Automated DNA extraction and multiplexed PCR assay to identify Escherichia coli O157:H7 from ground beef and kitchen surface swab samples

A comparison was made of the relative efficiencies of three enrichment media, RapidChek Escherichia coli O157:H7 enrichment broth (REB), R&F broth (RFB), and modified E. coli broth containing novobiocin (mEC+n), and four selective plating media for detection of cold- and freeze-stressed E. coli O157:H7 in raw ground beef. Ground beef (25 g) was inoculated with E. coli O157:H7 at ≤0.5 and ≤2 CFU/g, and samples were then enriched immediately or were stored at 4°C for 72 h or at −20°C for 2 weeks and then enriched. After 8 or 20 h of enrichment, the cultures were plated onto R&F E. coli O157: H7 chromogenic plating medium, cefixime-tellurite sorbitol MacConkey agar, CHROMagar O157, and Rainbow agar O157 and tested using the RapidChek E. coli O157 lateral flow immunoassay and a multiplex PCR assay targeting the E. coli O157: H7 eae, stx1, and stx2 genes. Recovery of E. coli O157:H7 on the four agar media was 4.0 to 7.9 log CFU/ml with the REB enrichment, 1.4 to 7.4 log CFU/ml with RFB, 1.7 to 6.7 log CFU/ml with mEC+n incubated at 42°C, and 1.3 to 3.3 log CFU/ml from mEC+n incubated at 35°C. The percentages of positive ground beef samples containing nonstressed, cold-stressed, and freeze-stressed E. coli O157:H7 as obtained by plating, the immunoassay, and the PCR assay were 97, 88, and 97%, respectively, with REB, 92, 81, and 78%, respectively, with RFB, 97, 58, and 53%, respectively, with mEC+n incubated at 42°C, and 22, 31, and 25%, respectively, with mEC+n incubated at 35°C. Logistic regression analyses of the data indicated significant main effects of treatment, type of medium, enrichment time, inoculum concentration, and detection method. In particular, a positive result was 1.1 times more likely to occur after 20 h of enrichment than after 8 h, 25 times more likely with RFB and REB than with mEC+nat35°C, 3.7 times more likely with an initial inoculum of ≤2.0 CFU/g than with ≤0.5 CFU/g, 2.5 to 3 times more likely using freeze-stressed or nonstressed bacteria than with cold-stressed bacteria, and 2.5 times more likely by plating than by the immunoassay or the PCR assay. REB had better overall performance for enrichment of cold- and freeze-stressed E. coli O157:H7 present in ground beef than did the other media examined.

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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.

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Efficacy of Enrichment Broths in the Recovery of Freeze-Injured Escherichia coli O157:H7 in Inoculated Ground Beef by PCR

Journal of Food Protection ◽

10.4315/0362-028x-66.10.1911 ◽

2003 ◽

Vol 66 (10) ◽

pp. 1911-1915 ◽

Cited By ~ 12

Author(s):

W. C. LIONBERG ◽

L. RESTAINO ◽

E. W. FRAMPTON ◽

W. M. BARBOUR

Keyword(s):

Escherichia Coli ◽

Ground Beef ◽

Immunomagnetic Separation ◽

Automated System ◽

Escherichia Coli O157 ◽

Pcr Assay ◽

E Coli ◽

Cultural Isolation ◽

Peptone Water ◽

Chromogenic Agar

Escherichia coli O157:H7 strains ATCC 35150 and ATCC 43894 and five pooled isolates from beef and pork freeze injured at −25°C in beef infusion were used to inoculate ground beef. Samples (25 g each) were added to 225 ml of buffered peptone water with vancomycin, cefsulodin, and cefixime (BPW-VCC), 225 ml of modified EC broth plus novobiocin (mEC+n), and 225 ml of R&F enrichment broth (R&F-EB) and aerobically incubated at 41 to 42°C. After 6, 7, 8, and 24 h of incubation, levels of E. coli O157:H7 recovered from each broth by a PCR assay with the BAX automated system as well as by conventional enrichment with the use of nonaerated mEC+n incubated at 35°C for 24 h were compared with levels recovered by cultural isolation with immunomagnetic separation and plating on BCM E. coli O157:H7 chromogenic agar. For ground beef inoculated with a mean of 4.23 ± 1.00 total cells (74% freeze injured) per 25 g, after 6 h the PCR assay identified 72.7, 57.6, and 66% of the samples for R&F-EB, BPW-VCC, and mEC+n, respectively, as presumptive positive, whereas the recovery rates after 7 and 8 h exceeded 90%, with the rate for R&F-EB being 100%. For ground beef inoculated with a mean of 1.50 ± 0.56 total cells (80% freeze injured) per 25 g, after 6 h the PCR assay identified 47.6, 19.1, and 9.5% of the samples for R&F-EB, BPW-VCC, and mEC+n, respectively, as presumptive positive. These values increased to 81.0, 61.9, and 52.4% after 7 h and to 95.2, 61.9, and 71.4% after 8 h. After 24 h, only 55 to 60% of the samples at both inoculum levels tested positive by PCR with conventional enrichment and incubation, whereas >95% of the samples tested positive with R&F-EB aerated at 41 to 42°C. Culture results for R&F-EB and mEC+n after 7 and 8 h of incubation were closely correlated with presumptive positive PCR results.

