Name: Colorimetric Detection of Bacteria Using Litmus Test (Video) | JoVE Journal
Uploaded: 2016-09-17T13:00:00.000+00:00
Duration: 10 min 5 s
Description: Learn a simple modified litmus test for rapid E. coli detection using an RNA-cleaving DNAzyme coupled with urease signal transduction. This method enables visible colorimetric readout for point-of-care diagnostics.

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5 minutes

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. Preparation of Reagents and Buffers
<ol>
	<li>0.5 M Ethylenediaminetetraacetic Acid (EDTA)
	<ol>
		<li>In a 2 L beaker, add 186.1 g EDTA to 800 ml of deionized-distilled water (ddH2O). Adjust the pH of the solution to 8.0 using NaOH pellets. Add ddH2O to a final volume of 1.0 L and transfer the solution to an autoclavable glass bottle for autoclaving and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>10&#215; Tris-borate EDTA (10x TBE)
	<ol>
		<li>In a 4 L plastic beaker, add 432 g Tris-base, 200 g boric acid, 80 ml of 0.5 M EDTA (pH 8.0) and ddH2O to a final volume of 4 L. Mix using a stir bar until all the components are dissolved. Transfer the solution to 1 L glass bottles for autoclaving and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>10% Denaturing Polyacrylamide Stock
	<ol>
		<li>In a 4 L plastic beaker, add 1681.7 g urea, 400 ml 10x TBE, 1 L of 40% acrylamide/bisacrylamide (29:1) solution and ddH2O until the final volume is 4 L. Mix using a stir bar until the urea is dissolved. Transfer the solution to 1 L amber glass bottles and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>2x Gel Loading Buffer (2x GLB)
	<ol>
		<li>In a 200 ml glass beaker, add 44 g urea, 8 g sucrose, 10 mg bromophenol blue, 10 mg xylene cyanol FF, 400 &#181;l of 10% sodium dodecyl sulfate, and 4 ml of 10&#215; TBE. Add ddH2O to a final volume of 40 ml and dissolve the components with mild heating at 50 &#176;C while mixing with a magnetic stir bar. Transfer 1 ml aliquot to 1.5 ml microfuge tubes and store at 4 &#176;C. 
		NOTE: Prior use requires brief heating at 90 &#176;C to redissolve any solids.</li>
	</ol>
	</li>
	<li>1 M Tris-hydrochloride (Tris-HCl) (pH 7.5)
	<ol>
		<li>In a glass bottle, add 12.1 g of Tris-base and 70 ml of ddH2O and mix until the solid is dissolved. Adjust the pH to 7.5 using 1 M hydrochloric acid (HCl). Add ddH2O to a final volume of 100 ml, autoclave and store at 4 &#176;C</li>
	</ol>
	</li>
	<li>5 M Sodium Chloride (NaCl)
	<ol>
		<li>In a glass bottle, dissolve 58.4 g of NaCl in 150 ml of ddH2O. Adjust the volume to 200 ml with ddH2O. Store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>DNA Elution Buffer
	<ol>
		<li>In a glass bottle, mix 2.0 ml of 1 M Tris-HCl (pH 7.5), 8.0 ml of 5 M NaCl and 0.4 ml of 0.5 M EDTA (pH 8.0). Adjust the volume to 200 ml with ddH2O, autoclave and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4)
	<ol>
		<li>In a glass bottle, dissolve 2.38 g of HEPES in 80 ml of ddH2O. Adjust the pH to 7.4 using 5 N NaOH and add ddH2O to a final volume of 100 ml.</li>
	</ol>
	</li>
	<li>1 M Magnesium Chloride (MgCl2)
	<ol>
		<li>In a glass bottle, add 2.03 g of MgCl2-6H2O and bring the volume to 100 ml with ddH2O.</li>
	</ol>
	</li>
	<li>Reaction Buffer (RB)
	<ol>
		<li>In a 50 ml conical tube, add 50 &#181;l of 1 M HEPES (pH 7.4), 1.5 ml of 5 M NaCl, 0.75 ml of 1 M MgCl2, and 5 &#181;l of Tween-20. Add ddH2O to a final volume of 50 ml. Mix the solution and filter into another conical tube using a syringe-driven filter (0.22 &#181;m) and store at 4 &#176;C until use.</li>
	</ol>
	</li>
	<li>Binding Buffer (BB)
	<ol>
		<li>In a 50 ml conical tube, add 500 &#181;l of 1 M Tris-HCl (pH 7.5), 8.8 g of NaCl, 50 &#181;l of 1 M of MgCl2, and 5 &#181;l of Tween 20. Add ddH2O to a final volume of 50 ml. Mix the solution and filter into another conical tube using a syringe-driven filter (0.22 &#181;m) and store at 4 &#176;C until use.</li>
	</ol>
	</li>
	<li>Substrate Solution (SS)
	<ol>
