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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Colorimetric Detection of Bacteria Using Litmus Test

Published: September 17th, 2016

DOI:

10.3791/54546

1Department of Biochemistry and Biomedical Sciences, McMaster University, 2Department of Chemistry and Chemical Biology, McMaster University, 3Department of Medicine, 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.

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.

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 ....

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1. Preparation of Reagents and Buffers

  1. 0.5 M Ethylenediaminetetraacetic Acid (EDTA)
    1. 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 °C.
  2. 10× Tris-borate EDTA (10x TBE)
    1. In a 4 L plastic beaker, add 432 g Tris-base, 200 .......

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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 .......

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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.......

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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.

....

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

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