Multiplex real-time PCR assay for detection of Escherichia coli O157:H7 and screening for non-O157 Shiga toxin-producing E. coli

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.

Download Full-text

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

Journal of Food Protection ◽

10.4315/0362-028x.jfp-17-396 ◽

2018 ◽

Vol 81 (3) ◽

pp. 490-496 ◽

Cited By ~ 2

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.

Download Full-text

Validation of the Thermo Scientific SureTect Escherichia coli O157:H7 Real-Time PCR Assay for Raw Beef and Produce Matrixes

Journal of AOAC International ◽

10.5740/jaoacint.15-043 ◽

2015 ◽

Vol 98 (5) ◽

pp. 1301-1314 ◽

Cited By ~ 1

Author(s):

Jonathan Cloke ◽

Erin Crowley ◽

Patrick Bird ◽

Ben Bastin ◽

Jonathan Flannery ◽

...

Keyword(s):

Escherichia Coli ◽

Food Safety ◽

Real Time ◽

Real Time Pcr ◽

Apple Juice ◽

Ground Beef ◽

Reference Method ◽

Escherichia Coli O157 ◽

Pcr Assay ◽

E Coli

Abstract The Thermo Scientific™ SureTect™ Escherichia coli O157:H7 Assay is a new real-time PCR assay which has been validated through the AOAC Research Institute (RI) Performance Tested MethodsSM program for raw beef and produce matrixes. This validation study specifically validated the assay with 375 g 1:4 and 1:5 ratios of raw ground beef and raw beef trim in comparison to the U.S. Department of Agriculture, Food Safety Inspection Service, Microbiology Laboratory Guidebook (USDS-FSIS/MLG) reference method and 25 g bagged spinach and fresh apple juice at a ratio of 1:10, in comparison to the reference method detailed in the International Organization for Standardization 16654:2001 reference method. For raw beef matrixes, the validation of both 1:4 and 1:5 allows user flexibility with the enrichment protocol, although which of these two ratios chosen by the laboratory should be based on specific test requirements. All matrixes were analyzed by Thermo Fisher Scientific, Microbiology Division, Vantaa, Finland, and Q Laboratories Inc, Cincinnati, Ohio, in the method developer study. Two of the matrixes (raw ground beef at both 1:4 and 1:5 ratios) and bagged spinach were additionally analyzed in the AOAC-RI controlled independent laboratory study, which was conducted by Marshfield Food Safety, Marshfield, Wisconsin. Using probability of detection statistical analysis, no significant difference was demonstrated by the SureTect kit in comparison to the USDA FSIS reference method for raw beef matrixes, or with the ISO reference method for matrixes of bagged spinach and apple juice. Inclusivity and exclusivity testing was conducted with 58 E. coli O157:H7 and 54 non-E. coli O157:H7 isolates, respectively, which demonstrated that the SureTect assay was able to detect all isolates of E. coli O157:H7 analyzed. In addition, all but one of the nontarget isolates were correctly interpreted as negative by the SureTect Software. The single isolate giving a positive result was an E. coli O157:NM isolate. Nonmotile isolates of E. coli O157 have been demonstrated to still contain the H7 gene; therefore, this result is not unexpected. Robustness testing was conducted to evaluate the performance of the SureTect assay with specific deviations to the assay protocol, which were outside the recommended parameters and which are open to variation. This study demonstrated that the SureTect assay gave reliable performance. A final study to verify the shelf life of the product, under accelerated conditions was also conducted.

Download Full-text

Fate ofEscherichia coliO157:H7 in irrigation water on soils and plants as validated by culture method and real-time PCR

Canadian Journal of Microbiology ◽

10.1139/w04-097 ◽

2004 ◽

Vol 50 (12) ◽

pp. 1007-1014 ◽

Cited By ~ 55

Author(s):

A Mark Ibekwe ◽

Pamela M Watt ◽

Peter J Shouse ◽

Catherine M Grieve

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Irrigation Water ◽

Real Time Pcr ◽

The United States ◽

Large Animal ◽

Escherichia Coli O157 ◽

Pcr Assay ◽

E Coli ◽

Rhizosphere Soils

One of the most common vehicles by which Escherichia coli O157:H7 may be introduced into crops is contaminated irrigation water. Water contamination is becoming more common in rural areas of the United States as a result of large animal operations, and up to 40% of tested drinking-water wells are contaminated with E. coli. In this study, 2 contrasting soil samples were inoculated with E. coli O157:H7 expressing green fluorescent protein through irrigation water. Real-time PCR and culture methods were used to quantify the fate of this pathogen in phyllosphere (leaf surface), rhizosphere (volume of soil tightly held by plant roots), and non-rhizosphere soils. A real-time PCR assay was designed with the eae gene of E. coli O157:H7. The probe was incorporated into real-time PCR containing DNA extracted from the phyllosphere, rhizosphere, and non-rhizosphere soils. The detection limit for E. coli O157:H7 quantification by real-time PCR was 1.2 × 103in the rhizosphere, phyllosphere, and non-rhizosphere samples. E. coli O157:H7 concentrations were higher in the rhizosphere than in the non-rhizosphere soils and leaf surfaces, and persisted longer in clay soil. The persistence of E. coli O157:H7 in phyllosphere, rhizosphere, and non-rhizosphere soils over 45 days may play a significant part in the recontamination cycle of produce in the environment. Therefore, the rapidity of the real-time PCR assay may be a useful tool for quantification and monitoring of E. coli O157:H7 in irrigation water and on contaminated fresh produce.Key words: real-time PCR, Escherichia coli O157:H7, irrigation, survival, quantification.