		<li>In a 50 ml conical tube, add 5.8 g of NaCl, 3 ml of 1 M MgCl2, 1.5 g urea, and 40 ml of ddH2O. Adjust the pH of the solution to 5.0 using 10 mM HCl. Because the solution is not buffered, adjust pH using HCl carefully through the addition of small HCl aliquots. Add ddH2O to a final volume of 50 ml.</li>
		<li>Mix the solution and filter into another conical tube using a syringe-driven filter (0.22 &#181;m) and store at 4 &#176;C until use.</li>
	</ol>
	</li>
	<li>Luria Bertani (LB) Broth
	<ol>
		<li>In a beaker, dissolve 20.0 g LB powder in 1 L of ddH2O. Transfer to a glass bottle, autoclave, and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>1.5% LB agar
	<ol>
		<li>In a 250 ml flask, add 1.5 g agar and 100 ml of LB broth. Autoclave and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Agar Plates
	<ol>
		<li>Redissolve the LB agar in a microwave and cool the solution to ~50 &#176;C. Pour the solution into petri dishes, making ~5 plates and allow them to solidify.</li>
	</ol>
	</li>
</ol>
2. Synthesis and Purification of E. coli-responsive DNAzyme EC1
<ol>
	<li>Synthesis of EC1 by Template Mediated Enzymatic Ligation
	<ol>
		<li>Purify commercially synthesized oligonucleotides BS1, DE1, and T1 (sequences provided in Table 1) by 10% denaturing polyacrylamide gel electrophoresis (dPAGE) according to standard protocols.</li>
		<li>Prepare a 100 &#181;M stock of BS1, DE1, and T1. Store at -20 &#176;C until use.</li>
		<li>In a 1.5 ml microfuge tube, add 38.5 &#181;l of ddH2O, 5 &#181;l of DE1, and 5 &#181;l of 10x T4 polynucleotide kinase reaction buffer provided by enzyme supplier (500 mM Tris-HCl (pH 7.6), 100 mM MgCl2, 50 mM dithiothreitol (DTT), 1.0 mM spermidine).</li>
		<li>Add 1 &#181;l of adenosine triphosphate (ATP) (100 mM). Add 5 units of T4 polynucleotide kinase (10 U/&#181;l). Mix by carefully pipetting the reaction mixture.</li>
		<li>Incubate the reaction at 37 &#176;C for 30 min.</li>
		<li>Quench the reaction by heating to 90 &#176;C for 5 min.</li>
		<li>Add 118 &#181;l of ddH2O, 5 &#181;l of BS1, and 5 &#181;l of T1. Heat the reaction to 90 &#176;C for 2 min and then cool to room temperature over 10 min.</li>
		<li>Add 20 &#181;l of 10&#215; T4 DNA ligase reaction buffer provided by the enzyme supplier (400 mM Tris-HCl (pH 7.8), 100 mM MgCl2, 100 mM DTT, 5 mM ATP). Add 10 units of T4 DNA ligase (5 U/&#181;l) and carefully mix by pipetting.</li>
		<li>Incubate at room temperature for 2 hr.</li>
		<li>Add 20 &#181;l of 3 M sodium acetate (NaOAc) (pH 5.2), 500 &#181;l of cold 100% ethanol. Mix the solution by vortexing and place the tube in a -20 &#176;C freezer for 30 min.</li>
		<li>Centrifuge the microfuge at 20,000 x g for 20 min at 4 &#176;C. Carefully remove the supernatant by pipetting.</li>
		<li>Wash the pellet with cold 70% ethanol and centrifuge again at 20,000 x g for 10 min at 4 &#176;C. Once again remove the supernatant and dry the pellet under vacuum for 10 min.</li>
		<li>Resuspend the DNA with 15 &#181;l of ddH2O and then add 15 &#181;l of 2x GLB.</li>
		<li>Vortex vigorously and heat to 90 &#176;C for 2 min. Purify the full-length DNA by 10% dPAGE as described below.</li>
	</ol>
	</li>
	<li>Setting Up 10% dPAGE
	<ol>
		<li>Clean two glass plates (one full plate and one notched), two 0.75 mm thick spacers, and a 12-well comb. Lay one glass plate on a flat surface with two spacers on each side and the notched plate on top. Clip the two plates together using the four clips provided by the supplier.</li>
		<li>In a 150 ml plastic beaker, pour 40 ml of 10% dPAGE, 40 &#181;l of tetramethylethylenediamine (TEMED), and 400 &#181;l of 10% ammonium persulfate (APS). Mix the components and pour the solution between the plates slowly.</li>
		<li>Insert the comb and then allow the gel to polymerize over 10 min. Once the gel is polymerized, slowly remove the comb and flush the wells with ddH2O.</li>
		<li>Mount the plates onto the gel electrophoresis apparatus. Use a metal plate to dissipate heat generated to prevent overheating.</li>
		<li>Pour 1x TBE onto the top and bottom chambers and ensure that the wells are well submerged in the buffer. Flush the wells with 1x TBE using a pipette or syringe.</li>
