Comparative Evaluation of a Phage Protein Ligand Assay with Real-Time PCR and a Reference Method for the Detection of Escherichia coli O157:H7 in Raw Ground Beef and Trimmings

2011 ◽  
Vol 74 (1) ◽  
pp. 6-12 ◽  
Author(s):  
F. SAVOYE ◽  
P. FENG ◽  
C. ROZAND ◽  
M. BOUVIER ◽  
A. GLEIZAL ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Ground Beef ◽  
Reference Method ◽  
Enterohemorrhagic Escherichia Coli ◽  
Detection Methods ◽  
Escherichia Coli O157 ◽  
Rt Pcr ◽  
Raw Meat ◽  
E Coli

Enterohemorrhagic Escherichia coli O157:H7 is an important pathogen associated with infections caused by consumption of undercooked raw meat. Sensitive and rapid detection methods for E. coli O157:H7 are essential for the meat industry to ensure a safe meat supply. This study was conducted to compare the sensitivity of the VIDAS ultraperformance E. coli test (ECPT UP) with a noncommercial real-time (RT) PCR method and the U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) reference method for detecting E. coli O157:H7 in raw ground beef. Optimal enrichment times and the efficacy of testing different types of raw meat, either as individual samples (25 g) or as composites (375 g), were examined. For 25-g samples of each type of raw ground beef tested, 6 h of enrichment was sufficient for both the VIDAS ECPT UP and RT-PCR methods, but for 375-g samples, 24 h of enrichment was required. Both the VIDAS ECPT UP and RT-PCR methods produced results similar to those obtained with the USDA-FSIS reference method after 18 to 24 h of enrichment. The primer specificity of the RT-PCR assay and the highly specific phage ligand used in the VIDAS ECPT UP for target recognition enabled the detection of low levels of E. coli O157:H7 in 25 g of various types of raw ground beef. The tests also allowed the detection of E. coli O157:H7 in composite raw ground beef and trimmings in samples of up to 375 g.

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

2015 ◽  
Vol 98 (5) ◽  
pp. 1301-1314 ◽  
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.

scholarly journals Modification and Matrix Extension of the Bio-Rad iQ-Check E. coli O157:H7, STEC VirX, and STEC SerO Test Kits for the Detection of Shiga Toxin–Producing Escherichia coli (STEC) and Escherichia coli O157 From a Single Enrichment

2020 ◽  
Vol 103 (1) ◽  
pp. 161-175
Author(s):  
Dane Brooks ◽  
Benjamin Bastin ◽  
Erin Crowley ◽  
James Agin ◽  
Mike Clark ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Ground Beef ◽  
Reference Method ◽  
Chicken Breast ◽  
E Coli ◽  
Free Dna ◽  
Target Analytes ◽  
Matrix Extension

