chemicals</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 concentrator</td>
			<td>Thermo Scientific</td>
			<td>Savant DNA SpeedVac 120</td>
		</tr>
		<tr>
			<td>Millex filter unit</td>
			<td>Millipore</td>
			<td>SLGP033RS</td>
		</tr>
		<tr>
			<td>Gel loading tips</td>
			<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 Vortexer</td>
			<td>VWR</td>
			<td>58816-121</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Pechiney Plastic Packaging</td>
			<td>PM996</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>Fisherbrand 08-757-12</td>
		</tr>
		<tr>
			<td>Stripettor Plus (Pipette gun)</td>
			<td>Corning</td>
			<td>07764714</td>
		</tr>
		<tr>
			<td>Quartz cuvettes</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 Variable mode</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Allegra X22-R</td>
		</tr>
		<tr>
			<td>UV Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>GenesysUV 10</td>
		</tr>
		<tr>
			<td>Fluorescence Spectrophotometer</td>
			<td>Varian Inc</td>
			<td>Cary Eclipse</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

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

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

A High-throughput Platform for the Screening of 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

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 ...