		<li>Set the apparatus to run at 35 mA and pre-run for 15 min before loading samples.</li>
	</ol>
	</li>
	<li>Elution of Ligated EC1 from 10% dPAGE
	<ol>
		<li>Following step 2.2.6, run the gel at 35 mA until the bottom dye (bromophenol blue) reaches the bottom of the glass plate. This should take approximately 1.5 hr. Turn off the power and remove the plates from the apparatus.</li>
		<li>Lay the plates on a few sheets of paper towels and carefully remove the spacers from the glass plates. Carefully remove the top glass plate and wrap the gel with plastic wrap.</li>
		<li>Flip the gel over to remove the second glass plate and cover with plastic wrap again. Take care to avoid wrinkles of the plastic wrap.</li>
		<li>Visualize the ligated product by using UV shadowing (260 nm), which will produce 4 distinct DNA bands (Fully ligated EC1, DE1, BS1, and T1).</li>
		<li>Excise the top band (EC1) with a sterile razor blade and transfer to a 1.5 ml microfuge tube. Crush the gel with a sterile pipette tip (200 &#181;l tip size) until it turns into a fine paste. Add 550 &#181;l of DNA elution buffer, cover the tube with aluminum foil to protect the embedded fluorophore, and shake for 15 min.</li>
		<li>Centrifuge the gel solution at 20,000 x g for 5 min and carefully transfer 400 &#181;l of the supernatant to another 1.5 ml microfuge tube. Avoid withdrawing gel pieces during pipetting.</li>
		<li>To the microfuge tube with the transferred supernatant add 40 &#181;l of 3 M NaOAc (pH 5.2), and 1.0 ml of cold 100% ethanol. Mix the solution by vortexing and place the tube in a -20 &#176;C freezer for 30 min.</li>
		<li>Centrifuge the microfuge at 20,000 x g for 20 min at 4 &#176;C. Carefully remove the supernatant by pipetting.</li>
		<li>Wash the pellet with cold 70% ethanol and centrifuge again at 20,000 x g for 10 min at 4 &#176;C. Once again, remove the supernatant and dry the pellet under vacuum for 10 min.</li>
		<li>Determine the concentration of EC1 by measuring UV absorbance at 260 nm. Make a 10 &#181;M stock and store the sample at -20 &#176;C until use.</li>
	</ol>
	</li>
</ol>
3. Conjugation of Urease to DNA
<ol>
	<li>Preparation of Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) Stock
	<ol>
		<li>Dissolve 1 mg SMCC in 676 &#181;l of DMSO. Vortex and place on ice until use.</li>
	</ol>
	</li>
	<li>Preparation of Urease Stock
	<ol>
		<li>Dissolve 1 mg urease in 1 ml of 1&#215; PBS (no Mg2+ or Ca2+). Place on ice until use. 
		NOTE: Crystallized urease is slow to dissolve and gentle mixing is needed to avoid denaturation.</li>
	</ol>
	</li>
	<li>Synthesis of Urease-DNA (UrDNA)
	<ol>
		<li>Add 10 &#181;l of 100 &#181;M LD1 to a 2.5 ml microfuge tube. Add 140 &#181;l of ddH2O, 40 &#181;l of 10x PBS, and vortex.</li>
		<li>Add 80.5 &#181;l of SMCC stock, 159.5 &#181;l of DMSO, vortex, and briefly centrifuge using a benchtop centrifuge.</li>
		<li>Incubate at 37 &#176;C for 60 min. Avoid condensation under the microfuge cap.</li>
		<li>Add 200 &#181;l of 1x PBS, 60 &#181;l of 3 M NaOAc (pH 5.2), and 1.5 ml of cold 100% ethanol. Mix the solution by vortexing and incubate at -20 &#176;C for 30 min.</li>
		<li>Centrifuge the solution at 20,000 x g for 20 min at 4 &#176;C. Remove the supernatant and dry the pellet under vacuum. Avoid over-drying.</li>
		<li>To the dried pellet add 400 &#181;l of urease stock and incubate at room temperature for 5 hr.</li>
		<li>Transfer 200 &#181;l of crude conjugate to a pre-washed 100k MWCO centrifugal filter column. Centrifuge the column at 14,000 x g for 5 min.</li>
		<li>Transfer the remaining 200 &#181;l of crude conjugate to the column and centrifuge at 14,000 x g for 5 min. Remove the column and place it upside down in a new 2.0 ml microfuge tube (&#34;collection tube&#34;).</li>
		<li>Centrifuge the collection tube (with the inverted column) at 1,000 x g for 2 min. Remove the collection tube and add 30 &#181;l of 1x PBS to the centrifugal column to wash the membrane for additional recovery of conjugates.</li>