Abstract Background: The iQ-Check Real-Time PCR kits use PCR technology based on gene amplification and detection by a real-time PCR thermalcycler for the detection of target analytes in select food matrices. The iQ-Check E. coli O157:H7 [Performance Tested MethodSM (PTM) 020801] and STEC VirX and STEC SerO (combined PTM 121203) methods were previously validated for different matrices under different enrichment schemes. Objective: To modify the current iQ-Check E. coli O157:H7 Kit for the detection of Escherichia coli O157:H7 from 25 to 375 g for raw ground beef (17% fat), raw beef trim, and fresh spinach. In addition, a matrix extension was validated for iQ-Check E. coli O157:H7 for raw chicken breast without skin (25 g), raw chicken thigh with skin (25 g), mechanically separated chicken (25 g), and raw ground pork (25 g). The study also included the modification of the iQ-Check STEC VirX and SerO Kits for the detection of non-O157 Shiga toxin–producing E. coli (STEC) for raw ground beef (375 g), raw beef trim (375 g), and fresh spinach (375 g) from STEC Enrichment Broth to buffered peptone water (BPW). All tests were carried out at 8–22 h (10–22 h for fresh spinach). Methods: Ground beef, beef trim, and spinach were co-inoculated with E. coli O157:H7, non-O157 STECs, and Salmonella spp. and analyzed for E. coli O157:H7 and non-O157 STECs after an 8-22 h enrichment in BPW for the beef matrices and after a 10–22 h enrichment in BPW for spinach. The chicken matrices were inoculated with E. coli O157:H7 only and analyzed after an 8–22 h enrichment in BPW. The iQ-Check Free DNA Removal Solution workflow was utilized for all matrices. Confirmations at the 22 h time point and method comparisons were conducted with the appropriate reference method as outlined in the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 4A or the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook Chapters 5.09 and 5B.05. For the iQ-Check STEC VirX and STEC SerO Kits, inclusivity and exclusivity were also performed. Results: The two inclusivity and exclusivity evaluations indicated that the test methods can accurately detect the target analytes and correctly excluded nontarget organisms after 8 h of enrichment. In the method comparison study, the iQ-Check E. coli O157:H7 and STEC VirX and STEC SerO test kits demonstrated no statistically significant differences between candidate and reference method results or between presumptive and confirmed results for all food matrices analyzed and the two time points (8 or 10 and 22 h). Both time points produced the same results, with no discrepancies. Conclusions: The iQ-Check real-time PCR kits are effective methods for the detection of E. coli O157 and non-O157 STECs (both the virulence factors and the O groups) from raw ground beef, raw beef trim, and fresh spinach in 375 g samples enriched in BPW for 8–22 h (10–22 h for fresh spinach). In addition, the iQ-Check E. coli O157 Kit is effective in detecting E. coli O157 in 25 g samples of raw chicken breast without skin, raw chicken thigh with skin, mechanically separated chicken, and raw ground pork. The iQ-Check test kits allow the end user to pair enrichments for multiple target analytes, allowing the user to prepare a single enrichment and perform a single DNA extraction. The Free DNA Removal Solution removes free DNA from samples prior to PCR analysis, protecting DNA from intact and living cells. Highlights: The method modifications were granted based on the data collected.

Rapid and Simultaneous Quantitation of Escherichia coli O157:H7, Salmonella, and Shigella in Ground Beef by Multiplex Real-Time PCR and Immunomagnetic Separation†

2007 ◽  
Vol 70 (6) ◽  
pp. 1366-1372 ◽  
Author(s):  
LUXIN WANG ◽  
YONG LI ◽  
AZLIN MUSTAPHA
Keyword(s):  
Escherichia Coli ◽  
Detection Limit ◽  
Real Time ◽  
Real Time Pcr ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Quantitative Detection ◽  
Escherichia Coli O157 ◽  
The Real ◽  
E Coli

The objective of this study was to establish a multiplex real-time PCR for the simultaneous quantitation of Escherichia coli O157:H7, Salmonella, and Shigella. Genomic DNA for the real-time PCR was extracted by the boiling method. Three sets of primers and corresponding TaqMan probes were designed to target these three pathogenic bacteria. Multiplex real-time PCR was performed with TaqMan Universal PCR Master Mix in an ABI Prism 7700 Sequence Detection System. Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log CFU per milliliter) via linear regression. With optimized conditions, the quantitative detection range of the real-time multiplex PCR for pure cultures was 102 to 109 CFU/ml for E. coli O157:H7, 103 to 109 CFU/ml for Salmonella, and 101 to 108 CFU/ml for Shigella. When the established multiplex real-time PCR system was applied to artificially contaminated ground beef, the detection limit was 105 CFU/g for E. coli O157:H7, 103 CFU/g for Salmonella, and 104 CFU/g for Shigella. Immunomagnetic separation (IMS) was further used to separate E. coli O157:H7 and Salmonella from the beef samples. With the additional use of IMS, the detection limit was 103 CFU/g for both pathogens. Results from this study showed that TaqMan real-time PCR, combined with IMS, is potentially an effective method for the rapid and reliable quantitation of E. coli O157:H7, Salmonella, and Shigella in food.

