A hydrogel based rapid test method for detection of Escherichia coli (E. coli) in contaminated water samples (original) (raw)
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DipTest: A litmus test for E. coli detection in water
PLOS ONE
We have developed a new litmus paper test (DipTest) for detecting Escherichia coli (E. coli) in water samples by performing enzymatic reactions directly on the porous paper substrate. The paper strip consists of a long narrow piece of cellulose blotting paper coated with chemoattractant (at bottom edge), wax hydrophobic barrier (at the top edge), and custom formulated chemical reagents (at reaction zone immediately below the wax hydrophobic barrier). When the paper strip is dipped in water, E. coli in the water sample is attracted toward the paper strip due to a chemotaxic mechanism followed by the ascent along the paper strip toward the reaction zone due to a capillary wicking mechanism, and finally the capillary motion is arrested at the top edge of the paper strip by the hydrophobic barrier. The E. coli concentrated at the reaction zone of the paper strip will react with custom formulated chemical reagents to produce a pinkish-red color. Such a color change on the paper strip when dipped into water samples indicates the presence of E. coli contamination in potable water. The performance of the DipTest device has been checked with different known concentrations of E. coli contaminated water samples using different dip and wait times. The DipTest device has also been tested with different interfering bacteria and chemical contaminants. It has been observed that the different interfering contaminants do not have any impact on the DipTest, and it can become a potential solution for screening water samples for E. coli contamination at the point of source.
The Detection Method ofEscherichia coliin Water Resources: A Review
Journal of physics, 2018
This article reviews several approaches for Escherichia coli (E. coli) bacteria detection from conventional methods, emerging method and goes to biosensor-based techniques. Detection and enumeration of E. coli bacteria usually required long duration of time in obtaining the result since laboratory-based approach is normally used in its assessment. It requires 24 hours to 72 hours after sampling to process the culturing samples before results are available. Although faster technique for detecting E. coli in water such as Polymerase Chain Reaction (PCR) and Enzyme-Linked Immunosorbent Assay (ELISA) have been developed, it still required transporting the samples from water resources to the laboratory, high-cost, complicated equipment usage, complex procedures, as well as the requirement of skilled specialist to cope with the complexity which limit their wide spread practice in water quality detection. Recently, development of biosensor device that is easy to perform, portable, highly sensitive and selective becomes indispensable in detecting extremely lower consolidation of pathogenic E. coli bacteria in water samples.
Applied and Environmental Microbiology, 2011
In this work, we used a rapid, simple, and efficient concentration-and-recovery procedure combined with a DNA enrichment method (dubbed CRENAME [concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment]), that we coupled to an Escherichia coli/Shigella-specific real-time PCR (rtPCR) assay targeting the tuf gene, to sensitively detect E. coli/Shigella in water. This integrated method was compared to U.S. Environmental Protection Agency (EPA) culture-based Method 1604 on MI agar in terms of analytical specificity, ubiquity, detection limit, and rapidity. None of the 179 non-E. coli/Shigella strains tested was detected by both methods, with the exception of Escherichia fergusonii, which was detected by the CRENAME procedure combined with the E. coli/Shigella-specific rtPCR assay (CRENAME ؉ E. coli rtPCR). DNA from all 90 E. coli/Shigella strains tested was amplified by the CRENAME ؉ E. coli rtPCR, whereas the MI agar method had limited ubiquity and detected only 65 (72.2%) of the 90 strains tested. In less than 5 h, the CRENAME ؉ E. coli rtPCR method detected 1.8 E. coli/Shigella CFU whereas the MI agar method detected 1.2 CFU/100 ml of water in 24 h (95% confidence). Consequently, the CRENAME method provides an easy and efficient approach to detect as little as one Gram-negative E. coli/Shigella cell present in a 100-ml potable water sample. Coupled with an E. coli/Shigella-specific rtPCR assay, the entire molecular procedure is comparable to U.S. EPA Method 1604 on MI agar in terms of analytical specificity and detection limit but provides significant advantages in terms of speed and ubiquity.
