colorimetric detection of a bacterial biomarker using a modified litmus test

Colorimetric Detection of a Bacterial Biomarker Using a Modified Litmus Test

After preparing E. coli cells according to the text protocol, pre-wash a 1.5-milliliter microfuge tube by adding and vortexing 100 microliters of RB. Then, discard the buffer.
Add 15 microliters of assembled EC1 to the washed tube, then, place the tube on the magnetic rack, and aspirate the supernatant. Remove the tube from the rack, add 100 microliters of RB, and carefully resuspend the magnetic beads.
After using RB to wash the beads two more times and removing the RB, add 10 microliters of the E. coli sample to the tube, and gently mix the contents by tapping on the tube. Then, incubate the reaction at room temperature for one hour.
After the incubation, add 90 microliters of doubly-distilled water, and place the tube on the magnetic rack. Following approximately three minutes of magnetic separation, carefully transfer 85 microliters of the supernatant to a 0.5-milliliter microfuge tube.
It is very important to pipette the solution carefully without taking up any of the magnetic beads. These beads are loaded with urease and will generate a false positive signal.
Add 15 microliters of 0.04% phenol red and 100 microliters of substrate solution. Then, take photographs at specific time intervals to record a color change.

colorimetric-detection-bacterial-biomarker-using-modified-litmus

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Imaging and Visualization Techniques

EoE Sub Articles

eoe sub articles

1. Preparing E. coli Cells for Testing
<ol>
	<li>For a desired cell suspension, transfer 1 ml of cultured stock to a 1.5 ml microfuge tube.</li>
	<li>Centrifuge the cells at 6,000 x g for 10 min at 4 &#176;C. Carefully remove the supernatant without disturbing the cell pellet.</li>
	<li>Add 10 &#181;l of reaction buffer to the cell pellet and resuspend the cells. Sonicate the cell suspension for 5 min. Transfer the cell suspension to an ice box for 5 min.</li>
	<li>Sonicate the cell suspension for another 5 min.</li>
	<li>Centrifuge the cell suspension at 13,000 x g for 10 min at 4 &#176;C. Use the supernatant for testing (10 &#181;l).</li>
</ol>
2. Litmus Test
<ol>
	<li>In a 1.5 ml microfuge tube, prewash the tube by adding and vortexing 100 &#181;l of reaction buffer (RB) in the microfuge tube and discarding the buffer.</li>
	<li>Transfer 15 &#181;l of assembled EC1 to the washed microfuge tube.</li>
	<li>Wash the magnetic beads by placing the microfuge tube on a magnetic rack. Remove the supernatant by pipetting. Remove the microfuge tube from the rack, add 100 &#181;l of RB, and carefully resuspend the magnetic beads.</li>
	<li>Wash the MB two more times by repeating step 2.3.</li>
	<li>Place the microfuge tube back on the magnetic rack, remove the supernatant, and add the 10 &#181;l E. coli sample prepared from step 1.3.</li>
	<li>Mix the sample and magnetic beads carefully by gently tapping on the microfuge tube.</li>
	<li>Incubate the reaction at room temperature for 1 hr.</li>
	<li>To the reaction, add 90 &#181;l of ddH2O and place the microfuge tube onto a magnetic rack.</li>
	<li>After approximately 3 min of magnetic separation, carefully transfer 85 &#181;l of the supernatant to a 0.5 ml microfuge tube. Withdraw the supernatant slowly to avoid collecting any magnetic beads.</li>
	<li>To the above microfuge tube add 15 &#181;l of 0.04% phenol red and 100 &#181;l of substrate solution.</li>
	<li>Take a photograph at specific time intervals to record color change. 
	NOTE: The change in pH can also be monitored using a pH meter with a microelectrode. The starting pH should be approximately 5.2-5.5 (the solution is yellow). If not, the solution can be adjusted by the addition of 1 mM Acetate Buffer pH 5.0).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>VWR AMRESCO</td>
			<td>105</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide (NaOH) pellets</td>
			<td>BIO BASIC CANADA INC</td>
			<td>SB6789</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>VWR AMRESCO</td>
			<td>497</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>VWR AMRESCO</td>
			<td>M123</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylenecyanol FF</td>
			<td>SIGMA-ALDRICH</td>
			<td>X-4126</td>
			<td></td>
		</tr>
		<tr>
			<td>0.04% Phenol red</td>
			<td>SIGMA-ALDRICH</td>
			<td>P3532</td>
			<td></td>
		</tr>
		<tr>
			<td>10x T4 polynucleotide kinase reaction buffer</td>
			<td>Lucigen</td>
			<td>30061-1</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli K12 (MG1655)</td>
			<td>ATCC</td>
			<td>ATCC700926</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Bead (BioMag)</td>
			<td>Bangs Laboratories Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Seperation Rack</td>
			<td>New England BioLabs</td>
			<td>S1506S</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfuge tubes</td>
			<td>Sarstedt</td>
			<td>72.69</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Eppendorf Scientific</td>
			<td>M13520000</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson Ultrasonic cleaner</td>
			<td>Branson</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Tram, K., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54546">Colorimetric Detection of Bacteria Using Litmus Test.</a> J. Vis. Exp. (2016).
This video demonstrates a colorimetric E. coli detection method, involving an E. coli biomarker activating a DNAzyme, leading to cleavage of RNA linkage and detachment of a DNAzyme-urease conjugate. A subsequent colorimetric assay detects the E. coli biomarker through ammonia-induced pH changes.

1. Preparing E. coli Cells for Testing

	For a desired cell suspension, transfer 1 ml of cultured stock to a 1.5 ml microfuge tube.
	Centrifuge the cells ...

Start with an RNA-cleaving DNAzyme, a catalytic DNA that, on binding to a specific bacterial biomarker, gets activated.
The DNAzyme is linked to a streptavidin-coated magnetic bead via biotin at its 5&#39; end. At the 3&#39; end, it is bound to an oligonucleotide-linked urease, forming a functional magnetic bead.
To a tube containing functional magnetic beads, add an E. coli lysate.
A specific E. coli biomarker from the lysate, activates the DNAzyme, triggering cleavage of the RNA linkage, causing the detachment of the DNAzyme-urease conjugate.
Use a magnetic stand to immobilize the beads and remove the bead-bound DNAzyme-urease conjugate.
Transfer the released urease-containing supernatant to another tube.
Add a pH indicator dye &#8212; phenol red, and a urea solution.
The urease hydrolyzes urea into ammonia and carbon dioxide.
Ammonia, a weak base, raises the pH of the solution, causing a color change, that indicates the presence of the E. coli biomarker.

32 ビュー. 修正リトマス試験紙を用いた細菌バイオマーカーの比色検出

ビデオ: 修正リトマス試験紙を用いた細菌バイオマーカーの比色検出 - 実験

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist&#160;of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (&#181;PADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the &#181;PAD. Alternatively, digital images of the reacted &#181;PADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24&#160;hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (&#181;PAD) imbedded with bacterial-indicative colorimetric substrates.

比色検出の紙ベース<em&gt;エシェリヒア·コリ</em&gt;<em&gt;サルモネラ</em&gt;属、および<em&gt;リステリア菌</em&gt;農業大量の水から

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of ...

JoVE Journal

環境学

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

超高感度単一分子の開発と検証配列人間のインターフェロン-α のデジタルの酵素免疫測定法

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

免疫・感染学

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.

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.

リトマス試験を用いて細菌の比色検出

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. ...

Colorimetric Detection of a Bacterial Biomarker Using a Modified Litmus Test

記録

Colorimetric Detection of a Bacterial Biomarker Using a Modified Litmus Test