Colorimetric Detection of Bacteria Using Litmus Test

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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 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.....

Results

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 .......

Discussion

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.......

Materials

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