Comparison of three different media for the detection of E. coli and coliforms in water
Water Science & Technology, 2006
The European Drinking Water Directive defines reference methods for the enumeration of microbiological parameters in drinking water. The method to be used for Escherichia coli and coliforms is the membrane filtration technique on Lactose TTC agar with Tergitol 7. Many technical drawbacks of the procedure, as well as its limitations regarding the recent taxonomy of coliforms, make it necessary to evaluate alternative methods. Two alternative assays, a chromogenic media (m-ColiBlu24 w ) and a defined substrate technology-DST test (Colilert 18/Quanty Traye) were compared with the ISO standard with attention to the phenotypic characteristic of the isolates. Results showed that the ISO method failed to detect an important percentage of coliforms and E. coli while m-ColiBlu24 w and Colilert 18 provided results in a shorter time allowing the simultaneous detection of E. coli and coliforms with no further confirmation steps.
On the track for an efficient detection of Escherichia coli in water: A review on PCR-based methods
Ecotoxicology and Environmental Safety, 2015
Ensuring water safety is an ongoing challenge to public health providers. Assessing the presence of faecal contamination indicators in water is essential to protect public health from diseases caused by waterborne pathogens. For this purpose, the bacteria Escherichia coli has been used as the most reliable indicator of faecal contamination in water. The methods currently in use for monitoring the microbiological safety of water are based on culturing the microorganisms. However, these methods are not the desirable solution to prevent outbreaks as they provide the results with a considerable delay, lacking on specificity and sensitivity. Moreover, viable but non-culturable microorganisms, which may be present as a result of environmental stress or water treatment processes, are not detected by culture-based methods and, thus, may result in false-negative assessments of E. coli in water samples. These limitations may place public health at significant risk, leading to substantial monetary losses in health care and, additionally, in costs related with a reduced productivity in the area affected by the outbreak, and in costs supported by the water quality control departments involved. Molecular methods, particularly polymerase chain reaction-based methods, have been studied as an alternative technology to overcome the current limitations, as they offer the possibility to reduce the assay time, to improve the detection sensitivity and specificity, and to identify multiple targets and pathogens, including new or emerging strains. The variety of techniques and applications available for PCR-based methods has increased considerably and the costs involved have been substantially reduced, which together have contributed to the potential standardization of these 3 techniques. However, they still require further refinement in order to be standardized and applied to the variety of environmental waters and their specific characteristics. The PCR-based methods under development for monitoring the presence of E. coli in water are here discussed. Special emphasis is given to methodologies that avoid preenrichment during the water sample preparation process so that the assay time is reduced and the required legislated sensitivity is achieved. The advantages and limitations of these methods are also reviewed, contributing to a more comprehensive overview toward a more conscious research in identifying E. coli in water.
Indirect detection of Escherichia coli and total coliforms can be based on the enzymatic activities of β-glucuronidase (β-glu) and β-galactosidase (β-gal). These enzymes utilize the substrates anthracene-β-d-glucuronide and pyrene d-galactopyranoside, respectively. Substrate cleavage by the enzyme releases the soluble fluorescent molecules 2-hydroxyanthracene and 1-hydroxypyrene, which can then be detected by a fluorometer. The Pathogen Detect® system is an automated portable unit that can measure fluorescent enzyme products. In this report, we investigated the utility of the Pathogen Detect® system for potential automation of water quality monitoring. The PDS unit has the ability to detect E. coli, mean 14.7 h at a standard deviation of 1.5, when the sample mean is 9.1 cells in 100 mL with a standard deviation of 12.6. Similarly, total coliforms may be detected at mean 14.7 h with a standard deviation of 1.4 when the sample mean is 59.6 cells in 100 mL, with a standard deviation of 144.5. The PDS unit has the ability to detect single cells of either total coliforms or E. coli in 100 mL water sample within 18 hours. Turbidity and color of water samples have no impact on the detection of E. coli and total coliforms.
