scholarly journals Detection of Escherichia coli O157:H7 Using Automated Immunomagnetic Separation and Enzyme-Based Colorimetric Assay

Sensors ◽  
2020 ◽  
Vol 20 (5) ◽  
pp. 1395 ◽  
Author(s):  
Ji Young Park ◽  
Kisang Park ◽  
Gyeongsik Ok ◽  
Hyun-Joo Chang ◽  
Tae Jung Park ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Colorimetric Assay ◽  
Colorimetric Detection ◽  
Enzymatic Reaction ◽  
Immunomagnetic Separation ◽  
Food Samples ◽  
Detection Methods ◽  
Immunomagnetic Beads ◽  
E Coli ◽  
Chlorophenol Red

The food industry requires rapid and simple detection methods for preventing harm from pathogenic bacteria. Until now, various technologies used to detect foodborne bacteria were time-consuming and laborious. Therefore, we have developed an automated immunomagnetic separation combined with a colorimetric assay for the rapid detection of E. coli O157:H7 in food samples. The colorimetric detection method using enzymatic reaction is fascinating because of its simplicity and rapidity and does not need sophisticated devices. Moreover, the proposed procedures for the detection of bacteria in food take less than 3 h including pre-enrichment, separation and detection steps. First, target-specific immunomagnetic beads were introduced to contaminated milk in a pre-enrichment step. Second, the pre-enriched sample solution containing target bacteria bound on immunomagnetic beads was injected into an automated pretreatment system. Subsequently, the immunomagnetic beads along with target bacteria were separated and concentrated into a recovery tube. Finally, released β-galactosidase from E. coli O157:H7 after lysis was reacted with chlorophenol red β-galactopyranoside (CPRG) used as a substrate and the colorimetric change of CPRG was determined by absorbance measuring or the naked eye. By the proposed approach in this study, we could detect 3 × 102 CFU/mL of E. coli O157:H7 from a milk sample within 3 h.

Immunomagnetic Separation in Microchannels: From MEMS to BioNEMS

2005 ◽  
Author(s):  
Ashok Sinha ◽  
Ranjan Ganguly ◽  
Ishwar K. Puri
Keyword(s):  
Pathogenic Bacteria ◽  
Pathogen Detection ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Contaminated Water ◽  
Selective Detection ◽  
Bacterial Detection ◽  
E Coli ◽  
Biological Environment ◽  
Innovative Concept

Conventional methods of monitoring and testing water quality involve collection of the sample to be tested and its subsequent analysis in a research laboratory for which some procedures may not be feasible or even accessible under certain field situations. Therefore, next generation sensors are required. Herein, an innovative concept that combines a micromixer and microparticle trap is proposed that should enable more rapid pathogen detection in contaminated water. In it, immunomagnetic separation (a procedure [1,2] that is well practiced in the field of immunochemistry) is scaled down from the benchtop to the microscale. Our design is generic, i.e., design is not limited to the detection of waterborne biological agents, but can be used for other forms of chemical analysis. Testing for waterborne bacteria requires analysis methods that must meet a number of challenging criteria. Time and sensitivity of analysis are the more important limitations. Bacterial detection methods have to be rapid and very sensitive since the presence of even a small pathogenic sample may sometimes constitute an infectious or otherwise harmful dose. Selective detection is also required because small numbers of pathogenic bacteria are often present in a complex biological environment along with many other nonpathogenic organisms. As an example, the infectious dosage of a pathogen such as E. coli O157:H7 or Salmonella is as low as 10 cells and the existing coliform standard for E. coli in water is 4 cells: 100 ml [3].

scholarly journals Paper-based analytical devices for colorimetric detection of S. aureus and E. coli and their antibiotic resistant strains in milk

The Analyst ◽  
2020 ◽  
Vol 145 (22) ◽  
pp. 7320-7329
Author(s):  
Muhammad Asif ◽  
Fazli Rabbi Awan ◽  
Qaiser Mahmood Khan ◽  
Bongkot Ngamsom ◽  
Nicole Pamme
Keyword(s):  
Pathogenic Bacteria ◽  
Colorimetric Detection ◽  
Antibiotic Resistant ◽  
Beta Lactamases ◽  
E Coli ◽  
Milk Samples ◽  
Extended Spectrum Beta Lactamases ◽  
Paper Microfluidic ◽  
Resistant Strains ◽  
Appropriate Prescription

We investigate paper microfluidic devices for detection of pathogenic bacteria and their sensitivity towards β-lactamase and Extended Spectrum Beta Lactamases (ESBLs) in milk samples to enable appropriate prescription of antibiotics for mastitis.

