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.

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.

Comparison of Rapid Enzyme-Linked Immunosorbent Assay and Immunomagnetic Separation Methods for Detection of Escherichia coli O157 in Fecal, Hide, Carcass, and Ground Beef Samples

2007 ◽  
Vol 70 (10) ◽  
pp. 2230-2234 ◽  
Author(s):  
T. W. THOMPSON ◽  
T. P. STEPHENS ◽  
G. H. LONERAGAN ◽  
M. F. MILLER ◽  
M. M. BRASHEARS
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Separation Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Escherichia Coli O157 ◽  
Perfect Agreement ◽  
Fecal Samples ◽  
E Coli ◽  
Beef Products

Rapid enzyme-linked immunosorbent assays (ELISAs) are approved for detection of Escherichia coli O157 in beef products. However, these kits have also been used in the industry to detect this pathogen on hides or in feces of cattle, although this use has not been validated. The objective of this study was to compare commercially available ELISAs (E. coli Now, Reveal, and VIP) with immunomagnetic separation along with selective media to detect E. coli O157 on hides, in feces, and in medium- and low-level-inoculated ground beef and carcasses (simulated by using briskets) samples. Naturally infected hide and fecal samples were subjected to both the immunomagnetic separation method and ELISAs for the detection of E. coli O157. Additionally, E. coli O157 inoculated and noninoculated ground beef and beef briskets were used to simulate meat and carcass samples. When comparing the detection results from the ELISAs (E. coli Now, Reveal, and VIP) to the immunomagnetic separation method, poor agreement was observed for fecal samples (kappa = 0.10, 0.02, and 0.03 for E. coli Now, Reveal, and VIP, respectively), and fair-to-moderate agreement was observed for hide samples (kappa = 0.30, 0.51, and 0.29 for E. coli Now, Reveal, and VIP, respectively). However, there was near-perfect agreement between the immunomagnetic separation method and ELISAs for ground beef (kappa = 1, 1, and 0.80 for E. coli Now, Reveal, and VIP, respectively) and brisket (kappa = 1, 1, and 1 for E. coli Now, Reveal, and VIP, respectively) samples. Assuming immunomagnetic separation is the best available method, these data suggest that the ELISAs are not useful in detecting E. coli O157 from hide or fecal samples. However, when ELISAs are used on ground beef and beef brisket samples they can be used with a high degree of confidence.

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.

Relative Sensitivity of Escherichia coli O157 Detection from Bovine Feces and Rectoanal Mucosal Swabs

2014 ◽  
Vol 77 (6) ◽  
pp. 972-976 ◽  
Author(s):  
K. J. WILLIAMS ◽  
M. P. WARD ◽  
O. DHUNGYEL ◽  
L. VAN BREDA
Keyword(s):  
Escherichia Coli ◽  
Confidence Interval ◽  
Human Health Risk ◽  
Relative Sensitivity ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Escherichia Coli O157 ◽  
Prevalence Studies ◽  
E Coli ◽  
The Difference

The need to quantify the potential human health risk posed by the bovine reservoir of Escherichia coli O157 has led to a wealth of prevalence studies and improvements in detection methods over the last two decades. Rectoanal mucosal swabs have been used for the detection of E. coli O157 fecal shedding, colonized animals, and those predisposed to super shedding. We conducted a longitudinal study to compare the detection of E. coli O157 from feces and rectoanal mucosal swabs (RAMS) from a cohort of dairy heifers. We collected 820 samples that were tested by immunomagnetic separation of both feces and RAMS. Of these, 132 were detected as positive for E. coli O157 from both samples, 66 were detected as positive from RAMS only, and 117 were detected as positive from feces only. The difference in results between the two sample types was statistically significant (P < 0.001). The relative sensitivities of detection by immunomagnetic separation were 53% (confidence interval, 46.6 to 59.3) from RAMS and 67% (confidence interval, 59.6 to 73.1) from fecal samples. No association between long-term shedding (P = 0.685) or super shedding (P = 0.526) and detection by RAMS only was observed.

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.

Magnetic Nanoparticle-Antibody Conjugates for the Separation of Escherichia coli O157:H7 in Ground Beef

2005 ◽  
Vol 68 (9) ◽  
pp. 1804-1811 ◽  
Author(s):  
MADHUKAR VARSHNEY ◽  
LIJU YANG ◽  
XIAO-LI SU ◽  
YANBIN LI
Keyword(s):  
Escherichia Coli ◽  
Magnetic Nanoparticles ◽  
Magnetic Nanoparticle ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Capture Efficiency ◽  
Escherichia Coli O157 ◽  
Mechanical Mixing ◽  
E Coli ◽  
Antibody Conjugates

