scholarly journals Intimin Gene (eae) Subtype-Based Real-Time PCR Strategy for Specific Detection of Shiga Toxin-Producing Escherichia coli Serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 in Cattle Feces

2013 ◽  
Vol 80 (3) ◽  
pp. 1177-1184 ◽  
Author(s):  
Delphine Bibbal ◽  
Estelle Loukiadis ◽  
Monique Kérourédan ◽  
Carine Peytavin de Garam ◽  
Franck Ferré ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Specific Detection ◽  
Content Type ◽  
E Coli ◽  
Cattle Feces ◽  
Pcr Assays

ABSTRACTShiga toxin-producingEscherichia coli(STEC) strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are known to be associated with particular subtypes of the intimin gene (eae), namely, γ1, β1, ε, θ, and γ1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targetedeaesubtypes. The simultaneous presence ofstx,eae, and one of the five O group markers was found in 58.0% of the samples, and the five targetedstxpluseaeplus O genetic combinations were detected 143 times. However, taking into consideration the association betweeneaesubtypes and O group markers, the resultingstxpluseaesubtype plus O combinations were detected only 46 times. The 46 isolation assays performed allowed recovery of 22E. colistrains belonging to one of the five targeted STEC serogroups. In contrast, only 2 of 39 isolation assays performed on samples that were positive forstx,eaeand an O group marker, but that were negative for the correspondingeaesubtype, were successful. Characterization of the 24E. coliisolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11, and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenicE. coli(aEPEC). Finally, the more discriminatingeaesubtype-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces.

scholarly journals Real-Time PCR Assay for Detection and Differentiation of Shiga Toxin-Producing Escherichia coli from Clinical Samples

2015 ◽  
Vol 53 (7) ◽  
pp. 2148-2153 ◽  
Author(s):  
Xuan Qin ◽  
Eileen J. Klein ◽  
Emmanouil Galanakis ◽  
Anita A. Thomas ◽  
Jennifer R. Stapp ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Primary Cultures ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Content Type ◽  
E Coli ◽  
Successful Recovery

Timely accurate diagnosis of Shiga toxin-producingEscherichia coli(STEC) infections is important. We evaluated a laboratory-developed real-time PCR (LD-PCR) assay targetingstx1,stx2, andrfbEO157with 2,386 qualifying stool samples submitted to the microbiology laboratory of a tertiary care pediatric center between July 2011 and December 2013. Broth cultures of PCR-positive samples were tested for Shiga toxins by enzyme immunoassay (EIA) (ImmunoCard STAT! enterohemorrhagicE. coli[EHEC]; Meridian Bioscience) and cultured in attempts to recover both O157 and non-O157 STEC.E. coliO157 and non-O157 STEC were detected in 35 and 18 cases, respectively. Hemolytic uremic syndrome (HUS) occurred in 12 patients (10 infected with STEC O157, one infected with STEC O125ac, and one with PCR evidence of STEC but no resulting isolate). Among the 59 PCR-positive STEC specimens from 53 patients, only 29 (54.7%) of the associated specimens were toxin positive by EIA. LD-PCR differentiated STEC O157 from non-O157 usingrfbEO157, and LD-PCR results prompted successful recovery ofE. coliO157 (n= 25) and non-O157 STEC (n= 8) isolates, although the primary cultures and toxin assays were frequently negative. A rapid “mega”-multiplex PCR (FilmArray gastrointestinal panel; BioFire Diagnostics) was used retrospectively, and results correlated with LD-PCR findings in 25 (89%) of the 28 sorbitol-MacConkey agar culture-negative STEC cases. These findings demonstrate that PCR is more sensitive than EIA and/or culture and distinguishes between O157 and non-O157 STEC in clinical samples and thatE. coliO157:H7 remains the predominant cause of HUS in our institution. PCR is highly recommended for rapid diagnosis of pediatric STEC infections.

