Name: colorimetric detection of bacteria using litmus test
Uploaded: 2016-09-17T13:00:00
Description: Watch this Scientific Journal Video about Colorimetric Detection of Bacteria Using Litmus Test at JoVE.com

The funding for this research project was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) via a Discovery Grant to YL.

1. Fremstilling af reagenser og buffere <ol><li> 0,5 M ethylendiamintetraeddikesyre (EDTA) <ol><li> I et 2 liters bægerglas, tilsættes 186,1 g EDTA til 800 ml deioniseret vand destilleret (Hedeselskabet 2 O). Indstil pH af opløsningen til 8,0 ved anvendelse af NaOH-pellets. Tilføj Hedeselskabet 2 O til et endeligt volumen på 1,0 l, og opløsningen overføres til en autoklaverbar glasflaske for autoklavering og opbevares ved 4 ° C. </li></ol></li><li> 10 × Tris-borat EDTA (10x TBE) <ol><li>...

<ol>
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Oversættelsen af virkningen af RNA-spaltning aktivitet af en bakterie-responsive DNAzym til en lakmusprøve er gjort mulig ved hjælp af urease og magnetisk separation, som illustreret af figur 1. Selv om demonstration af den modificerede lakmusprøve for bakteriel detektion er gøres med en E. coli -afhængige RNA-spaltende DNAzym, 5,19,20 designet kan generelt udvides til enhver RNA-spaltende DNAzym. I betragtning af den store tilgængelighed af RNA-spaltende DNAzymer til forskell...

Bacterial pathogens are one of the major causes of global morbidity and mortality. Outbreaks from hospital-acquired infections, food-borne pathogens, and bacterial contaminants in the environment pose serious and on-going threats to public health and safety. To prevent these outbreaks, effective tools are needed that permit pathogen detection in a timely fashion under a variety of settings. Simple but still effective tests that are portable and cost-effective are greatly coveted, especially in regions that are susceptible to outbreaks but cannot afford expensive testing facilities.1-3 Although there exists a multitude of methods to detect bacteria, many of them are not suitable as screening or on-site testing tools because they require long test times, expensive instruments and complicated testing procedures. 
Colorimetric tests are particularly attractive for point-of-care or field applications as color changes can be easily detected by the naked eye. The litmus test for pH is simple, quick, and effective. Although it is a very old technology, it is still widely used today because of its simplicity and effectiveness. Surprisingly, this simple test had never been modified to achieve the detection of other analytes before we recently developed an approach of modifying this test for E. coli testing.4
The expanded litmus test for E. coli employs three additional components: an E. coli activated RNA-cleaving DNAzyme (EC1), 5 urease, and magnetic beads. DNAzymes refer to synthetic single-stranded DNA molecules with catalytic activity.6 They can be isolated from random-sequence DNA pools using in vitro selection.7,8 They are highly stable and can be produced cost-effectively using high-efficiency automated DNA synthesis.9 For these reasons, DNAzymes, particularly RNA-cleaving DNAzymes, have been widely examined for biosensing applications.6,10,11 RNA-cleaving DNAzyme sensors have been developed to detect metal ions,12-16 small molecules,17,18 bacterial pathogens5,19-21 and cancer cells.22 Given the great availability of target-induced RNA-cleaving DNAzymes, any assay that utilizes a DNAzyme can be potentially expanded to detect a diverse range of analytes.
Urease is chosen for its ability to hydrolyze urea into ammonia,23,24 resulting in a pH increase. Urease is also highly efficient, stable and amenable for conjugation to other biomolecules. Therefore, we postulated that a conjugate of an RNA-cleavage DNAzyme with urease would allow the use of litmus test for the detection of other targets.5
The action of the RNA-cleaving DNAzyme is relayed to urease-mediated increase of pH through the use of magnetic beads that are immobilized with the DNAzyme-urease conjugate. Because the activity of the DNAzyme under investigation is strictly dependent on E. coli, the presence of this bacterium in the test solution will result in the release of urease from the magnetic beads to the solution, which is then taken and used to hydrolyze urea in a reporter solution that contains a pH-sensitive dye. The final outcome of this procedure is a color change that can be conveniently reported by the dye or pH paper.