		<li>Once again invert the column and place back into the collection tube. Centrifuge the collection tube (with the inverted column) at 1,000 x g for 2 min. Remove and dispose the column.</li>
		<li>Store the UrDNA at 4 &#176;C until use.</li>
	</ol>
	</li>
</ol>
4. Assembly of EC1 and UrDNA onto Magnetic Beads
<ol>
	<li>Mix the magnetic bead (MB) stock well and transfer 100 &#181;l of MB suspension to a 1.5 ml microfuge tube. Place the microfuge tube on a magnetic rack holder for isolating the MB.</li>
	<li>Remove the supernatant by pipetting and add 150 &#181;l of binding buffer (BB) to the tube. Remove the tube from the holder and carefully tap the tube to resuspend the MB to a homogeneous solution. Avoid splashing the suspension to the top of the tube or cap. If this happens, use a benchtop centrifuge to briefly spin the residue back to the suspension.</li>
	<li>Repeat step 4.2 two more times.</li>
	<li>To this suspension add 10 &#181;l of 10 &#181;M EC1. Carefully mix by tapping on the tube.</li>
	<li>Incubate the solution with mild shaking for 30 min. Tap the tube every 2-3 min to avoid the precipitation of the MB.</li>
	<li>Place the microfuge tube back on the magnetic rack to isolate the MB and remove the supernatant by pipetting. Wash the MB three times with 150 &#181;l of BB (as described in step 4.2).</li>
	<li>Once the washing is complete, suspend the MB in a total of 150 &#181;l of BB. To this solution, add 15 &#181;l of UrDNA and heat the solution to 45 &#176;C for 2 min. Cool the solution to room temperature and incubate for 2 hr.</li>
	<li>Place the microfuge tube back on the magnetic rack to isolate the MB and remove the supernatant by pipetting.</li>
	<li>Add 100 &#181;l of reaction buffer (RB). Remove the microfuge tube from the magnetic rack and carefully resuspend the MB.</li>
	<li>Wash the MB three more times by repeating step 4.9. 
	NOTE: The washed supernatant can be quickly tested to determine if unhybridized UrDNA is still present, which may result in a false positive signal. The test can be done by adding 10 &#181;l of 50 mM urea and 10 &#181;l of 0.04% phenol red to the wash solution. Continue to wash the MB until the wash solution does not cause a color change. The 100 &#181;l suspension is stored at 4 &#176;C until use.</li>
</ol>
5. Preparation of Bacterial Cells20
<ol>
	<li>Culturing E. coli from Stocks
	<ol>
		<li>Plate E. coli K12 (MG1655) onto LB agar plates from a glycerol stock under a flame or in a biological safety cabinet.</li>
		<li>Using a sterile pipette tip, gently touch the glycerol stock and lightly streak the surface of an agar plate to avoid puncturing the LB agar.</li>
		<li>Invert the streaked plate and incubate at 37 &#176;C for 14 hr.</li>
		<li>Seal the plate with Parafilm and store at 4 &#176;C for a maximum of 3 weeks.</li>
	</ol>
	</li>
	<li>Culturing E. coli for Cell Counting
	<ol>
		<li>In a sterile 14 ml culture tube, dispense 2 ml of LB broth.</li>
		<li>Using a sterile pipette tip, pick a single colony from the streaked agar plate prepared in step 5.1 and transfer it to the culture tube.</li>
		<li>Incubate the culture at 37 &#176;C and shake at 230 rpm for 14 hr.</li>
		<li>Serially dilute the bacterial culture in 10-fold intervals.</li>
		<li>For each diluted sample, evenly plate five 100 &#181;l aliquots on separate LB agar plates. Invert the plates and incubate at 37 &#176;C for 14 hr.</li>
		<li>Count the cells of each sample to obtain the average cell concentration of each dilution. 
		NOTE: 107 cells are often used to set up a reference litmus test for E. coli as this level of E. coli can trigger a quick color change. However, a properly executed litmus test can detect as low as 500 cells, as discussed in the Results section.</li>
	</ol>
	</li>
	<li>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>
	</li>
</ol>
6. 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 (protocol 4) 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 6.3.</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 5.3.</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 addition of 1 mM Acetate Buffer pH 5.0).</li>
</ol>