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Detection of Escherichia coli O157:H7 in ground beef using immunomagnetic separation and multiplex PCR

Food Control ◽

10.1016/j.foodcont.2008.06.002 ◽

2009 ◽

Vol 20 (4) ◽

pp. 357-361 ◽

Cited By ~ 34

Author(s):

Belgin Sarimehmetoglu ◽

Mihriban Hatun Aksoy ◽

Naim Deniz Ayaz ◽

Yildiz Ayaz ◽

Ozlem Kuplulu ◽

...

Keyword(s):

Escherichia Coli ◽

Multiplex Pcr ◽

Ground Beef ◽

Immunomagnetic Separation ◽

Escherichia Coli O157

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Comparison of Four Commercial DNA Extraction Kits for PCR Detection of Listeria monocytogenes, Salmonella, Escherichia coli O157:H7, and Staphylococcus aureus in Fresh, Minimally Processed Vegetables

Journal of Food Protection ◽

10.4315/0362-028x-71.10.2110 ◽

2008 ◽

Vol 71 (10) ◽

pp. 2110-2114 ◽

Cited By ~ 25

Author(s):

P. ELIZAQUÍVEL ◽

R. AZNAR

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Listeria Monocytogenes ◽

Dna Extraction ◽

Pcr Detection ◽

Escherichia Coli O157 ◽

Minimally Processed ◽

Green Pepper ◽

Minimally Processed Vegetables ◽

And Staphylococcus Aureus

Four commercial DNA extraction methods, PrepMan Ultra (Applied Biosystems), InstaGene Matrix (BioRad), DNeasy Tissue kit (Qiagen), and UltraClean (MoBio), were tested for PCR detection of Listeria monocytogenes, Escherichia coli O157: H7, Salmonella, and Staphylococcus aureus in fresh, minimally processed vegetables. For comparative purposes, sensitivity assays with specific PCRs were carried out after DNA extraction with the four methods in green pepper, broccoli, and onion artificially inoculated with the four pathogens separately. As confirmed by statistical analysis, the DNeasy Tissue kit rendered the highest sensitivity values in the three matrices assayed for Salmonella, L. monocytogenes, and E. coli O157:H7 and in onion for S. aureus. Despite being the most expensive of the methods compared, the DNeasy Tissue Kit can be successfully applied for any of the four most commonly studied pathogens, thus saving time and overall reducing the cost of the analysis.

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Validation of Time and Temperature Values as Critical Limits for the Control of Escherichia coli O157:H7 during the Production of Fresh Ground Beef

Journal of Food Protection ◽

10.4315/0362-028x-69.8.1978 ◽

2006 ◽

Vol 69 (8) ◽

pp. 1978-1982 ◽

Cited By ~ 8

Author(s):

J. E. MANN ◽

M. M. BRASHEARS

Keyword(s):

Escherichia Coli ◽

Room Temperature ◽

Hazard Analysis ◽

Control Point ◽

Ground Beef ◽

Escherichia Coli O157 ◽

Meat Processing ◽

E Coli ◽

Critical Control Point ◽

Critical Limits

In order to provide beef processors with valuable data to validate critical limits set for temperature during grinding, a study was conducted to determine Escherichia coli O157:H7 growth at various temperatures in raw ground beef. Fresh ground beef samples were inoculated with a cocktail mixture of streptomycin-resistant E. coli O157:H7 to facilitate recovery in the presence of background flora. Samples were held at 4.4, 7.2, and 10°C, and at room temperature (22.2 to 23.3°C) to mimic typical processing and holding temperatures observed in meat processing environments. E. coli O157:H7 counts were determined by direct plating onto tryptic soy agar with streptomycin (1,000 μg/ml), at 2-h intervals over 12 h for samples held at room temperature. Samples held under refrigeration temperatures were sampled at 4, 8, 12, 24, 48, and 72 h. Less than one log of E. coli O157:H7 growth was observed at 48 h for samples held at 10°C. Samples held at 4.4 and 7.2°C showed less than one log of E. coli O157:H7 growth at 72 h. Samples held at room temperature showed no significant increase in E. coli O157:H7 counts for the first 6 h, but increased significantly afterwards. These results illustrate that meat processors can utilize a variety of time and temperature combinations as critical limits in their hazard analysis critical control point plans to minimize E. coli O157:H7 growth during the production and storage of ground beef.