scholarly journals P1 Trisaccharide (Galα1,4Galβ1,4GlcNAc) Synthesis by Enzyme Glycosylation Reactions Using Recombinant Escherichia coli

2003 ◽  
Vol 69 (4) ◽  
pp. 2110-2115 ◽  
Author(s):  
Ziye Liu ◽  
Yuquan Lu ◽  
Jianbo Zhang ◽  
Keith Pardee ◽  
Peng George Wang
Keyword(s):  
Escherichia Coli ◽  
Sucrose Synthase ◽  
Pathogenic Bacteria ◽  
Enzymatic Reaction ◽  
Genetically Engineered ◽  
Reaction Volume ◽  
Recombinant Bacteria ◽  
Preparative Scale ◽  
E Coli ◽  
Oligosaccharide Moiety

ABSTRACT The frequency of Escherichia coli infection has lead to concerns over pathogenic bacteria in our food supply and a demand for therapeutics. Glycolipids on gut cells serve as receptors for the Shiga-like toxin produced by E. coli. Oligosaccharide moiety analogues of these glycolipids can compete with receptors for the toxin, thus acting as antibacterials. An enzymatic synthesis of the P1 trisaccharide (Galα1,4Galβ1,4GlcNAc), one of the oligosaccharide analogues, was assessed in this study. In the proposed synthetic pathway, UDP-glucose was generated from sucrose with an Anabaena sp. sucrose synthase and then converted with an E. coli UDP-glucose 4-epimerase to UDP-galactose. Two molecules of galactose were linked to N-acetylglucosamine subsequently with a Helicobacter pylori β-l,4-galactosyltransferase and a Neisseria meningitidis α-1,4-galactosyltransferase to produce one molecule of P1 trisaccharide. The four enzymes were coexpressed in a single genetically engineered E. coli strain that was then permeabilized and used to catalyze the enzymatic reaction. P1 trisaccharide was accumulated up to 50 mM (5.4 g in a 200-ml reaction volume), with a 67% yield based on the consumption of N-acetylglucosamine. This study provides an efficient approach for the preparative-scale synthesis of P1 trisaccharide with recombinant bacteria.

Relative Sensitivity of Escherichia coli O157 Detection from Bovine Feces and Rectoanal Mucosal Swabs

2014 ◽  
Vol 77 (6) ◽  
pp. 972-976 ◽  
Author(s):  
K. J. WILLIAMS ◽  
M. P. WARD ◽  
O. DHUNGYEL ◽  
L. VAN BREDA
Keyword(s):  
Escherichia Coli ◽  
Confidence Interval ◽  
Human Health Risk ◽  
Relative Sensitivity ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Escherichia Coli O157 ◽  
Prevalence Studies ◽  
E Coli ◽  
The Difference

The need to quantify the potential human health risk posed by the bovine reservoir of Escherichia coli O157 has led to a wealth of prevalence studies and improvements in detection methods over the last two decades. Rectoanal mucosal swabs have been used for the detection of E. coli O157 fecal shedding, colonized animals, and those predisposed to super shedding. We conducted a longitudinal study to compare the detection of E. coli O157 from feces and rectoanal mucosal swabs (RAMS) from a cohort of dairy heifers. We collected 820 samples that were tested by immunomagnetic separation of both feces and RAMS. Of these, 132 were detected as positive for E. coli O157 from both samples, 66 were detected as positive from RAMS only, and 117 were detected as positive from feces only. The difference in results between the two sample types was statistically significant (P < 0.001). The relative sensitivities of detection by immunomagnetic separation were 53% (confidence interval, 46.6 to 59.3) from RAMS and 67% (confidence interval, 59.6 to 73.1) from fecal samples. No association between long-term shedding (P = 0.685) or super shedding (P = 0.526) and detection by RAMS only was observed.

scholarly journals Evaluation of test-kits for the detection of Escherichia coli O157 in raw meats and cattle faeces.