Comparison of a New Enrichment Procedure for Shiga Toxin–Producing Escherichia coli with Five Standard Methods

2005 ◽  
Vol 68 (8) ◽  
pp. 1593-1599 ◽  
Author(s):  
MICHAEL A. GRANT
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Target Cells ◽  
Macconkey Agar ◽  
Standard Methods ◽  
E Coli ◽  
Canadian Health ◽  
Health Products

A new procedure for enrichment of Escherichia coli O157:H7 and other Shiga toxin–producing E. coli was compared to five standard methods: the British Public Health Laboratory Service, International Standard Method, U.S. Department of Agriculture, Canadian Health Products and Food Branch, and U.S. Food and Drug Administration. The new procedure was comparable to the standard methods in its ability to detect target cells inoculated into foods at approximately 1 CFU g−1. Comparisons were also made of the ability of the six enrichment procedures to detect E. coli O157:H7 against a large background of competitor microorganisms. In these experiments the new procedure yielded more target cells than the other five enrichments by two to three orders of magnitude as determined by enumeration on sorbitol MacConkey agar with tellurite and cefixime and Rainbow agar with tellurite and novobiocin and by verification of presumptive colonies by real-time PCR. For example, the population of enterohemorrhagic E. coli strain 6341 recovered on sorbitol MacConkey agar with tellurite and cefixime after enrichment with the experimental method was 2.42 × 108 CFU ml−1 and 1.80 × 106 CFU ml−1 after enrichment with the Canadian Health Products and Food Branch method, the second most effective in this experiment. In addition, broth cultures resulting from each of the six enrichment procedures were used to prepare templates for real-time PCR detection of stx1/stx2. Resulting threshold cycle (Ct) values after the experimental enrichment were similar to positive control values, whereas the five standard methods produced delayed Ct values or were not detected.

Potential pitfalls in the quantitative molecular detection ofEscherichia coliO157:H7 in environmental matrices

2006 ◽  
Vol 52 (5) ◽  
pp. 482-488 ◽  
Author(s):  
Rebekka R.E Artz ◽  
Lisa M Avery ◽  
Davey L Jones ◽  
Ken Killham
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Detection Sensitivity ◽  
Escherichia Coli O157 ◽  
Soil System ◽  
E Coli ◽  
Animal Wastes ◽  
Pcr Assays

The detection sensitivity and potential interference factors of a commonly used assay based on real-time polymerase chain reaction (PCR) for Escherichia coli O157:H7 using eae gene-specific primers were assessed. Animal wastes and soil samples were spiked with known replicate quantities of a nontoxigenic strain of E. coli O157:H7 in a viable or dead state and as unprotected DNA. The detection sensitivity and accuracy of real-time PCR for E. coli O157:H7 in animal wastes and soil is low compared to enrichment culturing. Nonviable cells and unprotected DNA were shown to produce positive results in several of the environmental samples tested, leading to potential overestimates of cell numbers due to prolonged detection of nonviable cells. This demonstrates the necessity for the specific calibration of real-time PCR assays in environmental samples. The accuracy of the eae gene–based detection method was further evaluated over time in a soil system against an activity measurement, using the bioluminescent properties of an E. coli O157:H7 Tn5luxCDABE construct. The detection of significant numbers of viable but nonculturable (VBNC) as well as nonviable and possibly physically protected cells as shown over a period of 90 days further complicates the use of real-time PCR assays for quick diagnostics in environmental samples and infers that enrichment culturing is still required for the final verification of samples found positive by real-time PCR methods.Key words: Escherichia coli O157:H7, real-time PCR, animal waste, soil, VBNC.

scholarly journals Evaluation of Enzyme Immunoassays and Real-Time PCR for Detecting Shiga Toxin-Producing Escherichia coli in Southern Alberta, Canada

2015 ◽  
Vol 53 (3) ◽  
pp. 1019-1023 ◽  
Author(s):  
Linda Chui ◽  
Laura Patterson-Fortin ◽  
Julie Kuo ◽  
Vincent Li ◽  
Valerie Boras
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Content Type ◽  
Enzyme Immunoassays ◽  
Southern Alberta

Two immunoassays (Shiga Toxin Chek and Shiga Toxin Quik Chek) and real-time PCR were used to detect Shiga toxin-producingEscherichia coli. For enriched culture, the sensitivity and specificity of the three methods ranged from 80.0% to 98.2% and 98.0% to 100.0%, respectively. STEC isolates were identified in 2.6% of the 784 samples.

scholarly journals High-resolution melting real-time PCR assays for detection of Escherichia coli O26 and O111 strains possessing Shiga toxin genes

LWT ◽  
2020 ◽  
Vol 131 ◽  
pp. 109785
Author(s):  
Prashant Singh ◽  
Gabriel Cubillos ◽  
Gabrielle Kirshteyn ◽  
Joseph M. Bosilevac
Keyword(s):  
Escherichia Coli ◽  
High Resolution ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Toxin Genes ◽  
Pcr Assays ◽  
Shiga Toxin Genes