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<li>Huang, P. J., Vazin, M., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+selection+of+a+new+lanthanide-dependent+DNAzyme+for+ratiometric+sensing+lanthanides%5BTitle%5D)+AND+%22Anal.+Chem%22%5BJournal%5D)">In vitro selection of a new lanthanide-dependent DNAzyme for ratiometric sensing lanthanides.</a> Anal. Chem. 86, 9993-9999 (2014).</li>
<li>Chiuman, W., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simple+fluorescent+sensors+engineered+with+catalytic+DNA+%27MgZ%27+based+on+a+non-classic+allosteric+design%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Simple fluorescent sensors engineered with catalytic DNA 'MgZ' based on a non-classic allosteric design.</a> PLoS One. 2, e1224(2007).</li>
<li>Xiang, Y., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+personal+glucose+meters+and+functional+DNA+sensors+to+quantify+a+variety+of+analytical+targets%5BTitle%5D)+AND+%22Nat.+Chem%22%5BJournal%5D)">Using personal glucose meters and functional DNA sensors to quantify a variety of analytical targets.</a> Nat. Chem. 3, 697-703 (2011).</li>
<li>Aguirre, S. D., Ali, M. M., Salena, B. J., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+sensitive+DNA+enzyme-based+fluorescent+assay+for+bacterial+detection%5BTitle%5D)+AND+%22Biomolecules%22%5BJournal%5D)">A sensitive DNA enzyme-based fluorescent assay for bacterial detection.</a> Biomolecules. 3, 563-577 (2013).</li>
<li>Aguirre, S. D., Ali, M. M., Kanda, P., Li, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+Bacteria+Using+Fluorogenic+DNAzymes%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Detection of Bacteria Using Fluorogenic DNAzymes.</a> J. Vis. Exp. (63), e3961(2012).</li>
<li>Shen, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+catalytic+DNA+activated+by+a+specific+strain+of+bacterial+pathogen%5BTitle%5D)+AND+%22Angew.+Chem.+Int.+Ed%22%5BJournal%5D)">A catalytic DNA activated by a specific strain of bacterial pathogen.</a> Angew. Chem. Int. Ed. 54, (2015).</li>
<li>He, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+specific+recognition+of+breast+tumors+by+an+RNA-cleaving+fluorogenic+DNAzyme+probe%5BTitle%5D)+AND+%22Anal.+Chem%22%5BJournal%5D)">Highly specific recognition of breast tumors by an RNA-cleaving fluorogenic DNAzyme probe.</a> Anal. Chem. 87, 569-577 (2015).</li>
<li>Sumner, J. B., Hand, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isoelectric+point+of+crystalline+urease%5BTitle%5D)+AND+%22J.+Am.+Chem.+Soc%22%5BJournal%5D)">Isoelectric point of crystalline urease.</a> J. Am. Chem. Soc. 51, 1255-1260 (1929).</li>
<li>Karplus, P. A., Pearson, M., Hausinger, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((70+Years+of+crystalline+urease%3A+What+have+we+learned%5BTitle%5D)+AND+%22Acc.+Chem.+Res%22%5BJournal%5D)">70 Years of crystalline urease: What have we learned.</a> Acc. Chem. Res. 30, 330-337 (1997).</li>
<li>Omiccioli, E., Amagliani, G., Brandi, G., Magnani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+platform+for+Real-Time+PCR+detection+of+Salmonella+spp.%2C+Listeria+monocytogenes+and+Escherichia+coli+O157+in+milk%5BTitle%5D)+AND+%22Food+Microbiol%22%5BJournal%5D)">A new platform for Real-Time PCR detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 in milk.</a> Food Microbiol. 26, 615-622 (2009).</li>
<li>Cui, S., Schroeder, C. M., Zhang, D. Y., Meng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+sample+preparation+method+for+PCR-based+detection+of+Escherichia+coli+O157%3AH7+in+ground+beef%5BTitle%5D)+AND+%22J.+Appl.+Microbiol%22%5BJournal%5D)">Rapid sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef.</a> J. Appl. Microbiol. 95, 129-134 (2003).</li>
<li>Ibekwe, A. M., Watt, P. M., Grieve, C. M., Sharma, V. K., Lyons, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplex+fluorogenic+real-time+PCR+for+detection+and+quantification+of+Escherichia+coli+O157%3AH7+in+dairy+wastewater+wetlands%5BTitle%5D)+AND+%22Appl.+Environ.+Microbiol%22%5BJournal%5D)">Multiplex fluorogenic real-time PCR for detection and quantification of Escherichia coli O157:H7 in dairy wastewater wetlands.</a> Appl. Environ. Microbiol. 68, 4853-4862 (2002).</li>
<li>Strachan, N. J., Ogden, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+sensitive+microsphere+coagulation+ELISA+for+Escherichia+coli+O157%3AH7+using+Russell%27s+viper+venom%5BTitle%5D)+AND+%22FEMS+Microbiol+Lett%22%5BJournal%5D)">A sensitive microsphere coagulation ELISA for Escherichia coli O157:H7 using Russell's viper venom.</a> FEMS Microbiol Lett. 186, 79-84 (2000).</li>
<li>de Boer, E., Beumer, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methodology+for+detection+and+typing+of+foodborne+microorganisms%5BTitle%5D)+AND+%22Int.+J.+Food+Microbiol%22%5BJournal%5D)">Methodology for detection and typing of foodborne microorganisms.</a> Int. J. Food Microbiol. 50, 119-130 (1999).</li>
<li>Gracias, K. S., McKillip, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+review+of+conventional+detection+and+enumeration+methods+for+pathogenic+bacteria+in+food%5BTitle%5D)+AND+%22Can.+J.+Microbiol%22%5BJournal%5D)">A review of conventional detection and enumeration methods for pathogenic bacteria in food.</a> Can. J. Microbiol. 50, 883-890 (2004).</li>
</ol>

