Evaluation of Three Commercially Available Enzyme-Linked Immunosorbent Assay Kits for Detection of Shiga Toxin

2009 ◽  
Vol 72 (4) ◽  
pp. 741-747 ◽  
Author(s):  
JOHN WILLFORD ◽  
KENNETH MILLS ◽  
LAWRENCE D. GOODRIDGE
Keyword(s):  
Escherichia Coli ◽  
Immunosorbent Assay ◽  
Shiga Toxin ◽  
Screening Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Microplate Assay ◽  
E Coli ◽  
Elisa Kit ◽  
Pure Cultures ◽  
Order Of Magnitude

Three commercially available Shiga toxin (Stx) enzyme-linked immunosorbent assay (ELISA) kits were evaluated for their ability to detect Stx in pure cultures of Stx-producing Escherichia coli (specificity). The detection limits (sensitivity) of each ELISA kit were also evaluated. Seventy-eight Stx-producing E. coli (STEC) isolates that produced Stx1, Stx2, or Stx1 and Stx2 variants were examined in this study. The specificities of the tests were comparable, and the sensitivities of two of the tests (Premier EHEC and rBiopharm Ridascreen Verotoxin Enzyme Immunoassay) were within the same order of magnitude. The ProSpecT Shiga Toxin E. coli Microplate Assay was approximately 10-fold less sensitive. The inability of all three tests to detect the Stx2d and Stx2e variants indicated that some STEC strains may not be detected by Stx ELISA. The ability of the Premier EHEC ELISA to detect toxin in artificially inoculated bovine fecal samples (following enrichment) indicated that this kit may be used to screen cattle for the presence of Stx as an indicator of the presence of STEC. In particular, such a screening method could be useful during the summer, when the number of STEC-positive animals and the number of STEC that they shed increase.

Use of a Shiga Toxin (Stx)-Enzyme-Linked Immunosorbent Assay and Immunoblot for Detection and Isolation of Stx-Producing Escherichia coli from Naturally Contaminated Beef

2000 ◽  
Vol 63 (9) ◽  
pp. 1167-1172 ◽  
Author(s):  
HEBA NASHED ATALLA ◽  
ROGER JOHNSON ◽  
SCOTT MCEWEN ◽  
R. W. USBORNE ◽  
C. L. GYLES
Keyword(s):  
Escherichia Coli ◽  
Vero Cell ◽  
Immunosorbent Assay ◽  
Shiga Toxin ◽  
Total Coliform ◽  
Complete Agreement ◽  
Enzyme Linked Immunosorbent Assay ◽  
Spearman Correlation ◽  
Southern Ontario ◽  
E Coli

The purpose of this study was to evaluate an enzyme-linked immunosorbent assay (ELISA) and an immunoblot procedure for detection and isolation of Shiga toxin-producing Escherichia coli (STEC) from beef, and to correlate the presence of STEC in beef with E. coli and total coliform counts. A total of 120 samples of boneless beef supplied to a meat processor in southern Ontario were tested for the presence of STEC, E. coli, and total coliforms. Following enrichment in modified tryptic soy broth, samples were screened for Shiga toxin (Stx) by a Stx-ELISA and a Vero cell assay (VCA). Samples that were positive in the Stx-ELISA were subjected to the Stx-immunoblot for STEC isolation. Overall, 33.3% of samples were positive in the VCA, and 34.2% were positive in the Stx-ELISA. There was almost complete agreement between the Stx-ELISA and the VCA results (kappa = 0.98). The sensitivity and specificity of the Stx-ELISA with respect to the VCA were 100% and 98.75%, respectively. STEC were isolated by the Stx-immunoblot from 87.8% of the samples that were positive in the Stx-ELISA. The STEC isolates belonged to 19 serotypes, with serotype O113:H21 accounting for 10 of 41 isolates. No STEC of serotype O157:H7 were isolated. There was a significant correlation between E. coli counts and total coliform counts (Spearman correlation coefficient = 0.68, P < 0.01). The E. coli count was positively correlated with detection of STEC by both the Stx-ELISA and the VCA (P < 0.01).