Incidence of Enterohemorrhagic Escherichia coli, Escherichia coli O157, Salmonella, and Listeria monocytogenes in Retail Fresh Ground Beef, Sprouts, and Mushrooms

2006 ◽  
Vol 69 (2) ◽  
pp. 441-443 ◽  
Author(s):  
M. SAMADPOUR ◽  
M. W. BARBOUR ◽  
T. NGUYEN ◽  
T.-M. CAO ◽  
F. BUCK ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Ground Beef ◽  
Food Samples ◽  
Enterohemorrhagic Escherichia Coli ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Pcr Assays ◽  
Retail Food

The objective of this study was to determine the prevalence of enterohemorrhagic Escherichia coli (EHEC), E. coli O157, Salmonella, and Listeria monocytogenes in retail food samples from Seattle, Wash. A total of 2,050 samples of ground beef (1,750 samples), mushrooms (100 samples), and sprouts (200 samples) were collected over a 12-month period and analyzed for the presence of these pathogens. PCR assays, followed by culture confirmation were used to determine the presence or absence of each organism. Of the 1,750 ground beef samples analyzed, 61 (3.5%) were positive for EHEC, and 20 (1.1%) of these were positive for E. coli O157. Salmonella was present in 67 (3.8%) of the 1,750 ground beef samples. Of 512 ground beef samples analyzed, 18 (3.5%) were positive for L. monocytogenes. EHEC was found in 12 (6.0%) of the 200 sprout samples, and 3 (1.5%) of these yielded E. coli O157. Of the 200 total sprout samples, 14 (7.0%) were positive for Salmonella and none were positive for L. monocytogenes. Among the 100 mushroom samples, 4 (4.0%) were positive for EHEC but none of these 4 samples were positive for E. coli O157. Salmonella was detected in 5 (5.0%) of the mushroom samples, and L. monocytogenes was found in 1 (1.0%) of the samples.

Antibacterial Effect of Lactoferricin B on Escherichia coli O157:H7 in Ground Beef

1999 ◽  
Vol 62 (7) ◽  
pp. 747-750 ◽  
Author(s):  
KUMAR S. VENKITANARAYANAN ◽  
TONG ZHAO ◽  
MICHAEL P. DOYLE
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Effect ◽  
Ground Beef ◽  
Enterohemorrhagic Escherichia Coli ◽  
Plate Count ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Significant Difference ◽  
Sufficient Magnitude ◽  
Peptone Medium

The antibacterial activity of lactoferricin B on enterohemorrhagic Escherichia coli O157:H7 in 1% peptone medium and ground beef was studied at 4 and 10°C. In 1% peptone medium, 50 and 100 μg of lactoferricin B per ml reduced E. coli O157:H7 populations by approximately 0.7 and 2.0 log CFU/ml, respectively. Studies comparing the antibacterial effect of lactoferricin B on E. coli O157:H7 in 1% peptone at pH 5.5 and 7.2 did not reveal any significant difference (P > 0.5) at the two pH values. Lactoferricin B (100 μg/g) reduced E. coli O157:H7 population in ground beef by about 0.8 log CFU/g (P < 0.05). No significant difference (P > 0.5) was observed in the total plate count between treatment and control ground beef samples stored at 4 and 10°C. The antibacterial effect of lactoferricin B on E. coli O157:H7 observed in this study is not of sufficient magnitude to merit its use in ground beef for controlling the pathogen.

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.

Novel Real-Time PCR Method To Detect Escherichia coli O157:H7 in Raw Milk Cheese and Raw Ground Meat

2012 ◽  
Vol 75 (8) ◽  
pp. 1373-1381 ◽  
Author(s):  
STÉPHANE D. MISZCZYCHA ◽  
SARAH GANET ◽  
LYSIANE DUNIERE ◽  
CHRISTINE ROZAND ◽  
ESTELLE LOUKIADIS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Meat Products ◽  
Reference Method ◽  
Raw Milk ◽  
Escherichia Coli O157 ◽  
Ground Meat ◽  
Uida Gene ◽  
E Coli

Raw milk, raw milk cheeses, and raw ground meat have been implicated in Escherichia coli O157:H7 outbreaks. Developing methods to detect these bacteria in raw milk and meat products is a major challenge for food safety. The aim of our study was to develop a real-time PCR assay to detect E. coli O157:H7 in raw milk cheeses and raw ground meat. Well-known primers targeting a mutation at position +93 of the uidA gene in E. coli O157:H7 were chosen, and a specific TaqMan–minor groove binder probe was designed. This probe targets another mutation, at position +191 of the uidA gene in E. coli O157:H7. The first step in the study was to evaluate the specificity of this probe with 156 different O157:H7/NM strains and 48 non-O157:H7/NM strains of E. coli. The sensitivity of the method was evaluated by pre- and postinoculation of cheeses and meat enrichments with different E. coli O157:H7 strains. All the E. coli O157:H7 isolates tested were positive, and none of the other bacteria were detected. Our results indicate that this method is sensitive enough to detect 102 E. coli O157:H7 isolates per ml of cheese or meat enrichment broth (24 h at 41.5°C) and is more sensitive than the International Organization for Standardization reference method. We can conclude that this new real-time PCR protocol is a useful tool for rapid, specific, and sensitive detection of E. coli O157:H7 in raw milk and raw ground meat products.