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SENSITIVE AND RAPID DETECTION OF ESCHERICHIA COLI O157:H7 IN GROUND BEEF BY NESTED PCR INCORPORATING IMMUNOMAGNETIC SEPARATION

Food Biotechnology ◽

10.1081/fbt-120014325 ◽

2002 ◽

Vol 16 (2) ◽

pp. 145-154 ◽

Cited By ~ 1

Author(s):

J. Guan ◽

R. E. Levin

Keyword(s):

Escherichia Coli ◽

Nested Pcr ◽

Rapid Detection ◽

Ground Beef ◽

Immunomagnetic Separation ◽

Escherichia Coli O157

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Thermal Inactivation of Escherichia coli O157: H7, Salmonella senftenberg, and Enzymes with Potential as Time-Temperature Indicators in Ground Beef

Journal of Food Protection ◽

10.4315/0362-028x-60.5.471 ◽

1997 ◽

Vol 60 (5) ◽

pp. 471-475 ◽

Cited By ~ 39

Author(s):

ALICIA ORTA-RAMIREZ ◽

JAMES F. PRICE ◽

YIH-CHIH HSU ◽

GIRIDARAN J. VEERAMUTHU ◽

JAMIE S. CHERRY-MERRITT ◽

...

Keyword(s):

Escherichia Coli ◽

Thermal Inactivation ◽

Ground Beef ◽

Escherichia Coli O157 ◽

E Coli ◽

Triose Phosphate ◽

Beef Products ◽

Z Value ◽

D Values ◽

Salmonella Senftenberg

The USDA has established processing schedules for beef products based on the destruction of pathogens. Several enzymes have been suggested as potential indicators of heat processing. However, no relationship between the inactivation rates of these enzymes and those of pathogenic microorganisms has been determined. Our objective was to compare the thermal inactivation of Escherichia coli O157:H7 and Salmonella senftenberg to those of endogenous muscle proteins. Inoculated and noninoculated ground beef samples were heated at four temperatures for predetermined intervals of time in thermal-death-time studies. Bacterial counts were determined and enzymes were assayed for residual activity. The D values for E. coli O157:H7 were 46.10, 6.44, 0.43, and 0.12 min at 53, 58, 63, and 68°C, respectively, with a z value of 5.60°C. The D values for S. senftenberg were 53.00, 15.17, 2.08, and 0.22 min at 53, 58, 63, and 68°C, respectively, with a z value of 6.24°C. Apparent D values at 53, 58, 63, and 68°C were 352.93, 26.31, 5.56, and 3.33 min for acid phosphatase; 6968.64, 543.48, 19.61, and 1.40 min for lactate dehydrogenase; and 3870.97, 2678.59, 769.23, and 42.92 min for peroxidase; with z values of 7.41,3.99, and 7.80°C, respectively. Apparent D values at 53, 58, 63, and 66°C were 325.03, 60.07, 3.07, and 1.34 min for phosphoglycerate mutase; 606.72, 89.86, 4.40, and 1.28 min for glyceraldehyde-3-phosphate dehydrogenase; and 153.06, 20.13, 2.25, and 0.74 min for triose phosphate isomerase; with z values of 5.18, 4.71, and 5.56°C, respectively. The temperature dependence of triose phosphate isomerase was similar to those of both E. coli O157 :H7 and S. senftenberg, suggesting that this enzyme could be used as an endogenous time-temperature indicator in beef products.

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Viability of Acid-Adapted Escherichia coli O157:H7 in Ground Beef Treated with Acidic Calcium Sulfate

Journal of Food Protection ◽

10.4315/0362-028x-67.3.591 ◽

2004 ◽

Vol 67 (3) ◽

pp. 591-595 ◽

Cited By ~ 5

Author(s):

LARRY R. BEUCHAT ◽

ALAN J. SCOUTEN

Keyword(s):

Escherichia Coli ◽

Acetic Acid ◽

Calcium Sulfate ◽

Ground Beef ◽

Storage Period ◽

Acid Tolerance ◽

Escherichia Coli O157 ◽

E Coli ◽

Acidic Environments ◽

Subsequent Acid

The effects of lactic acid, acetic acid, and acidic calcium sulfate (ACS) on viability and subsequent acid tolerance of three strains of Escherichia coli O157:H7 were determined. Differences in tolerance to acidic environments were observed among strains, but the level of tolerance was not affected by the acidulant to which cells had been exposed. Cells of E. coli O157:H7 adapted to grow on tryptic soy agar acidified to pH 4.5 with ACS were compared to cells grown at pH 7.2 in the absence of ACS for their ability to survive after inoculation into ground beef treated with ACS, as well as untreated beef. The number of ACS-adapted cells recovered from ACS-treated beef was significantly (α = 0.05) higher than the number of control cells recovered from ACS-treated beef during the first 3 days of a 10-day storage period at 4°C, suggesting that ACS-adapted cells might be initially more tolerant than unadapted cells to reduced pH in ACS-treated beef. Regardless of treatment of ground beef with ACS or adaptation of E. coli O157:H7 to ACS before inoculating ground beef, the pathogen survived in high numbers.

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