2012 ◽  
Vol 1 (2) ◽  
Author(s):  
Hilda Nyati ◽  
Annet Heuvelink ◽  
Caroliene Van Heerwaarden ◽  
Ans Zwartkruis
Keyword(s):  
Escherichia Coli ◽  
Real Time Pcr ◽  
Immunomagnetic Separation ◽  
Escherichia Coli O157 ◽  
Immunomagnetic Beads ◽  
Tryptone Soya Broth ◽  
Detection Rates ◽  
E Coli ◽  
Peptone Water ◽  
Cattle Faeces

Escherichia coli O157 detection limits in artificially contaminated beef and cattle faeces samples were determined using Dynabeads anti E. coli O157 immunomagnetic beads, VIDAS-UP, VIDAS-ICE, and real-time PCR (GeneDisc and LightCycler) systems. Dynabeads anti-E. coli O157 immunomagnetic separation (IMS) and the GeneDisc cycler were the most sensitive methods, and could detect an initial 1 CFU in 25g beef samples after 6h of incubation in modified tryptone soya broth with novobiocin (mTSB+n) or buffered peptone water (BPW). The VIDAS-UP method could detect an initial 10 CFU, while VIDAS-ICE and the LightCycler methods could only detect an initial 100 CFU. Higher detection rates were achieved with 18 hour incubations, where an initial 1 CFU in a 25g sample could be detected with all five methods. For cattle faeces enrichments, Dynabeads anti-E. coli O157 IMS could detect an initial 1 CFU after a 6 h incubation in mTSB+n, while the VIDAS-UP and VIDAS-ICE methods could detect an initial 10 CFU and both PCR methods could only detect an initial 100 CFU. Detection rates were lower in BPW, compared to mTSB+n, with thresholds of 100 CFU for VIDAS-ICE, VIDAS-UP and GeneDisc methods, and >100 CFU for the LightCycler method.

Simultaneous Immunomagnetic Separation Method for the Detection of Escherichia coli O26, O111, and O157 from Food Samples

2014 ◽  
Vol 77 (1) ◽  
pp. 15-22 ◽  
Author(s):  
MASASHI KANKI ◽  
KAZUKO SETO ◽  
YUKO KUMEDA
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Food Samples ◽  
Food Culture ◽  
Cell Detection ◽  
Labor Costs ◽  
Single Strain ◽  
E Coli ◽  
Detection Medium

We performed a simultaneous immunomagnetic separation (IMS) assay on Shiga toxin–producing Escherichia coli (STEC) serogroups O26, O111, and O157 with immunobeads coated with O26, O111, or O157 antibodies that were simultaneously added to an aliquot of food culture. We also compared the usefulness of CHROMagar STEC medium against various selective isolation agars designed to test for the three serogroups. Samples of sliced beef, ground beef, and radish sprout were artificially contaminated with STEC O26, O111, and O157 strains after incubation in enrichment broth and were subjected to conventional and simultaneous IMS assays. Simultaneous IMS did not affect the sensitivity of target cell detection. For STEC O26, O111, and O157 inoculated into the enriched samples of sliced beef and radish sprout, the detection ability of CHROMagar STEC was similar to or exceeded that of other isolation agars. However for STEC O157 inoculated into ground beef cultures, cefixime tellurite sorbitol MacConkey agar (CT-SMAC) was the superior detection medium. The performance of simultaneous IMS combined with CT-SMAC and CHROMagar STEC detection is similar to that of the Japanese standard method for isolating E. coli O26, O111, and O157. However, the proposed approach involves the same time, materials, and labor costs as the standard E. coli O157 reference procedure but allows detection of three E. coli serotypes rather than a single strain.

Limitations of Immunomagnetic Separation for Detection of the Top Seven Serogroups of Shiga Toxin–Producing Escherichia coli

2017 ◽  
Vol 80 (4) ◽  
pp. 598-603 ◽  
Author(s):  
J. Hallewell ◽  
T. Alexander ◽  
T. Reuter ◽  
K. Stanford
Keyword(s):  
Escherichia Coli ◽  
Quantitative Pcr ◽  
Foodborne Pathogens ◽  
Shiga Toxin ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Conventional Pcr ◽  
E Coli ◽  
Pure Cultures

ABSTRACT Shiga toxin–producing Escherichia coli (STEC) strains are foodborne pathogens that negatively impact human health and compromise food safety. Serogroup O157 is the most frequently isolated and studied STEC serogroup, but six others (O26, O45, O103, O111, O121, and O145) have also been identified as significant sources of human disease and collectively have been referred to as the “top six” pathogenic serogroups. Because detection methods for non-O157 serogroups are not yet refined, the objective of this study was to compare the effectiveness of immunomagnetic separation (IMS) for recovery of serogroup O157 isolates with that for each of the top six E. coli serogroups in pure and mixed cultures of STEC at 103 to 107 CFU/mL. After serogroup-specific IMS, DNA was extracted from cultured isolates to analyze the specificity of each IMS assay using conventional and quantitative PCR. In pure cultures, DNA copy number obtained after IMS was lower for O111 and O157 (P < 0.01) than for other serogroups. Based on quantitative PCR (qPCR) analyses, specificity was reduced for all IMS assays when STEC isolates were mixed at 7 log CFU/mL, although the O157 IMS assays recovered only O157 over a wider range of concentrations than did assays for non-O157 serogroups. At the lowest dilution tested, conventional PCR was specific for all serogroups except O121 and O145. For these two serogroups, no dilution tested recovered only O121 or O145 when evaluated with conventional PCR. Refinements to IMS assays, development of selective media, and determination of optimal enrichment times to reduce background microflora or competition among serogroups would be especially beneficial for recovery of O111, O121, and O145 serogroups to improve STEC detection and isolation.