A rapid (one day), sensitive real-time polymerase chain reaction assay for detecting Escherichia coli O157:H7 in ground beef

2006 ◽  
Vol 52 (10) ◽  
pp. 992-998 ◽  
Author(s):  
Jane Holicka ◽  
Rebecca A Guy ◽  
Anita Kapoor ◽  
David Shepherd ◽  
Paul A Horgen
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Polymerase Chain Reaction Assay ◽  
Ground Beef ◽  
Automatic Process ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
E Coli ◽  
Polymerase Chain

The purpose of this study was to apply our rapid, integrated double enrichment 5′ nuclease real-time polymerase chain reaction assay for the detection of Escherichia coli O157:H7 and evaluate its efficacy. The assay targeted ground beef, an important vehicle in disease epidemiology. The assay reliably determined in 8 h the presence of E. coli O157:H7 in ground beef at the level of 1 colony-forming unit (CFU)/g. The sensitivity and specificity of the assay were compared with that of standard enrichment diagnostic techniques. A correlation of 100% in detection was achieved to the limit of 1 CFU/g. This assay can be used as a rapid, automatic process for identification of E. coli O157:H7 in ground beef or can be integrated with standard culture procedures, resulting in considerable cost and time savings.Key words: real-time PCR, E. coli O157:H7, ground beef, molecular diagnostics, rapid O157:H7 assay.

scholarly journals Evaluation of Escherichia coli O157:H7 Growth Media for Use in Test-and-Hold Procedures for Ground Beef Processing†

2006 ◽  
Vol 69 (5) ◽  
pp. 1007-1011 ◽  
Author(s):  
MICHAEL N. GUERINI ◽  
TERRANCE M. ARTHUR ◽  
STEVEN D. SHACKELFORD ◽  
MOHAMMAD KOOHMARAIE
Keyword(s):  
Escherichia Coli ◽  
Time Course ◽  
Ground Beef ◽  
Threshold Level ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Growth Media ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Peptone Water

Since the mid-1990s, the beef industry has used a process called test and hold, wherein beef trim and ground beef are tested to keep products contaminated with Escherichia coli O157:H7 out of commerce. Current O157:H7 detection methods rely on a threshold level of bacterial growth for detection, which is dependent on the growth medium used. Twelve media were examined for growth and doubling time: buffered peptone water (BPW), SOC (which contains tryptone, yeast extract, KCl, MgCl2, and glucose), buffered peptone water plus SOC (BPW-SOC), Bacto-NZYM, RapidChek E. coli O157:H7 medium, BioControl EHEC8 culture medium, Neogen Reveal for E. coli O157:H7—Eight Hour medium (Neogen Reveal 8), BAX System medium for E. coli O157:H7 (BAX), BAX System medium for E. coli O157:H7 MP (BAX-MP), modified E. coli broth, nutrient medium, and tryptic soy broth (TSB). All media were tested at 37 or 42°C under static or shaking conditions. The eight media with the highest total CFU per milliliter and most rapid doubling times were BPW-SOC, NZYM, RapidChek, EHEC8, Neogen Reveal 8, BAX, BAX-MP, and TSB. The ability of these eight media to enrich E. coli O157:H7 in ground beef was further evaluated through time-course experiments using immunomagnetic separation. Of these media, TSB was the easiest to prepare, had a wide application base, and was the least expensive. In the test-and-hold process, the normal ratio of medium to product is 1:10. In this study, a 1:3 ratio worked as well as a 1:10 ratio. Processors using test-and-hold procedures could use 1 liter of TSB to enrich for E. coli O157:H7 in a 375-g sample instead of the usual 3.375 liters, thus saving reagents, time, and labor while maintaining accuracy.

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

2006 ◽  
Vol 69 (8) ◽  
pp. 1978-1982 ◽  
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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