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:275px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:183px;">VWR AMRESCO</td>
			<td style="width:163px;">0105</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Hydroxide (NaOH) pellets</td>
			<td>BIO BASIC CANADA INC.</td>
			<td>SB6789</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Tris-base</td>
			<td>VWR AMRESCO</td>
			<td>0497</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Boric acid</td>
			<td>AMRESCO</td>
			<td>0588</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Urea</td>
			<td>VWR AMRESCO</td>
			<td>M123</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">40% acrylamide/bisacrylamide (29:1) solution</td>
			<td>BIO BASIC CANADA INC.</td>
			<td>A0007</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sucrose</td>
			<td>Bioshop Canada inc.</td>
			<td>SUC507</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Bromophenol blue</td>
			<td>Bioshop Canada inc.</td>
			<td>BRO777</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Xylenecyanol FF</td>
			<td>SIGMA-ALDRICH</td>
			<td>X-4126</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10% sodium dodecyl sulfate</td>
			<td>Bioshop Canada inc.</td>
			<td>SDS001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Hydrochloric Acid (HCl)</td>
			<td>CALEDON LABORATORIES LTD</td>
			<td>6026</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Chloride (NaCl)</td>
			<td>Bioshop Canada inc.</td>
			<td>SOD001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>Bioshop Canada inc.</td>
			<td>HEP001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Magnesium Chloride (II) hexahydrate</td>
			<td>VWR AMRESCO</td>
			<td>0288</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Tween 20</td>
			<td>Bioshop Canada inc.</td>
			<td>TW508</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Adenosine Triphospahte (ATP)</td>
			<td>AMRESCO</td>
			<td>0220</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Acetate trihydrate (NaOAc)</td>
			<td>SIGMA-ALDRICH</td>
			<td>S8625</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Ethanol</td>
			<td>Commercial Alcohols</td>
			<td>P016EAAN</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Tetramethyleneethylenediamine (TEMED)</td>
			<td>AMRESCO</td>
			<td>0761</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10% Ammonium persulfate (APS)</td>
			<td>BIO BASIC CANADA INC.</td>
			<td>AB0072</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC)</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>22360</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dimethyl sulfoxide (DMSO)</td>
			<td>CALEDON LABORATORIES</td>
			<td>803540</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Urease</td>
			<td>SIGMA-ALDRICH</td>
			<td>U0251</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">1&#215; Phosphate Buffered Saline (PBS)</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>70011-069</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">0.04% Phenol red</td>
			<td>SIGMA-ALDRICH</td>
			<td>P3532</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10&#215;T4 polynucleotide kinase reaction buffer</td>
			<td>Lucigen</td>
			<td>30061-1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10&#215; T4 DNA ligase reaction buffer</td>
			<td>Bio Basics Canada</td>
			<td>B1122-B</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">T4 DNA ligase (5 U/uL)</td>
			<td>Thermo Fischer Scientific</td>
			<td>B1122</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Luria Bertani (LB) Broth</td>
			<td>AMRESCO</td>
			<td>J106</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Agar</td>
			<td>AMRESCO</td>
			<td>J637</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">T4 polynucleotide kinase (10 U/uL)</td>
			<td>Lucigen</td>
			<td>30061-1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">E. coli K12 (MG1655)</td>
			<td>ATCC</td>
			<td>ATCC700926</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Centrifuge</td>
			<td>Beckman Coulter, Inc.</td>
			<td>392187</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Glass plates</td>
			<td>CBS scientific</td>
			<td>ngp-250nr</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">0.75 mm thick spacers</td>
			<td>CBS scientific</td>
			<td>VGS-0725r</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">12-well comb</td>
			<td>CBS scientific</td>
			<td>VGC-7512</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">UV Lamp</td>
			<td>UVP</td>
			<td>95-0017-09</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Spectrophotometer (NanoVue)</td>
			<td>GE Healthcare</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Metal plate</td>
			<td>CBS scientific</td>
			<td>CPA165-250</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Vortex</td>
			<td>VWR International</td>
			<td>58816-123</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Gel electrophoresis apparatus</td>
			<td>CBS scientific</td>
			<td>ASG-250</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Petri dishes</td>
			<td>VWR International</td>
			<td>25384-342</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">100 kDa MWCO centrifugal filters</td>
			<td>EMD Millipore</td>
			<td>UFC510024</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Magnetic Bead (BioMag)</td>
			<td>Bangs Laboratories Inc</td>
			<td>BM568</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Magnetic Seperation Rack</td>
			<td>New England BioLabs</td>
			<td>S1506S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Microfuge tubes</td>
			<td>Sarstedt</td>
			<td>72.69</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Syringe filter (0.22 um)</td>
			<td>VWR International</td>
			<td>28145-501</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">14 mL culture tube</td>
			<td>VWR International</td>
			<td>60818-725</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Cell culture incubator</td>
			<td>Eppendorf Scientific</td>
			<td>M13520000</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Branson Ultrasonic cleaner</td>
			<td>Branson</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Camera (Canon Powershot G11)</td>
			<td>Canon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">50 mL conical tube</td>
			<td>VWR International</td>
			<td>89004-364</td>
			<td></td>
		</tr>
	</tbody>
</table>

colorimetric detection of bacteria using litmus test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

Princippet i den bakterielle lakmusprøve er forklaret i figur 1 Testen anvender tre centrale materialer:. En RNA-spaltende DNAzym, der aktiveres af en særlig bakterie, urease og magnetiske perler. Den DNAzym anvendes som molekylær genkendelse element at opnå meget specifik påvisning af en bakterie af interesse. Urease og magnetiske perler bruges til at opnå signaltransduktion af RNA-spaltning aktivitet af DNAzym. Dette indebærer oprettelsen af ​​magnetiske per...

Watch this Scientific Journal Video about Colorimetric Detection of Bacteria Using Litmus Test at JoVE.com

Colorimetric Detection of Bacteria Using Litmus Test (Video) | JoVE

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

Kolorimetrisk påvisning af bakterier Brug lakmusprøve

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

Research

JoVE Journal

Biology

Colorimetric Detection of Bacteria Using Litmus Test

Detection of Protein Ubiquitination

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C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

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Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

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Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

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Udvikling af en kolloid guld-baserede immunokromatografisk teststrimmel til påvisning af Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

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Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...