A biosensor platform for rapid detection of E. coli in drinking water
Enzyme and microbial technology, 2016
The need for rapid, specific and sensitive assays that provide a detection of bacterial indicators are important for monitoring water quality. Rapid detection using biosensor is a novel approach for microbiological testing applications. Besides, validation of rapid methods is an obstacle in adoption of such new bio-sensing technologies. In this study, the strategy developed is based on using the compound 4methylumbelliferyl glucuronide (MUG), which is hydrolyzed rapidly by the action of E. coli β-D-glucuronidase (GUD) enzyme to yield a fluorogenic product that can be quantified and directly related to the number of E. coli cells present in water samples. The detection time required for the biosensor response ranged from 30 to 120 minutes, depending on the number of bacteria. The specificity of the MUG based biosensor platform assay for the detection of E. coli was examined by pure cultures of non-target bacterial genera and also non-target substrates. GUD activity was found to be specific for E. coli and no such enzymatic activity was detected in other species. Moreover, the sensitivity of rapid enzymatic assays was investigated and repeatedly determined to be less than 10 E. coli cells per reaction vial concentrated from 100 mL of water samples. The applicability of the method was tested by performing fluorescence assays under pure and mixed bacterial flora in environmental samples. In addition, the procedural QA/QC for routine monitoring of drinking water samples have been validated by comparing the performance of the biosensor platform for the detection of E. coli and culture-based standard techniques such as Membrane Filtration (MF). The results of this study Absar Alum and Valerie Stout for serving on my committee.
Advances in Microbiology, 2012
The aims of this study were to evaluate two methods, qPCR and a chemiluminescent assay (ColiLight II), for rapid detection of E. coli in water, and to examine the survival and persistence of clinical E. coli in drinking water and biofilm using qPCR and ColiLight II. qPCR and ColiLight II were compared with a cultivation-based method (MPN), and survival and persistence of four clinical E. coli strains in water and biofilms on stainless steel (SS) and polyethylene (PE) surfaces were studied in a flow-through reactor with non-disinfected drinking water using ColiLight II, qPCR, ATP bioluminescence, and MPN. ColiLight II and qPCR correlated well with MPN. In drinking water, some clinical E. coli strains showed prolonged survival in drinking water flow-through systems, and persisted 3-3.4 times longer than the theoretical washout due to incorporation into biofilms. Strain specific attributes can significantly affect detection and persistence of E. coli in drinking water matrices.
Rapid enzymatic detection of Escherichia coli contamination in polluted river water
Letters in Applied Microbiology, 2001
The relationship between the rate of b-D D-glucuronidase hydrolysis (GLUase-HR) and the E. coli concentration in rivers differing in the extent of faecal pollution was investigated. It was hypothesized that the rate of GLUase-HR is a better surrogate parameter for E. coli concentrations than estimated numbers of faecal coliforms (FC). Methods and Results: The GLUase-HR of the water sample ®lter residues was determined as the rate of cleavage of 4-methylumbelliferyl-b-D D-glucuronide. FC and E. coli concentrations were enumerated using mFC and Chromocult Coliform agar, respectively. Regression analysis revealed that a 90% variation of the variable log GLUase-HR was directly related to the variable log E. coli concentrations. The observed relationship between the log of the FC count and the log of the GLUase activity could be explained by the hydrolysis activity of the E. coli population, as E. coli is a part of the FC group. Conclusions: The data suggest that the log of the GLUase-HR can be used as a surrogate parameter for the log of the E. coli concentrations. Signi®cance and Impact of the Study: GLUase-HR determination may provide a rapid alternative technique to estimate E. coli concentrations in freshwaters.
Rapid Detection of the Escherichia coli Genospecies in Water by Conventional and Real-Time PCR
Methods in Molecular Biology, 2012
The presence of Escherichia coli has long been established as the most reliable microbiological indication of fecal contamination in water. Current recommended culture-based methods for assessing water quality by the detection of E. coli are lengthy and lack ubiquity (ability to detect most if not all strains of a target microorganism). We describe rapid and sensitive conventional and real-time PCR assays speci fi c to E. coli and Shigella, based on the nucleotide sequence of the highly conserved elongation factor Tu ( tuf ) gene enabling the detection of all members of the genospecies.