Evaluation of a Multiplex Real-Time PCR Method for Detecting Shiga Toxin–Producing Escherichia coli in Beef and Comparison to the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook Method†

2014 ◽  
Vol 77 (2) ◽  
pp. 180-188 ◽  
Author(s):  
PINA M. FRATAMICO ◽  
JAMIE L. WASILENKO ◽  
BRADLEY GARMAN ◽  
DANIEL R. DeMARCO ◽  
STEPHEN VARKEY ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Virulence Genes ◽  
Microbiology Laboratory ◽  
Pcr Assay ◽  
Department Of Agriculture ◽  
E Coli ◽  
System Method ◽  
Inspection Service ◽  
The U.S

The “top-six” non-O157 Shiga toxin–producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) most frequently associated with outbreaks and cases of foodborne illnesses have been declared as adulterants in beef by the U.S. Department of Agriculture Food Safety and Inspection Service (FSIS). Regulatory testing in beef began in June 2012. The purpose of this study was to evaluate the DuPont BAX System method for detecting these top six STEC strains and strains of E. coli O157:H7. For STEC, the BAX System real-time STEC suite was evaluated, including a screening assay for the stx and eae virulence genes and two panel assays to identify the target serogroups: panel 1 detects O26, O111, and O121, and panel 2 detects O45, O103, O145. For E. coli O157:H7, the BAX System real-time PCR assay for this specific serotype was used. Sensitivity of each assay for the PCR targets was ≥1.23 × 103 CFU/ml in pure culture. Each assay was 100% inclusive for the strains tested (20 to 50 per assay), and no cross-reactivity with closely related strains was observed in any of the assays. The performance of the BAX System methods was compared with that of the FSIS Microbiology Laboratory Guidebook (MLG) methods for detection of the top six STEC and E. coli O157:H7 strains in ground beef and beef trim. Generally, results of the BAX System method were similar to those of the MLG methods for detecting non-O157 STEC and E. coli O157:H7. Reducing or eliminating novobiocin in modified tryptic soy broth (mTSB) may improve the detection of STEC O111 strains; one beef trim sample inoculated with STEC O111 produced a negative result when enriched in mTSB with 8 mg/liter novobiocin but was positive when enriched in mTSB without novobiocin. The results of this study indicate the feasibility of deploying a panel of real-time PCR assay configurations for the detection and monitoring of the top six STEC and E. coli O157:H7 strains in beef. The approach could easily be adapted for additional multiplex assays should regulations expand to include other O serogroups or virulence genes.

Detection and Isolation of the “Top Seven” Shiga Toxin–Producing Escherichia coli in Ground Beef: Comparison of RapidFinder Kits to the U.S. Department of Agriculture Microbiology Laboratory Guidebook Method

2017 ◽  
Vol 80 (5) ◽  
pp. 829-836 ◽  
Author(s):  
Pina M. Fratamico ◽  
Lori K. Bagi ◽  
Aisha Abdul-Wakeel
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
Microbiology Laboratory ◽  
Department Of Agriculture ◽  
E Coli ◽  
The U.S