Assessment of Microbiological Safety and Quality of Marinades Used To Treat Beef and That Were Collected over a 12-Month Period from Specialty Retailers Near Raleigh, North Carolina

2018 ◽  
Vol 81 (3) ◽  
pp. 490-496 ◽  
Author(s):  
Yangjin Jung ◽  
Christopher L. Rupert ◽  
Benjamin Chapman ◽  
Anna C. S. Porto Fett ◽  
John B. Luchansky
Keyword(s):  
Escherichia Coli ◽  
North Carolina ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Plate Count ◽  
Pcr Assay ◽  
E Coli ◽  
Aerobic Plate Count

ABSTRACT In total, 115 marinade samples (58 fresh marinades and 57 spent marinades) were collected over 12 months from specialty retailers (four individual stores) near Raleigh, NC. These marinades were screened for total mesophilic aerobic plate count (M-APC), total psychrotrophic aerobic plate count (P-APC), and Enterobacteriaceae. These marinades were also screened for the seven regulated serogroups of Shiga toxin–producing Escherichia coli. Stores A and B used immersion to marinade raw beef cuts, whereas stores C-1 and C-2 used vacuum tumbling. In general, marinade temperatures at the stores ranged from 1.8 to 6.6°C, and beef cuts were marinated from a few minutes to up to 3 days. Regardless of the process used to marinade meat, levels of M-APC and P-APC in fresh marinades ranged from 3.4 to 4.7 and 1.4 to 1.8 log CFU/mL, respectively, whereas Enterobacteriaceae were not detected in any fresh marinades, even after enrichment. However, levels of M-APC, P-APC, and Enterobacteriaceae in spent marinades collected from stores C-1 and C-2 (ca. 3.6 to 7.1 log CFU/mL) were significantly higher (P < 0.05) compared with levels of these same types of bacteria enumerated from spent marinades collected at stores A and B (ca. ≤0.7 to 4.9 log CFU/mL). None of the 115 marinade samples tested positive for Shiga toxin–producing E. coli by using a BAX system real-time PCR assay. No significant (P > 0.05) association was observed between microbial levels (i.e., M-APC, P-APC, and Enterobacteriaceae) and the temperature or duration of the marination process. Levels of M-APC, P-APC, and Enterobacteriaceae in spent marinades were significantly affected by the marination method (P < 0.05), with levels, in general, being higher in marinades used for tumbling. Thus, retailers must continue to keep marinade solutions and meat at a safe temperature (i.e., ≤4°C) and to properly and frequently sanitize the equipment and environment in both the processing area and deli case.

scholarly journals Real-Time PCR Methodology for Selective Detection of Viable Escherichia coli O157:H7 Cells by Targeting Z3276 as a Genetic Marker

2012 ◽  
Vol 78 (15) ◽  
pp. 5297-5304 ◽  
Author(s):  
Baoguang Li ◽  
Jin-Qiang Chen
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Genetic Marker ◽  
Real Time Pcr ◽  
Accurate Method ◽  
Selective Detection ◽  
Pcr Assay ◽  
Content Type ◽  
E Coli ◽  
Viable Cells

ABSTRACTThe goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viableEscherichia coliO157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection ofE. coliO157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific forE. coliO157:H7 strains (n= 298). Using this assay, we can detect amounts of genomic DNA ofE. coliO157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)–real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 × 107dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viableE. coliO157:H7 cells with an 8-h enrichment. In conclusion, this PMA–real-time PCR assay offers a sensitive and specific means to selectively detect viableE. coliO157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viableE. coliO157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources.

Rapid and Reliable Detection of Shiga Toxin–Producing Escherichia coli by Real-Time Multiplex PCR

2012 ◽  
Vol 75 (4) ◽  
pp. 643-650 ◽  
Author(s):  
KELLY S. ANKLAM ◽  
KAUSHI S. T. KANANKEGE ◽  
TINA K. GONZALES ◽  
CHARLES W. KASPAR ◽  
DÖRTE DÖPFER
Keyword(s):  
Public Health ◽  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Real Time ◽  
Shiga Toxin ◽  
Virulence Genes ◽  
Health Concern ◽  
E Coli ◽  
Reliable Detection ◽  
Pcr Assays