The authors have nothing to disclose.

The translation of the action of the RNA cleavage activity of a bacterium-responsive DNAzyme to a litmus test is made possible through the use of urease and magnetic separation, as illustrated by Figure 1. Although the demonstration of the modified litmus test for bacterial detection is done with an E. coli-dependent RNA-cleaving DNAzyme,5,19,20 the design can be generally extended for any RNA-cleaving DNAzyme. Given the great availability of RNA-cleaving DNAzymes for different analyt...

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><tbody><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>VWR AMRESCO</td><td>0105</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>0497</td><td></td></tr><tr><td>Boric acid</td><td>AMRESCO</td><td>0588</td><td></td></tr><tr><td>Urea</td><td>VWR AMRESCO</td><td>M123</td><td></td></tr><tr><td>40% acrylamide/bisacrylamide (29:1) solution</td><td>BIO BASIC CANADA INC.</td><td>A0007</td><td></td></tr><tr><td>Sucrose</td><td>Bioshop Canada inc.</td><td>SUC507</td><td></td></tr><tr><td>Bromophenol blue</td><td>Bioshop Canada inc.</td><td>BRO777</td><td></td></tr><tr><td>Xylenecyanol FF</td><td>SIGMA-ALDRICH</td><td>X-4126</td><td></td></tr><tr><td>10% sodium dodecyl sulfate</td><td>Bioshop Canada inc.</td><td>SDS001</td><td></td></tr><tr><td>Hydrochloric Acid (HCl)</td><td>CALEDON LABORATORIES LTD</td><td>6026</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Bioshop Canada inc.</td><td>SOD001</td><td></td></tr><tr><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td><td>Bioshop Canada inc.</td><td>HEP001</td><td></td></tr><tr><td>Magnesium Chloride (II) hexahydrate</td><td>VWR AMRESCO</td><td>0288</td><td></td></tr><tr><td>Tween 20</td><td>Bioshop Canada inc.</td><td>TW508</td><td></td></tr><tr><td>Adenosine Triphospahte (ATP)</td><td>AMRESCO</td><td>0220</td><td></td></tr><tr><td>Sodium Acetate trihydrate (NaOAc)</td><td>SIGMA-ALDRICH</td><td>S8625</td><td></td></tr><tr><td>Ethanol</td><td>Commercial Alcohols</td><td>P016EAAN</td><td></td></tr><tr><td>Tetramethyleneethylenediamine (TEMED)</td><td>AMRESCO</td><td>0761</td><td></td></tr><tr><td>10% Ammonium persulfate (APS)</td><td>BIO BASIC CANADA INC.</td><td>AB0072</td><td></td></tr><tr><td>Succinimidyl 4-(&lt;em&gt;N&lt;/em&gt;-maleimidomethyl) cyclohexane-1-carboxylate (SMCC)</td><td>ThermoFisher SCIENTIFIC</td><td>22360</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>CALEDON LABORATORIES</td><td>803540</td><td></td></tr><tr><td>Urease</td><td>SIGMA-ALDRICH</td><td>U0251</td><td></td></tr><tr><td>1x Phosphate Buffered Saline (PBS)</td><td>ThermoFisher SCIENTIFIC</td><td>70011-069</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>10x T4 DNA ligase reaction buffer</td><td>Bio Basics Canada</td><td>B1122-B</td><td></td></tr><tr><td>T4 DNA ligase (5 U/&amp;#956;l)</td><td>Thermo Fischer Scientific</td><td>B1122</td><td></td></tr><tr><td>Luria Bertani (LB) Broth</td><td>AMRESCO</td><td>J106</td><td></td></tr><tr><td>Agar</td><td>AMRESCO</td><td>J637</td><td></td></tr><tr><td>T4 polynucleotide kinase (10 U/&amp;#956;l)</td><td>Lucigen</td><td>30061-1</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; K12 (MG1655)</td><td>ATCC</td><td>ATCC700926</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman Coulter, Inc.</td><td>392187</td><td></td></tr><tr><td>Glass plates</td><td>CBS scientific</td><td>ngp-250nr</td><td></td></tr><tr><td>0.75 mm thick spacers</td><td>CBS scientific</td><td>VGS-0725r</td><td></td></tr><tr><td>12-well comb</td><td>CBS scientific</td><td>VGC-7512</td><td></td></tr><tr><td>UV Lamp</td><td>UVP</td><td>95-0017-09</td><td></td></tr><tr><td>Spectrophotometer (NanoVue)</td><td>GE Healthcare</td><td>N/A</td><td></td></tr><tr><td>Metal plate</td><td>CBS scientific</td><td>CPA165-250</td><td></td></tr><tr><td>Vortex</td><td>VWR International</td><td>58816-123</td><td></td></tr><tr><td>Gel electrophoresis apparatus</td><td>CBS scientific</td><td>ASG-250</td><td></td></tr><tr><td>Petri dishes</td><td>VWR International</td><td>25384-342</td><td></td></tr><tr><td>100 kDa MWCO centrifugal filters</td><td>EMD Millipore</td><td>UFC510024</td><td></td></tr><tr><td>Magnetic Bead (BioMag)</td><td>Bangs Laboratories Inc</td><td>BM568</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>Syringe filter (0.22 &amp;#956;m)</td><td>VWR International</td><td>28145-501</td><td></td></tr><tr><td>14 ml culture tube</td><td>VWR International</td><td>60818-725</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><tr><td>Camera (Canon Powershot G11)</td><td>Canon</td><td>N/A</td><td></td></tr><tr><td>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