Download Full-text

High Resolution Meltcurve PCR assay for detection of Salmonella and Escherichia coli o157:h7 in foods

10.32469/10355/62057 ◽

2017 ◽

Author(s):

◽

Yuejiao Liu

Keyword(s):

Escherichia Coli ◽

High Resolution ◽

Real Time ◽

Real Time Pcr ◽

Escherichia Coli O157 ◽

Single Mutation ◽

Pcr Assay ◽

E Coli ◽

Hrm Analysis ◽

Pcr Targeting

Foodborne illnesses associated with Salmonella and Escherichia coli O157:H7 have become world-wide public-health problems. Conventional methods for the identification of foodborne pathogens are tedious, expensive, and time-consuming. Alternatively, real-time PCR (RT-PCR) as a promising method to detect pathogens in food samples, has recently been widely applied in food safety areas. High Resolution Meltcurve (HRM) analysis, performed immediately at the end of a real-time PCR, is able to yield a higher resolution plot compared with SYBR Green I PCR. HRM dyes completely saturate all amplicons without showing preferential bindings, making the results more clear and distinct. In this research, a multiplex real-time PCR targeting the invA, fimA and stn genes were developed to efficiently detect Salmonella in foods. Furthermore, HRM analysis is sensitive to any single mutation in PCR products, thus it was also applied in this study to distinguish E. coli O157 from other serogroups of E. coli by targeting the uidA gene. The specificity of primers used in this study was checked using many different strains. Results of artificially contaminated foods presented a high sensitivity of the HRM detection methods. Due to its low cost, simplicity of the approach and rapidness, HRM technology is highly competitive with relaxed-condition PCR and probe-based PCR. Besides, an HRM assay can be performed on generic real-time PCR instrumentations found in many laboratories. In conclusion, HRM-based PCR assay are proved to be efficient methods in foodborne pathogen detections.

Download Full-text

Promising Nucleic Acid Lateral Flow Assay Plus PCR for Shiga Toxin–Producing Escherichia coli

Journal of Food Protection ◽

10.4315/0362-028x.jfp-14-495 ◽

2015 ◽

Vol 78 (8) ◽

pp. 1560-1568 ◽

Cited By ~ 6

Author(s):

YOSHITAKA TERAO ◽

KANA TAKESHITA ◽

YASUTAKA NISHIYAMA ◽

NAOKI MORISHITA ◽

TAKASHI MATSUMOTO ◽

...

Keyword(s):

Escherichia Coli ◽

Detection Limit ◽

Nucleic Acid ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Pcr Assay ◽

E Coli ◽

Lower Detection Limit ◽

Lower Detection

Shiga toxin (Stx)–producing Escherichia coli (STEC) is a frequent cause of foodborne infections, and methods for rapid and reliable detection of STEC are needed. A nucleic acid lateral flow assay (NALFA) plus PCR was evaluated for detecting STEC after enrichment. When cell suspensions of 45 STEC strains, 14 non-STEC strains, and 13 non–E. coli strains were tested with the NALFA plus PCR, all of the STEC strains yielded positive results, and all of the non-STEC and non–E. coli strains yielded negative results. The lower detection limit for the STEC strains ranged from 0.1 to 1 pg of genomic DNA (about 20 to 200 CFU) per test, and the NALFA plus PCR was able to detect Stx1- and Stx2-producing E. coli strains with similar sensitivities. The ability of the NALFA plus PCR to detect STEC in enrichment cultures of radish sprouts, tomato, raw ground beef, and beef liver inoculated with 10-fold serially diluted STEC cultures was comparable to that of a real-time PCR assay (at a level of 100 to 100,000 CFU/ml in enrichment culture). The bacterial inoculation test in raw ground beef revealed that the lower detection limit of the NALFA plus PCR was also comparable to that obtained with a real-time PCR assay that followed the U.S. Department of Agriculture guidelines. Although further evaluation is required, these results suggest that the NALFA plus PCR is a specific and sensitive method for detecting STEC in a food manufacturing plant.

Download Full-text

Multiplex Real-Time PCR Method To Identify Shiga Toxin Genes stx1 and stx2 and Escherichia coli O157:H7/H− Serotype

Applied and Environmental Microbiology ◽

10.1128/aem.69.10.6327-6333.2003 ◽

2003 ◽

Vol 69 (10) ◽

pp. 6327-6333 ◽

Cited By ~ 81

Author(s):

Karen C. Jinneman ◽

Ken J. Yoshitomi ◽

Stephen D. Weagant

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Real Time Pcr ◽

Escherichia Coli O157 ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

E Coli ◽

Pcr Method ◽

Shiga Toxin Genes

ABSTRACT A multiplex real-time PCR method to simultaneously detect the stx1 and stx2 genes of Shiga toxin-producing Escherichia coli and a unique conserved single-nucleotide polymorphism in the E. coli O157:H7/H− uidA gene has been developed. There is more than 98.6% sensitivity and 100% specificity for all three gene targets based on a panel of 138 isolates. The PCR efficiencies were ≥1.89, and as few as 6 CFU/reaction could be detected.

Download Full-text

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

Journal of Food Protection ◽

10.4315/0362-028x-68.8.1593 ◽

2005 ◽

Vol 68 (8) ◽

pp. 1593-1599 ◽

Cited By ~ 13

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.

Download Full-text

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

Canadian Journal of Microbiology ◽

10.1139/w05-149 ◽

2006 ◽

Vol 52 (5) ◽

pp. 482-488 ◽

Cited By ~ 25

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.

Download Full-text