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>

<ol>
	<li>Daar, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top+ten+biotechnologies+for+improving+health+in+developing+countries.">Top ten biotechnologies for improving health in developing countries.</a> Nat. Genet. 32, 229-232 (2002).</li><li>Newman, J. D., Turner, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Home+blood+glucose+biosensors:+a+commercial+perspective.">Home blood glucose biosensors: a commercial perspective.</a> Biosens. Bioelectron. 20, 2435-2453 (2005).</li><li>Turner, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosensors:+sense+and+sensibility.">Biosensors: sense and sensibility.</a> Chem. Soc. Rev. 42, 3184-3196 (2013).</li><li>Tram, K., Kanda, P., Salena, B. J., Huan, S. Y., Li, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+Bacterial+Detection+by+DNAzymes+into+a+Litmus+Test.">Translating Bacterial Detection by DNAzymes into a Litmus Test.</a> Angew. Chem. Int. Ed. 53, 12799-12802 (2014).</li><li>Ali, M. M., Aguirre, S. D., Lazim, H., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorogenic+DNAzyme+probes+as+bacterial+indicators.">Fluorogenic DNAzyme probes as bacterial indicators.</a> Angew. Chem. Int. Ed. 50, 3751-3754 (2011).</li><li>Schlosser, K., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologically+inspired+synthetic+enzymes+made+from+DNA.">Biologically inspired synthetic enzymes made from DNA.</a> Chem. Biol. 16, 311-322 (2009).</li><li>Tuerk, C., Gold, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+evolution+of+ligand+by+exponential+enrichment:+RNA+ligands+to+bacteriophage+T4+DNA+polymerase.">Systematic evolution of ligand by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.</a> Science. 249, 505-510 (1990).</li><li>Ellington, A. D., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+selection+of+RNA+molecules+that+bind+specific+ligands.">In vitro selection of RNA molecules that bind specific ligands.</a> Nature. 346, 818-822 (1990).</li><li>Breaker, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+catalytic+DNAs.">Making catalytic DNAs.</a> Science. 290, 2095-2096 (2000).</li><li>Liu, J., Cao, Z., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+nucleic+acid+sensors.">Functional nucleic acid sensors.</a> Chem. Rev. 109, 1948-1998 (2009).</li><li>Navani, N. K., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acid+aptamers+and+enzymes+as+sensors.">Nucleic acid aptamers and enzymes as sensors.</a> Curr. Opin. Chem. Biol. 10, 272-281 (2006).</li><li>Li, J., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+sensitive+and+selective+catalytic+DNA+biosensor+for+lead+ions.">A highly sensitive and selective catalytic DNA biosensor for lead ions.</a> J. Am. Chem. Soc. 122, 10466-10467 (2000).</li><li>Liu, J., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+colorimetric+lead+biosensor+using+DNAzyme-directed+assembly+of+gold+nanoparticles.">A colorimetric lead biosensor using DNAzyme-directed assembly of gold nanoparticles.</a> J. Am. Chem. Soc. 125, 6642-6643 (2003).</li><li>Liu, Z., Mei, S. H. J., Brennan, J. D., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assemblage+of+signaling+DNA+enzymes+with+intriguing+metal+specificity+and+pH+dependence.">Assemblage of signaling DNA enzymes with intriguing metal specificity and pH dependence.</a> J. Am. Chem. Soc. 125, 7539-7545 (2003).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+catalytic+beacon+sensor+for+uranium+with+parts-per-trillion+sensitivity+and+millionfold+selectivity.">A catalytic beacon sensor for uranium with parts-per-trillion sensitivity and millionfold selectivity.</a> Proc. Natl. Acad. Sci. USA. 104, 2056-2061 (2007).</li><li>Huang, P. J., Vazin, M., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+selection+of+a+new+lanthanide-dependent+DNAzyme+for+ratiometric+sensing+lanthanides.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+fluorescent+sensors+engineered+with+catalytic+DNA+'MgZ'+based+on+a+non-classic+allosteric+design.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+personal+glucose+meters+and+functional+DNA+sensors+to+quantify+a+variety+of+analytical+targets.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+DNA+enzyme-based+fluorescent+assay+for+bacterial+detection.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Bacteria+Using+Fluorogenic+DNAzymes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+catalytic+DNA+activated+by+a+specific+strain+of+bacterial+pathogen.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+specific+recognition+of+breast+tumors+by+an+RNA-cleaving+fluorogenic+DNAzyme+probe.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoelectric+point+of+crystalline+urease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=70+Years+of+crystalline+urease:+What+have+we+learned.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+platform+for+Real-Time+PCR+detection+of+Salmonella+spp.,+Listeria+monocytogenes+and+Escherichia+coli+O157+in+milk.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+sample+preparation+method+for+PCR-based+detection+of+Escherichia+coli+O157:H7+in+ground+beef.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+fluorogenic+real-time+PCR+for+detection+and+quantification+of+Escherichia+coli+O157:H7+in+dairy+wastewater+wetlands.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+microsphere+coagulation+ELISA+for+Escherichia+coli+O157:H7+using+Russell's+viper+venom.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology+for+detection+and+typing+of+foodborne+microorganisms.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+conventional+detection+and+enumeration+methods+for+pathogenic+bacteria+in+food.">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 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.

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 ...

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

Colorimetric Detection of Bacteria Using 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.

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-bacteria-using-litmus-test

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Biology

9.1K Views.  McMaster University. 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. 

Video: Colorimetric Detection of Bacteria Using Litmus Test

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

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 egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

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 ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

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

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.

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

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

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

Detection of Tissue-resident Bacteria in Bladder Biopsies by 16S rRNA Fluorescence In Situ Hybridization

Visualization of the interaction of bacteria with host mucosal surfaces and tissues can provide valuable insight into mechanisms of pathogenesis. While visualization of bacterial pathogens in animal models of infection can rely on bacterial strains engineered to express fluorescent proteins such as GFP, visualization of bacteria within the mucosa of biopsies or tissue obtained from human patients requires an unbiased method. Here, we describe an efficient method for the detection of tissue-associated bacteria in human biopsy sections. This method utilizes fluorescent in situ hybridization (FISH) with a fluorescently labeled universal oligonucleotide probe for 16S rRNA to label tissue-associated bacteria within bladder biopsy sections acquired from patients suffering from recurrent urinary tract infection. Through use of a universal 16S rRNA probe, bacteria can be detected without prior knowledge of species, genera, or biochemical characteristics, such as lipopolysaccharide (LPS), that would be required for detection by immunofluorescence experiments. We describe a complete protocol for 16S rRNA FISH from biopsy fixation to imaging by confocal microscopy. This protocol can be adapted for use in almost any type of tissue and represents a powerful tool for the unbiased visualization of clinically-relevant bacterial-host interactions in patient tissue. Furthermore, using species or genera-specific probes, this protocol can be adapted for the detection of specific bacterial pathogens within patient tissue.

This protocol is for the unbiased detection of tissue-associated bacteria in patient biopsies by 16S rRNA in situ hybridization and confocal microscopy.

Visualization of the interaction of bacteria with host mucosal surfaces and tissues can provide valuable insight into mechanisms of pathogenesis. While ...