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.

The principle of the bacterial litmus test is explained in Figure 1. The test uses three key materials: an RNA-cleaving DNAzyme that is activated by a specific bacterium, urease and magnetic beads. The DNAzyme is used as the molecular recognition element to achieve highly specific detection of a bacterium of interest. Urease and magnetic beads are used to achieve signal transduction of the RNA-cleavage activity of the DNAzyme. This involves the creation of magnetic beads ...

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.

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. ...

The overall goal of this experiment is to demonstrate a simple modified litmus test for bacterial detection. This method describes how an E.coli-activated RNA-cleaving DNA enzyme can be coupled with a urease-based signal trandsduction platform to generate a color change in response to E.coli. The two main advantages of this technique are the simplicity of the test and its applicability to other analyte-responsive RNA-cleaving DNA enzymes.Demonstrating the procedure will be Seperh Manchehry and Dingran Chang, graduate students from Dr.Yingfu Li's lab at McMaster University. After preparing reagents and buffers according to the text protocol, prepare a stock solution of succinimidyl 4-cyclohexane-1-carboxylate, or SMCC, by dissolving one milligram of SMCC in 676 microliters of DMSO. Vortex and place on ice until use.Dissolve one milligram of urease in milliliter of 1x PBS and also place that on ice until use. To synthesize urease DNA, or UrDNA, add 10 microliters of 100 micromolar LD1 to a 2.5 milliliter microfuge tube. Then, add 140 microliters of doubly-distilled water and 40 microliters of 10x PBS and vortex.Next, add 80.5 microliters of SMCC stock and 159.5 microliters of DMSO. Then, vortex again and use a benchtop centrifuge to briefly spin. Incubate the reaction at 37 degrees Celsius in an incubator or a heat block for 60 minutes.Following the incubation, add 200 microliters of 1x PBS, 60 microliters of three-molar sodium acetate, and 1.5 milliliters of cold 100%ethanol to the tube. Mix by vortexing, and incubate the reaction at minus 20 degrees Celsius for 30 minutes. Centrifuge the solution at 20, 000 times g and four degrees Celsius for 20 minutes.Then, remove the supernatant and dry the pellet under vacuum. To the dried pellet, add 400 microliters of urease stock and incubate at room temperature for 5 hours. Transfer 200 microliters of crude conjugate to a pre-washed 100, 000 molecular weight cutoff centrifugal filter column.Centrifuge the column at 14, 000 times g for five minutes. Transfer the remaining 200 microliters of crude conjugate to the column and centrifuge at 14, 000 times g for five minutes. Then, remove the column and place it upside down in a new two-milliliter microfuge collection tube.Centrifuge the inverted column and tube at 1, 000 times g for two minutes. After removing the collection tube, add 30 microliters of 1x PBS to the centrifugal column to wash the membrane for additional recovery of conjugates. Invert the column again and place it back into the collection tube.Centrifuge the column at 1, 000 times g for two minutes. Remove and dispose of the column. Store the UrDNA at four degrees Celsius until use.To assemble an E.coli-activated RNA-cleaving DNAzyme, or EC1, and UrDNA onto magnetic beads, mix the magnetic bead stock well and transfer 100 microliters of bead suspension to a 1.5 milliliter microfuge tube. Then, place the tube on a magnetic rack holder for isolating the beads. Remove the supernatant by pipetting and add 150 microliters of binding buffer, or BB, to the tube.Remove the tube from the holder and carefully tap the tube to re-suspend the beads into a homogeneous solution. After repeating the BB wash two more times with the beads in suspension with BB, add 10 microliters of 10 micromolar EC1. Carefully mix by tapping on the tube.Incubate the solution with mild shaking for 30 minutes. To avoid aggregation of the beads, tap on the tube every two to three minutes. Next, place the microfuge tube back on the magnetic rack to isolate the beads and remove the supernatant by pipetting.Then, use 150 microliters of BB to wash the beads three times. Once the washing is complete, suspend the beads in a total of 150 microliters of BB.To this solution, add 15 microliters of UrDNA and heat the sample at 45 degrees Celsius for two minutes, then cool the solution to room temperature and incubate for two hours. Place the microfuge tube back on the magnetic rack to isolate the beads and remove the supernatant by pipetting.Add 100 microliters of reaction buffer, or RB, then remove the tube from the magnetic rack and carefully re-suspend the beads. 