ABSTRACT Shiga toxin–producing Escherichia coli (STEC) O157:H7 and serogroups O26, O45, O103, O111, O121, and O145 are often referred to as the “top seven” STEC, and these have been declared to be adulterants in beef by the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS). The aim of this work was to compare the methods described in the USDA FSIS Microbiology Laboratory Guidebook (MLG) to a two-stage Applied Biosystems RapidFinder STEC real-time PCR method to test for the top seven STEC in raw ground beef. The specificity of the RapidFinder workflow that targets non-O157 STEC O-antigen genes, stx1, stx2, and eae, and E. coli O157–specific targets was determined with 132 top seven STEC strains and 283 exclusion strains. All inclusion strains were positive, and all exclusion strains gave negative results with the RapidFinder assay. Strains carrying all of the known variants of stx1 and stx2, including stx2f and stx2g, were also detected. For RapidFinder analysis, 375-g ground beef samples spiked with ≥4 CFU of representative STEC strains were enriched in 1 L of tryptic soy broth (TSB) for 10 h at 42 ± 1°C, and for the MLG method, 325-g samples were similarly spiked and enriched in 975 mL of modified TSB for 15 h at 42 ± 1°C. Following DNA extraction, real-time PCR was performed using RapidFinder Express software, and for the MLG method, the BAX Real-Time PCR STEC Suite and the BAX Real-Time E. coli O157:H7 assay were used with the BAX System Q7 software. Following immunomagnetic separation, presumptive colonies from modified Rainbow agar O157 plates were confirmed by the real-time PCR assays. Results of the RapidFinder and BAX assays were similar; all samples were positive after 10 and 15 h of enrichment, respectively. Isolation and confirmation of isolates was possible on all samples, except that two O111:NM strains could not be isolated from a portion of the inoculated samples. Thus, the RapidFinder system can be used for routine and rapid detection of the top seven STEC in beef.

scholarly journals Validation of iQ-Check E. coli O157:H7 Real-Time PCR Test Kit for Detection of Escherichia coli O157:H7 in Selected Foods

2009 ◽  
Vol 92 (4) ◽  
pp. 1095-1104 ◽  
Author(s):  
Wendy F Lauer ◽  
Sylvie Tymciu ◽  
Caroline D Sidi ◽  
Pierre Sonigo
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Ground Beef ◽  
Reference Method ◽  
Target Sequence ◽  
Apple Cider ◽  
E Coli ◽  
Test Kit ◽  
Significant Difference ◽  
The U.S

Abstract iQ-Check E. coli O157:H7 (Bio-Rad Laboratories, Hercules, CA) is a real-time PCR kit for detection of E. coli O157:H7 from selected foods. Specific fluorescent oligonucleotide probes are used to detect target DNA during the amplification, by hybridizing to the amplicons. These fluorescent probes are linked to a fluorophore which fluoresces only when hybridized to the target sequence. Three foods (ground beef, apple cider, fresh spinach) were selected to compare the performance of iQ-Check E. coli O157:H7 to the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook (MLG) reference method for ground beef and the U.S. Food and Drug Administration/Bacteriological Analytical Manual reference method for apple cider and fresh spinach. Three protocols were tested in this study: a shortened 8 h primary enrichment in buffered peptone water (BPW), a 24 h enrichment in BPW, and an enrichment in appropriate reference method enrichment broth. The iQ-Check E. coli O157:H7 method was able to identify more true/confirmed positive samples than the reference method. Inclusivity and exclusivity rates of the method were 100. iQ-Check E. coli O157:H7 performed as expected when minor procedural variations were introduced, validating the ruggedness of the method. There was no significant difference observed in performance over the shelf life of the kit.

Contamination Revealed by Indicator Microorganism Levels during Veal Processing

2016 ◽  
Vol 79 (8) ◽  
pp. 1341-1347 ◽  
Author(s):  
JOSEPH M. BOSILEVAC ◽  
RONG WANG ◽  
BRANDON E. LUEDTKE ◽  
TOMMY L. WHEELER ◽  
MOHAMMAD KOOHMARAIE
Keyword(s):  
Escherichia Coli ◽  
Plate Count ◽  
Department Of Agriculture ◽  
E Coli ◽  
Veal Calves ◽  
Indicator Microorganism ◽  
Analysis Of Results ◽  
Aerobic Plate Count ◽  
Inspection Service ◽  
The U.S