The immunomagnetic separation with magnetic nanoparticle-antibody conjugates (MNCs) was investigated and evaluated for the detection of Escherichia coli O157:H7 in ground beef samples. MNCs were prepared by immobilizing biotin-labeled polyclonal goat anti–E. coli antibodies onto streptavidin-coated magnetic nanoparticles. For bacterial separation, MNCs were mixed with inoculated ground beef samples, then nanoparticle-antibody–E. coli O157:H7 complexes were separated from food matrix with a magnet, washed, and surface plated for microbial enumeration. The capture efficiency was determined by plating cells bound to nanoparticles and unbound cells in the supernatant onto sorbitol MacConkey agar. Key parameters, including the amount of nanoparticles and immunoreaction time, were optimized with different concentrations of E. coli O157:H7 in phosphate-buffered saline. MNCs presented a minimum capture efficiency of 94% for E. coli O157:H7 ranging from 1.6 × 101 to 7.2 × 107 CFU/ml with an immunoreaction time of 15 min without any enrichment. Capture of E. coli O157:H7 by MNCs did not interfere with other bacteria, including Salmonella enteritidis, Citrobacter freundii, and Listeria monocytogenes. The capture efficiency values of MNCs increased from 69 to 94.5% as E. coli O157:H7 decreased from 3.4 × 107 to 8.0 × 100 CFU/ml in the ground beef samples prepared with minimal steps (without filtration and centrifugation). An enrichment of 6 h was done for 8.0 × 100 and 8.0 × 101 CFU/ml of E. coli O157:H7 in ground beef to increase the number of cells in the sample to a detectable level. The results also indicated that capture efficiencies of MNCs for E. coli O157:H7 with and without mechanical mixing during immunoreaction were not significantly different (P > 0.05). Compared with microbeads based immunomagnetic separation, the magnetic nanoparticles showed their advantages in terms of higher capture efficiency, no need for mechanical mixing, and minimal sample preparation.

scholarly journals Improvement of Immunomagnetic Separation for Escherichia coli O157:H7 Detection by the PickPen Magnetic Particle Separation Device†

2006 ◽  
Vol 69 (12) ◽  
pp. 2870-2874 ◽  
Author(s):  
XIANGWU NOU ◽  
TERRANCE M. ARTHUR ◽  
JOSEPH M. BOSILEVAC ◽  
DAYNA M. BRICHTA-HARHAY ◽  
MICHAEL N. GUERINI ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pathogen Detection ◽  
Magnetic Particles ◽  
Magnetic Particle ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Escherichia Coli O157 ◽  
High Background ◽  
E Coli ◽  
Significant Difference

Conventional immunomagnetic separation (IMS) procedures, which use an external magnetic source to capture magnetic particles against the side of a test tube, are labor-intensive and can have poor sensitivity for the target organism because of high background microflora that is not effectively washed away during the IMS process. This report compares the conventional IMS procedure to a new IMS procedure with an intrasolution magnetic particle transfer device, the PickPen. The IMS target for the majority of these studies is Escherichia coli O157:H7 in various types of samples, including cattle feces, hides, carcasses, and ground beef. Comparison of the two IMS methods showed a significant difference (P < 0.05) in the efficiency of detecting E. coli O157:H7 from cattle carcass surface, cattle hide, and cattle fecal samples. No significant improvement (P > 0.05) in E. coli O157:H7 detection was observed when the PickPen IMS procedure was used to isolate this pathogen from ground beef samples. Use of the PickPen IMS greatly increases the throughput of the IMS procedure and may be more compatible with various emerging technologies for pathogen detection. In addition, the efficacy of sequential IMS for multiple pathogens is reported herein.

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

2003 ◽  
Vol 66 (10) ◽  
pp. 1911-1915 ◽  
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.

Survival of Escherichia coli O157:H7 in Bovine Feces over Time under Various Temperature Conditions

2006 ◽  
Vol 69 (12) ◽  
pp. 2851-2855 ◽  
Author(s):  
A. ECHEVERRY ◽  
G. H. LONERAGAN ◽  
M. M. BRASHEARS
Keyword(s):  
Escherichia Coli ◽  
Pathogen Detection ◽  
Storage Temperature ◽  
Immunomagnetic Separation ◽  
Detection Methods ◽  
Microbiological Analysis ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Bovine Feces ◽  
Over Time

Although Escherichia coli O157:H7 prevalence estimates in cattle have increased over time due to improvements in detection methods, fecal sample transport conditions from farm to microbiological laboratories for further analysis may be a factor for prevalence underestimation. The objective of this study was to compare and determine the survival characteristics of E. coli O157:H7 in bovine feces under various storage conditions that could be encountered during transport. Fecal pats were inoculated with a four-strain cocktail of antibiotic-resistant E. coli O157:H7 to contain ∼1 × 105 CFU/g. Inoculated and control samples were taken after 0, 24, 48, 120, and 168 h at each storage temperature and examined for presence and numbers of E. coli O157:H7. Each sample was subdivided and placed at each of the four following temperatures: 37, 23, 4.4°C, and in a plastic cooler with refrigerant packs (0, 4, 4, 21, and 23°C at five sampling times, respectively) to simulate transportation conditions. A statistically significant decrease in the population of the pathogen was observed after 48 h in samples held at 37°C(P < 0.01) and after 168 h at 4.4°C(P = 0.02). At 37°C, E. coli O157:H7 was not detected after 48 h, either by direct plating (P < 0.01) or by immunomagnetic separation. Overall, the results of this study showed that E. coli O157:H7 survived without significant detriment in bovine fecal material inside the cooler for up to 168 h. These results indicate that shipment and storage under these conditions before microbiological analysis would be acceptable and should not affect pathogen detection.

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

Food Control ◽  
2009 ◽  
Vol 20 (4) ◽  
pp. 357-361 ◽  
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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