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 re-suspend 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.The starting color of the litmus test must be yellow, reflecting a solution pH below 5.5. If necessary, the starting pH of the litmus test can be adjusted with a weak buffer, such as one millimolar sodium acetate buffer at pH five. This figure shows the results of the bacterial litmus test, where E.coli-activated RNA-cleaving DNAzyme EC1 was used as the DNAzyme and Phenol red was used as the pH indicator.As shown by the lack of color change in the absence of E.coli, EC1 is highly specific and exhibits minimal activity towards other bacteria. The depth of color change is dependent both on the number of E.coli cells used in the DNAzyme activation step and on the time allowed for the urea hydrolysis step. More E.coli cells result in a stronger color change, and a longer time for urea hydrolysis allows for the detection of a smaller number of cells.This graph shows the pH change as monitored using a handheld pH meter. The presence of 10 to the seven E.coli cells resulted in a gradual increase in pH by three units within 10 minutes. In contrast, the absence of E.coli did not cause detectable pH changes in the same conditions.Once mastered, this technique can be done in two hours if it is performed properly. While attempting this procedure, it is important to remember that urease is very active, and poor washing or contamination will lead to a false positive. Following this procedure, this method can be performed to test for E.coli in more relevant conditions, such as testing for environmental water quality.After watching this video, you should have a good understanding for taking advantage of a simple litmus test for E.coli testing. Don't forget that this test can be used to detect pathogenic E.coli, which is extremely hazardous. You need to take biosafety training before conducting any of the tests for pathogenic bacteria.

colorimetric-detection-of-bacteria-using-litmus-test

Research

JoVE Journal

Biology

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

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...

Detection of Bacteria Using Fluorogenic DNAzymes

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Bacterial Detection &amp; Identification Using Electrochemical Sensors

This video describes a colorimetric assay for lactate measurement in cell lysates by using the lactate dehydrogenase enzyme. This enzyme converts lactate into pyruvate, along with the conversion of NAD to NADH that reacts with tetrazolium dyes and gives a colored product that can be measured spectrophotometrically to quantitate the lactate.

Colorimetric Assay for Quantification of Lactate in Test Samples

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.

Colorimetric Detection of a Bacterial Biomarker Using a Modified Litmus Test

Source:Rhea Kang1, Gabby Mora1, Sherry Dunbar11Luminex, A DiaSorin Company, Austin.This video demonstrates multiplexed quantitative bacterial detection employing carboxylated magnetic polystyrene microspheres dyed into distinct sets. The procedure encompasses the formation of bead-bacteria complexes, magnetic separation, and flow-based analysis, enabling precise and simultaneous quantification of multiple bacteria in the same reaction well.

Multiplexed Quantitative Bacterial Detection using Dye-Labeled Magnetic Microspheres

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells.  This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

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 (µPAD) imbedded with bacterial-indicative colorimetric substrates.

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

Here, we present a protocol to detect bacterial motility based on a color reaction. Key advantages of this method are that it is easy to evaluate and more accurate, and does not require specialized equipment.

Visualizing Bacterial Motility Based on a Color Reaction

Learn a simple modified litmus test for rapid E. coli detection using an RNA-cleaving DNAzyme coupled with urease signal transduction. This method enables visible colorimetric readout for point-of-care diagnostics.

Colorimetric Detection of Bacteria Using Litmus Test

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