Escherichia coli O26, O45, O103, O111, O121, O145, and O157 are the predominant Shiga toxin–producing E. coli (STEC) serogroups implicated in outbreaks of human foodborne illness worldwide. The increasing prevalence of these pathogens has important public health implications. Beef products have been considered a main source of foodborne human STEC infections. Robust and sensitive methods for the detection and characterization of these pathogens are needed to determine prevalence and incidence of STEC in beef processing facilities and to improve food safety interventions aimed at eliminating STEC from the food supply. This study was conducted to develop Taqman real-time multiplex PCR assays for the screening and rapid detection of the predominant STEC serogroups associated with human illness. Three serogroup-specific assays targeted the O-antigen gene clusters of E. coli O26 (wzy), O103 (wzx), and O145 (wzx) in assay 1, O45 (wzy), O111 (manC), and O121 (wzx) in assay 2, and O157 (rfbE) in assay 3. The uidA gene also was included in the serogroup-specific assays as an E. coli internal amplification control. A fourth assay was developed to target selected virulence genes for Shiga toxin (stx1 and stx2), intimin (eae), and enterohemolysin (ehxA). The specificity of the serogroup and virulence gene assays was assessed by testing 100 and 62 E. coli strains and non–E. coli control strains, respectively. The assays correctly detected the genes in all strains examined, and no cross-reactions were observed, representing 100% specificity. The detection limits of the assays were 103 or 104 CFU/ml for pure cultures and artificially contaminated fecal samples, and after a 6-h enrichment period, the detection limit of the assays was 100 CFU/ml. These results indicate that the four real-time multiplex PCR assays are robust and effective for the rapid and reliable detection of the seven predominant STEC serogroups of major public health concern and the detection of their virulence genes.

Validation and Application of a Real-Time PCR Assay Based on the CRISPR Array for Serotype-Specific Detection and Quantification of Enterohemorrhagic Escherichia coli O157:H7 in Cattle Feces†

2018 ◽  
Vol 81 (7) ◽  
pp. 1157-1164 ◽  
Author(s):  
LANCE W. NOLL ◽  
RACHEL CHALL ◽  
PRAGATHI B. SHRIDHAR ◽  
XUMING LIU ◽  
JIANFA BAI ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Culture Method ◽  
Qpcr Assay ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Cattle Feces ◽  
Crispr Array ◽  
Detection And Quantification

ABSTRACT Several real-time quantitative PCR (qPCR) assays have been developed for detection and quantification of Escherichia coli O157:H7 in complex matrices by targeting genes for serogroup-specific O-antigen (rfbEO157), H7 antigen, and one or more major virulence factors (Shiga toxin and intimin). A major limitation of such assays is that coamplification of H7 and virulence genes in a sample does not signal association of those genes with the O157 serogroup. Clusters of regularly interspaced short palindromic repeats (CRISPR) polymorphisms are highly correlated with certain enterohemorrhagic E. coli (EHEC) serotypes, including O157:H7, and the presence of genes for Shiga toxin (stx1 and stx2) and intimin (eae). Our objectives were to develop and validate a qPCR assay targeting the CRISPR array for the detection and quantification of EHEC O157:H7 in cattle feces and to evaluate the applicability of the assay for detection of and comparison with a four-plex qPCR assay targeting rfbEO157, stx1, stx2, and eae genes and a culture method. Detection limits of the CRISPRO157:H7 qPCR assay for cattle feces spiked with pure cultures were 2.1 × 103 and 2.3 × 100 CFU/g before and after enrichment, respectively. Detection of E. coli O157 in feedlot cattle fecal samples (n = 576) was compared among the CRISPRO157:H7 qPCR assay, culture method, and four-plex qPCR assay. The CRISPRO157:H7 qPCR detected 42.2% of the samples (243 of 576 samples) as positive for E. coli O157:H7, compared with 30.4% (175 samples) by the culture method. Nearly all samples (97.2%; 560 samples) were positive for rfbEO157 by the four-plex PCR, but 21.8% (122 of 560 samples) were negative for the stx and/or eae genes, making it unlikely that EHEC O157:H7 was present in these samples. Cohen's kappa statistic indicated a fair and poor agreement beyond that due to chance between the CRISPR assay and the culture method and four-plex assay, respectively. This novel qPCR assay can detect the EHEC O157:H7 serotype in cattle feces by targeting CRISPR polymorphisms.

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