ABSTRACT During site visits of veal processors, the U.S. Department of Agriculture, Food Safety Inspection Service (FSIS) has reported processing deficiencies that likely contribute to increased levels of veal contamination. Here, we report the results of measuring aerobic plate count bacteria (APC), Enterobacteriaceae, coliforms (CF), and Escherichia coli during eight sample collections at five veal processors to assess contamination during the harvest of bob veal and formula-fed veal before (n = 5 plants) and after (n = 3 plants) changes to interventions and processing practices. Hides of veal calves at each plant had mean log CFU/100 cm2 APC, Enterobacteriaceae, CF, and E. coli of 6.02 to 8.07, 2.95 to 5.24, 3.28 to 5.83, and 3.08 to 5.59, respectively. Preintervention carcasses had mean log CFU/100 cm2 APC, Enterobacteriaceae, CF, and E. coli of 3.08 to 5.22, 1.16 to 3.47, 0.21 to 3.06, and −0.07 to 3.10, respectively, before and 2.72 to 4.50, 0.99 to 2.76, 0.69 to 2.26, and 0.33 to 2.12, respectively, after changes were made to improve sanitary dressing procedures. Final veal carcasses had mean log CFU/100 cm2 APC, Enterobacteriaceae, CF, and E. coli of 0.36 to 2.84, −0.21 to 1.59, −0.23 to 1.59, and −0.38 to 1.45 before and 0.44 to 2.64, −0.16 to 1.33, −0.42 to 1.20, and −0.48 to 1.09 after changes were made to improve carcass-directed interventions. Whereas the improved dressing procedures resulted in improved carcass cleanliness, the changes to carcass-directed interventions were less successful, and veal processors are urged to use techniques that ensure uniform and consistent delivery of antimicrobials to carcasses. Analysis of results comparing bob veal to formula-fed veal found bob veal hides, preintervention carcasses, and final carcasses to have increased (P < 0.05) APC, Enterobacteriaceae, CF, and E. coli (with the exception of hide Enterobacteriaceae; P > 0.05) relative to formula fed veal. When both veal categories were harvested at the same plant on the same day, similar results were observed. Since identification by FSIS, the control of contamination during veal processing has started to improve, but challenges still persist.

Developing a multiplex real-time PCR with a new pre-enrichment to simultaneously detect four foodborne bacteria in milk

2019 ◽  
Vol 14 (10) ◽  
pp. 885-898 ◽  
Author(s):  
Moezi Parichehr ◽  
Kargar Mohammad ◽  
Doosti Abbas ◽  
Khoshneviszadeh Mehdi
Keyword(s):  
Real Time ◽  
Foodborne Pathogens ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Pcr Assay ◽  
Reliable Technique ◽  
E Coli ◽  
Foodborne Bacteria ◽  
Multiple Pathogens ◽  
Enrichment Step

Aim: The aim of this study is to formulate a new single nonselective pre-enrichment medium (ELSS) that can support the concurrent growth of four major foodborne pathogens containing E. coli O157: H7, L. monocytogenes, S. aureus and S. enterica serovar Entertidis to develop a multiplex TaqMan Real-time PCR (mRT-PCR). Methods: The mRT-PCR with a new pre-enrichment was carried out for simultaneous detection and quantification of these foodborne bacteria. Results: By using mRT-PCR after 16 h pre-enrichment in ELSS, the detection limit of each pathogen was 1 CFU/25 ml contaminated milk, as well as inclusivity and exclusivity reached 100%. Conclusion: The mRT-PCR assay with pre-enrichment step is a fast and reliable technique for detecting single or multiple pathogens in food products.

Crystal Diagnostics MultiPath System™

2014 ◽  
Vol 97 (6) ◽  
pp. 1592-1600 ◽  
Author(s):  
Curtis H Stumpf ◽  
Weidong Zhao ◽  
Brian Bullard ◽  
Christine Ammons ◽  
Karl I Devlin ◽  
...  
Keyword(s):  
Liquid Crystal ◽  
Laboratory Tests ◽  
Ground Beef ◽  
Escherichia Coli O157 ◽  
Department Of Agriculture ◽  
E Coli ◽  
Independent Laboratory ◽  
Diagnostics System ◽  
Inspection Service ◽  
The U.S