Use of the ecf1 Gene To Detect Shiga Toxin–Producing Escherichia coli in Beef Samples

2015 ◽  
Vol 78 (4) ◽  
pp. 675-684 ◽  
Author(s):  
KRISTIN W. LIVEZEY ◽  
BETTINA GROSCHEL ◽  
MICHAEL M. BECKER
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
False Positive ◽  
Screening Method ◽  
Severe Illness ◽  
Specific Gene ◽  
E Coli ◽  
Detection Assay ◽  
Meat Samples ◽  
Inspection Service

Escherichia coli O157:H7 and six serovars (O26, O103, O121, O111, O145, and O45) are frequently implicated in severe clinical illness worldwide. Standard testing methods using stx, eae, and O serogroup–specific gene sequences for detecting the top six non-O157 STEC bear the disadvantage that these genes may reside, independently, in different nonpathogenic organisms, leading to false-positive results. The ecf operon has previously been identified in the large enterohemolysin-encoding plasmid of eae-positive Shiga toxin–producing E. coli (STEC). Here, we explored the utility of the ecf operon as a single marker to detect eae-positive STEC from pure broth and primary meat enrichments. Analysis of 501 E. coli isolates demonstrated a strong correlation (99.6%) between the presence of the ecf1 gene and the combined presence of stx, eae, and ehxA genes. Two large studies were carried out to determine the utility of an ecf1 detection assay to detect non-O157 STEC strains in enriched meat samples in comparison to the results using the U.S. Department of Agriculture Food Safety and Inspection Service (FSIS) method that detects stx and eae genes. In ground beef samples (n = 1,065), the top six non-O157 STEC were detected in 4.0% of samples by an ecf1 detection assay and in 5.0% of samples by the stx- and eae-based method. In contrast, in beef samples composed largely of trim (n = 1,097), the top six non-O157 STEC were detected at 1.1% by both methods. Estimation of false-positive rates among the top six non-O157 STEC revealed a lower rate using the ecf1 detection method (0.5%) than using the eae and stx screening method (1.1%). Additionally, the ecf1 detection assay detected STEC strains associated with severe illness that are not included in the FSIS regulatory definition of adulterant STEC.

scholarly journals Enzyme-linked immunosorbent assay for detection of Escherichia coli heat-labile enterotoxin

1977 ◽  
Vol 6 (5) ◽  
pp. 439-444 ◽  
Author(s):  
R H Yolken ◽  
H B Greenberg ◽  
M H Merson ◽  
R B Sack ◽  
A Z Kapikian
Keyword(s):  
Escherichia Coli ◽  
Immunosorbent Assay ◽  
Radioactive Isotope ◽  
Enzyme Linked Immunosorbent Assay ◽  
Adrenal Cell ◽  
Elisa System ◽  
Culture Techniques ◽  
E Coli ◽  
Heat Labile ◽  
Tissue Culture Techniques

The development of an enzyme-linked immunosorbent assay (ELISA) for the detection of heat-labile Escherichia coli enterotoxin is described. The assay, which is based on the immunological similarity between Vibrio cholerae toxin and heat-labile E. coli enterotoxin, is similar in design to a radioimmunoassay but utilizes enzyme-labeled rather than radioactive isotope-labeled reagents. The ELISA system is as sensitive as both radioimmunoassay and the y-1 adrenal cell assay for the detection of heat-labile E. coli enterotoxin but requires neither radioactive reagents nor tissue culture techniques. The ELISA is easy to perform and is adaptable for use in small laboratories.

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.

Comparison of the Presence of Shiga Toxin 1 in Food Matrices as Determined by an Enzyme-Linked Immunosorbent Assay and a Biological Activity Assay

2012 ◽  
Vol 75 (6) ◽  
pp. 1036-1042 ◽  
Author(s):  
STEPHEN E. LUMOR ◽  
NEAL R. FREDRICKSON ◽  
IAN RONNINGEN ◽  
BRONWYN D. DEEN ◽  
KENNETH SMITH ◽  
...  
Keyword(s):  
Biological Activity ◽  
Immunosorbent Assay ◽  
Shiga Toxin ◽  
Orange Juice ◽  
Enzyme Linked Immunosorbent Assay ◽  
Activity Assay ◽  
Elisa Kit ◽  
Residual Toxicity ◽  
Highly Correlated ◽  
Phosphate Buffered Saline