A Multiplex PCR for Rapid Identification of Shiga-Like Toxin-Producing Escherichia coli O157:H7 Isolated from Foods‡

1996 ◽  
Vol 59 (6) ◽  
pp. 570-576 ◽  
Author(s):  
MING Y. DENG ◽  
PINA M. FRATAMICO
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Dna Sequences ◽  
Bacterial Species ◽  
Colorimetric Detection ◽  
Food Samples ◽  
Enterohemorrhagic Escherichia Coli ◽  
Experimental Conditions ◽  
Serological Tests ◽  
E Coli

For rapid and specific identification of enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 isolated from food samples, experimental conditions for a multiplex polymerase chain reaction (PCR) were optimized and a multiple digoxigenin (DIG)-labeled oligonucleotide probe hybridization (DLOPH) assay was developed. A suspect colony from MacConkey sorbitol agar containing 5-bromo-4-chloro-3-indoxyl-β-d-glucuronide (MSA-BCIG) was used for the multiplex PCR. Three different DNA sequences of E. coli O157:H7 were amplified simultaneously in the PCR: a specific fragment of an attaching and effacing gene (eae gene), conserved sequences of Shiga-like toxins (SLT) I and II, and a fragment of the 60-MDa plasmid. The identities of PCR products were confirmed by hybridization using DIG-labeled internal oligonucleotide probes and colorimetric detection with anti-DIG Fab fragments conjugated to alkaline phosphatase. This method yielded positive results with all reference strains of EHEC serogroup O157, including serotypes O157:H7, O157:NM, and O157:H−, and negative results were obtained with all strains of nontoxigenic E. coli serogroup O157, other serotypes of E. coli, and other bacterial species. The detection limit of the method was 65 colony-forming units (CFU) of E. coli O157:H7. All 29 cultures of EHEC O157:H7 isolated from meat samples and identified by biochemical and serological tests were positive in the multiplex PCR. EHEC O157:H7 was identified from all of 70 experimentally inoculated ground beef and milk samples which had initial inocula of 4 to 9 CFU/g (ml) and were subjected to a 6-h enrichment culturing. The multiplex PCR procedure could be very useful for routine examinations of food samples for the presence of EHEC O157.

Evaluation of Culture- and PCR-Based Detection Methods for Escherichia coli O157:H7 in Inoculated Ground Beef†

2005 ◽  
Vol 68 (8) ◽  
pp. 1566-1574 ◽  
Author(s):  
TERRANCE M. ARTHUR ◽  
JOSEPH M. BOSILEVAC ◽  
XIANGWU NOU ◽  
MOHAMMAD KOOHMARAIE
Keyword(s):  
Escherichia Coli ◽  
False Positive ◽  
False Negative ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Perishable Product ◽  
Specificity And Sensitivity

Currently, several beef processors employ test-and-hold systems for increased quality control of ground beef. In such programs, each lot of product must be tested and found negative for Escherichia coli O157:H7 prior to release of the product into commerce. Optimization of three testing attributes (detection time, specificity, and sensitivity) is critical to the success of such strategies. Because ground beef is a highly perishable product, the testing methodology used must be as rapid as possible. The test also must have a low false-positive result rate so product is not needlessly discarded. False-negative results cannot be tolerated because they would allow contaminated product to be released and potentially cause disease. In this study, two culture-based and three PCR-based methods for detecting E. coli O157:H7 in ground beef were compared for their abilities to meet the above criteria. Ground beef samples were individually spiked with five genetically distinct strains of E. coli O157: H7 at concentrations of 17 and 1.7 CFU/65 g and then subjected to the various testing methodologies. There was no difference (P > 0.05) in the abilities of the PCR-based methods to detect E. coli O157:H7 inoculated in ground beef at 1.7 CFU/65 g. The culture-based systems detected more positive samples than did the PCR-based systems, but the detection times (21 to 48 h) were at least 9 h longer than those for the PCR-based methods (7.5 to 12 h). Ground beef samples were also spiked with potentially cross-reactive strains. The PCR-based systems that employed an immunomagnetic separation step prior to detection produced fewer false-positive results.

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