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.

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.

Comparison of Methods for Detection and Isolation of Cold- and Freeze-Stressed Escherichia coli O157:H7 in Raw Ground Beef†

2007 ◽  
Vol 70 (7) ◽  
pp. 1663-1669 ◽  
Author(s):  
PINA M. FRATAMICO ◽  
LORI K. BAGI
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Escherichia Coli O157 ◽  
Pcr Assay ◽  
Initial Inoculum ◽  
E Coli ◽  
Multiplex Pcr Assay ◽  
Treatment Type ◽  
Agar Media ◽  
Main Effects

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.

Prevalence and Numbers of Escherichia coli O157 on Bovine Hides at a Beef Slaughter Plant

2005 ◽  
Vol 68 (4) ◽  
pp. 660-665 ◽  
Author(s):  
S. B. O[apos]BRIEN ◽  
G. DUFFY ◽  
E. CARNEY ◽  
J. J. SHERIDAN ◽  
D. A. McDOWELL ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Early Stage ◽  
Immunomagnetic Separation ◽  
Pcr Analysis ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Hemolysin Gene ◽  
Peptone Water ◽  
Encoding Gene ◽  
Genetic Profiles

In this study, we investigated the prevalence and numbers of Escherichia coli O157 on bovine hides. Samples (n = 1,500) were collected over a 17-month period (30 samples per week) by sponge swabbing approximately 122-cm2 areas of the bovine rump of slaughtered cattle at an early stage of carcass processing (first legging). Sponge samples (n = 1,500) were stomached in buffered peptone water supplemented with novobiocin, directly plated on sorbitol MacConkey with Cefixime tellurite (SMAC-CT), enriched for 24 h, extracted by immunomagnetic separation, and plated onto SMAC-CT agar. Presumptive E. coli O157 colonies from SMAC-CT plates were confirmed by PCR for the presence of eaeA, hlyA, fliCh7, vt1, vt2, and portions of the rfb (O-antigen encoding) region of E. coli O157. Overall, E. coli O157 was recovered from 109 samples (7.3%) at concentrations ranging from less than 0.13 to 4.24 log CFU/100 cm2. PCR analysis revealed a wide diversity of genetic profiles among recovered isolates of verocytotoxigenic E. coli. Of the isolates recovered, 99 of 109 contained the attaching and effacing gene (eaeA) and the hemolysin gene (hlyA), and 78 of 109 had the flagellar H7 antigen–encoding gene (fliCh7). Only 6 of 109 isolates contained both verotoxin-producing genes (vt1 and vt2); 91 of 109 contained the vt2 gene only, whereas 1 of 109 contained the vt1 gene only. The remaining 11 of 109 contained neither vt1 nor vt2.

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 Evaluation of test-kits for the detection of Escherichia coli O157 in raw meats and cattle faeces.

2012 ◽  
Vol 1 (2) ◽  
Author(s):  
Hilda Nyati ◽  
Annet Heuvelink ◽  
Caroliene Van Heerwaarden ◽  
Ans Zwartkruis
Keyword(s):  
Escherichia Coli ◽  
Real Time Pcr ◽  
Immunomagnetic Separation ◽  
Escherichia Coli O157 ◽  
Immunomagnetic Beads ◽  
Tryptone Soya Broth ◽  
Detection Rates ◽  
E Coli ◽  
Peptone Water ◽  
Cattle Faeces

Escherichia coli O157 detection limits in artificially contaminated beef and cattle faeces samples were determined using Dynabeads anti E. coli O157 immunomagnetic beads, VIDAS-UP, VIDAS-ICE, and real-time PCR (GeneDisc and LightCycler) systems. Dynabeads anti-E. coli O157 immunomagnetic separation (IMS) and the GeneDisc cycler were the most sensitive methods, and could detect an initial 1 CFU in 25g beef samples after 6h of incubation in modified tryptone soya broth with novobiocin (mTSB+n) or buffered peptone water (BPW). The VIDAS-UP method could detect an initial 10 CFU, while VIDAS-ICE and the LightCycler methods could only detect an initial 100 CFU. Higher detection rates were achieved with 18 hour incubations, where an initial 1 CFU in a 25g sample could be detected with all five methods. For cattle faeces enrichments, Dynabeads anti-E. coli O157 IMS could detect an initial 1 CFU after a 6 h incubation in mTSB+n, while the VIDAS-UP and VIDAS-ICE methods could detect an initial 10 CFU and both PCR methods could only detect an initial 100 CFU. Detection rates were lower in BPW, compared to mTSB+n, with thresholds of 100 CFU for VIDAS-ICE, VIDAS-UP and GeneDisc methods, and >100 CFU for the LightCycler method.

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.

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.

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