This study was conducted to compare the identification of Shiga toxin 1 (Stx1) based on its specific biological activity and based on results of a commercial enzyme-linked immunosorbent assay (ELISA) kit. Stx1 was thermally treated for various periods in phosphate-buffered saline, milk, and orange juice. The residual Stx1 concentration was determined with the commercial ELISA kit, and its residual enzymatic activity (amount of adenine released from a 2,551-bp DNA substrate) was determined with a biological activity assay (BAA). Regression analysis indicated that the inactivation of Stx1 as a function of time followed first-order kinetics. The half-lives determined at 60, 65, 70, 75, 80, and 85°C were 9.96, 3.19, 2.67, 0.72, 0.47, and 0.29 min, respectively, using the BAA. The half-lives determined by the ELISA with thermal treatments at 70, 75, 80, and 85°C were 40.47, 11.03, 3.64, and 1.40 min, respectively. The Z, Q10, and Arrhenius activation energy values derived by both assays were dissimilar, indicating that the rate of inactivation of the active site of Stx1 was less sensitive to temperature change than was denaturation of the epitope(s) used in the ELISA. These values were 10.28°C and 9.40 and 54.70 kcal/mol, respectively, with the ELISA and 16°C and 4.11 and 34 kcal/mol, respectively, with the BAA. Orange juice enhanced Stx1 inactivation as a function of increasing temperature, whereas inactivation in 2% milk was not very much different from that in phosphate-buffered saline. Our investigation indicates that the ELISA would be a reliable method for detecting the residual toxicity of heat-treated Stx1 because the half-lives determined with the ELISA were greater than those determined with the BAA (faster degradation) at all temperatures and were highly correlated (R2 = 0.994) with those determined with the BAA.

scholarly journals Enhancement of Susceptibility to Shiga Toxin-Producing Escherichia coli O157:H7 by Protein Calorie Malnutrition in Mice

1998 ◽  
Vol 66 (4) ◽  
pp. 1726-1734 ◽  
Author(s):  
Takaaki Kurioka ◽  
Yoshihisa Yunou ◽  
Eiji Kita
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Protein Diet ◽  
Immunocytochemical Staining ◽  
Enzyme Linked Immunosorbent Assay ◽  
Renal Tubules ◽  
Infected Control ◽  
E Coli ◽  
Intestinal Epithelia ◽  
Protein Calorie Malnutrition

ABSTRACT Infection with Shiga toxin (Stx)-producing enterohemorrhagicEscherichia coli is increasing among children. In this study, 5-week-old C57BL/6 mice with protein calorie malnutrition (PCM) that had been fed a 5% protein diet for 2 weeks since ablactation were inoculated intragastrically with 2 × 106 CFU of Stx-producing E. coli O157:H7. More than 75% of infected mice with PCM died by 10 days postinfection. Infected mice with PCM developed neurologic symptoms 5 days after infection, while well-nourished control mice receiving a 25% protein diet did not. In the intestinal tracts of infected mice with PCM, inoculated E. coli O157:H7 multiplied between days 2 and 4 of infection, with a peak of growth at day 4. Although the pathogens were not culturable from the stool after day 7, O157 lipopolysaccharide was detectable in the stool by enzyme-linked immunosorbent assay even after day 8. Stx was detectable in the stool after day 2 of infection and increased in proportion to the growth of inoculated organisms. The maximal production of Stx occurred at 4 days postchallenge, and Stx was detectable in the blood on days 3 to 5. In contrast, well-nourished control mice survived the infection, and all of them remained well even after 3 weeks of infection. In these control mice, inoculated E. coli O157:H7 disappeared from the stool before day 3. Stx was not detectable in the stool and blood of infected control mice at any time from day 1 through day 8. Histologically, cerebral hemorrhages seemed to be the cause of acute death of infected mice with PCM. Immunocytochemical staining demonstrated the positive immunoreaction to Stx at the alveus and stratum pyramidale of the hippocampus and in renal tubules of infected malnourished mice. Such immunoreactions were not found in tissues from infected control mice. Histological study of the intestinal epithelium before infection showed that PCM severely affected the development of intestinal epithelia. These findings strongly indicate that PCM-induced nondevelopment of intestinal physical barrier is one of the predisposing factors for infection with Stx-producing E. coli O157:H7 in mice and suggest that our mouse model may explain the high incidence of infection with Stx-producing E. coli O157:H7 in the children whose intestinal epithelia have not yet completely developed.