Abstract The Crystal Diagnostics MultiPath System™ provides rapid detection of Escherichia coli O157 in fresh raw ground beef, raw beef trim, and spinach. The Crystal Diagnostics system combines patented Liquid Crystal technology with antibody-coated paramagnetic microspheres to selectively capture and detect E. coli O157 in food matrixes. This is the only liquid crystal-based biosensor commercially available for the detection of pathogens. The Crystal Diagnostics system expeditiously provides the sensitivity and accuracy of the U.S. Department of Agriculture Food Safety Inspection Service (USDA-FSIS) and the U.S. Food and Drug Administration Bacteriological Analytical Manual (FDA-BAM) methods for detecting as low as one CFU of E. coli O157 per 375 g of raw ground beef and raw beef trim, or 200 g of raw spinach. An internal inclusivity validation demonstrated detection of all 50 tested strains of E. coli O157. The internal and independent laboratory tests demonstrate that the method is rapid and sensitive for detecting of E. coli O157 in fresh raw ground beef, beef trim, and spinach.

A Scorpion Probe–Based Real-Time PCR Assay for Detection of E. coli O157:H7 in Dairy Products

2009 ◽  
Vol 6 (3) ◽  
pp. 395-400 ◽  
Author(s):  
Jitender Singh ◽  
Virender K. Batish ◽  
Sunita Grover
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dairy Products ◽  
Pcr Assay ◽  
E Coli

Performance Comparison of a fliCh7 Real-Time PCR Assay with an H7 Latex Agglutination Test for Confirmation of the H Type of Escherichia coli O157:H7†

2009 ◽  
Vol 72 (10) ◽  
pp. 2195-2197 ◽  
Author(s):  
NEELAM NARANG ◽  
PINA M. FRATAMICO ◽  
GLENN TILLMAN ◽  
KITTY PUPEDIS ◽  
WILLIAM C. CRAY
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Latex Agglutination ◽  
Agglutination Test ◽  
Latex Agglutination Test ◽  
Escherichia Coli O157 ◽  
Pcr Assay ◽  
E Coli ◽  
Reference Strains ◽  
Pcr Test

Escherichia coli O157:H7 is a foodborne pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. Positive identification of E. coli O157:H7 is made using biochemical tests and specific antisera or latex agglutination reagents for the O157 and H7 antigens. However, under certain conditions, some E. coli O157:H7 isolates can appear to be nonreactive with H7 antisera and may require multiple passages on motility medium to restore H7 antigenicity. In this study, we compared the performance of a real-time PCR test with that of a method using latex agglutination reagents to detect the presence of the fliCh7 gene or the H7 antigen, respectively, in E. coli O157:H7 isolates. One hundred twenty-six E. coli strains were tested including reference strains and strains isolated from meat. Lyophilized E. coli O157:H7 isolates were rehydrated and were plated on sheep blood agar without passage on motility medium. All strains were analyzed in parallel by a real-time PCR test targeting the fliCh7 gene and by a latex agglutination test that detects the H7 antigen. The real-time PCR assay showed 100% agreement with the H7 status reported for reference strains and E. coli O157:H7 meat isolates. The latex agglutination test results agreed with the H7 status reported for the E. coli O157:H7 reference strains and non-O157:H7 strains, except for one, E. coli O117:H7; however, 42% (42 of 100) of the E. coli O157:H7 meat isolates tested negative for the H7 antigen by latex agglutination. The real-time fliCh7 PCR test can be used to confirm E. coli O157:H7 strains that are not expressing the immunoreactive H7 antigen.

scholarly journals Real-Time PCR Assay for Detection and Differentiation of Shiga Toxin-Producing Escherichia coli from Clinical Samples

2015 ◽  
Vol 53 (7) ◽  
pp. 2148-2153 ◽  
Author(s):  
Xuan Qin ◽  
Eileen J. Klein ◽  
Emmanouil Galanakis ◽  
Anita A. Thomas ◽  
Jennifer R. Stapp ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Primary Cultures ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Content Type ◽  
E Coli ◽  
Successful Recovery

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.

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

2018 ◽  
Vol 81 (3) ◽  
pp. 490-496 ◽  
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.

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