scholarly journals Evaluation of home-mode ELISA system for thedetection of antibodies against Escherichia coli O157:H7 using purified lipopolysaccharide

2008 ◽  
Vol 5 (4) ◽  
pp. 563-568
Author(s):  
Baghdad Science Journal
Keyword(s):  
Escherichia Coli ◽  
Immunosorbent Assay ◽  
Gel Filtration ◽  
Laboratory Animals ◽  
Partial Purification ◽  
Nucleic Acid Content ◽  
Enzyme Linked Immunosorbent Assay ◽  
Biochemical Basis ◽  
Elisa System ◽  
E Coli

An enzyme linked immunosorbent assay (ELISA) for the detection and quantitation of human immunoglobulin G (IgG) antibodies against vero- cytotoxine (VT) producing Escherichia coli serogroup O157:H7 was produced. E. coli O157: H7 lipopolysaccharide was extracted from locally isolated strains by using hot phenol- water method, followed by partial purification using gel filtration chromatography by sepharose- 4B. The purity of the lipopolysaccharide was checked by measuring the protein and nucleic acid content and then used as antigen. Four isolates of vero- cytotoxin producing E. coli serogroup O157:H7 was obtained by culturing 350 stool samples from children suffering from bloody diarrhea. These isolates were identified on bacteriological, serological and biochemical basis. Toxin production was confirmed on laboratory animals as well as by cytopathic effect on tissue culture. The possibility of using E. coli O157:H7 lipopolysaccharide in an enzyme- linked immunosorbent assay for the routine diagnostic testing of serum from patients for evidence of O157:H7 infection is discussed.

Spiral Plating Method To Quantify the Six Major Non-O157 Escherichia coli Serogroups in Cattle Feces

2017 ◽  
Vol 80 (5) ◽  
pp. 848-856 ◽  
Author(s):  
Pragathi B. Shridhar ◽  
Lance W. Noll ◽  
Charley A. Cull ◽  
Xiaorong Shi ◽  
Natalia Cernicchiaro ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Foodborne Illnesses ◽  
Fecal Samples ◽  
Fecal Shedding ◽  
E Coli ◽  
Cattle Feces ◽  
Plating Method ◽  
Pure Cultures ◽  
High Concentrations

ABSTRACT Cattle are a major reservoir of the six major Shiga toxin–producing non-O157 Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) responsible for foodborne illnesses in humans. Besides prevalence in feces, the concentrations of STEC in cattle feces play a major role in their transmission dynamics. A subset of cattle, referred to as super shedders, shed E. coli O157 at high concentrations (≥4 log CFU/g of feces). It is not known whether a similar pattern of fecal shedding exists for non-O157. Our objectives were to initially validate the spiral plating method to quantify the six non-O157 E. coli serogroups with pure cultures and culture-spiked fecal samples and then determine the applicability of the method and compare it with multiplex quantitative PCR (mqPCR) assays for the quantification of the six non-O157 E. coli serogroups in cattle fecal samples collected from commercial feedlots. Quantification limits of the spiral plating method were 3 log, 3 to 4 log, and 3 to 5 log CFU/mL or CFU/g for individual cultures, pooled pure cultures, and cattle fecal samples spiked with pooled pure cultures, respectively. Of the 1,152 cattle fecal samples tested from eight commercial feedlots, 122 (10.6%) and 320 (27.8%) harbored concentrations ≥4 log CFU/g of one or more of the six serogroups of non-O157 by spiral plating and mqPCR methods, respectively. A majority of quantifiable samples, detected by either spiral plating (135 of 137, 98.5%) or mqPCR (239 of 320, 74.7%), were shedding only one serogroup. Only one of the quantifiable samples was positive for a serogroup carrying Shiga toxin (stx1) and intimin (eae) genes; 38 samples were positive for serogroups carrying the intimin gene. In conclusion, the spiral plating method can be used to quantify non-O157 serogroups in cattle feces, and our study identified a subset of cattle that was super shedders of non-O157 E. coli. The method has the advantage of quantifying non-O157 STEC, unlike mqPCR that quantifies serogroups only.

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