BIOTECON Diagnostics foodproof®E. coli O157 Detection Kit, 5′ Nuclease for E. coli O157 in Combination with foodproof ShortPrep II Kit

2011 ◽  
Vol 94 (6) ◽  
pp. 1846-1852
Author(s):  
Benjamin Junge ◽  
Cordt Grönewald ◽  
Kornelia Berghof-Jäger
Keyword(s):  
Real Time ◽  
Food Samples ◽  
Food Groups ◽  
Rapid Preparation ◽  
E Coli ◽  
Cultural Methods ◽  
The Matrix ◽  
Inspection Service ◽  
High Level ◽  
The U.S

Abstract The method describes the detection of Escherichia coli O157 in food. The method is based on real-time PCR using hydrolysis probes (5′ Nuclease). This advanced PCR method was designed to reduce the time necessary to achieve results from PCR reactions and enable the user to monitor the amplification of the PCR product simultaneously in real time. After DNA isolation using the BIOTECON foodproof® ShortPrep II Kit designed for the rapid preparation of E. coli O157 DNA for direct use in PCR, the real-time detection of E. coli O157 DNA is carried out using the foodproof E. coli O157 Detection Kit. The kit provides primers and hydrolysis probes for sequence-specific detection, convenient premixed reagents, and controls for reliable interpretation of results. For repeatability studies three different foods (egg salad, large bockwurst/frankfurter, and apple juice) were analyzed, chosen from the 15 food groups recommended by the AOAC Research Institute for E. coli O157 detection. From each food, 20 samples were inoculated with a low level (1–10 CFU/25 g) and 20 samples with a high level (10–50 CFU/25 g) of E. coli O157. Additionally, five nonspiked samples were prepared from each food. Depending on the matrix, the food samples were examined with the test kits and compared with the cultural methods according to the U.S. Food and Drug Administration Bacteriological Analytical Manual or the U.S. Department of Agriculture/Food Safety and Inspection Service Microbiology Laboratory Guidebook.

BIOTECON Diagnostics foodproof®Listeria monocytogenes Detection Kit, 5′ Nuclease in Combination with the foodproof ShortPrep II Kit

2012 ◽  
Vol 95 (1) ◽  
pp. 92-99 ◽  
Author(s):  
Benjamin Junge ◽  
Cordt Grönewald ◽  
Kornelia Berghof-Jäger
Keyword(s):  
Listeria Monocytogenes ◽  
Real Time ◽  
Food Samples ◽  
Food Groups ◽  
Rapid Preparation ◽  
Cultural Methods ◽  
The Matrix ◽  
Inspection Service ◽  
High Level ◽  
The U.S

Abstract A method was developed for the detection of Listeria monocytogenes in food. The method is based on real-time PCR using hydrolysis probes (5′ Nuclease). This advanced PCR method was designed to reduce the time necessary to achieve results from PCR reactions and to enable the user to monitor the amplification of the PCR product simultaneously, in real-time. After DNA isolation using the BIOTECON food proof® ShortPrep II Kit designed for the rapid preparation of L. monocytogenes DNA for direct use in PCR, the real- time detection of L. monocytogenes DNA is carried out using the food proof Listeria monocytogenes Detection Kit. The kit provides primers and hydrolysis probes for sequence-specific detection, convenient premixed reagents, and controls for reliable interpretation of results. For the internal comparison study, three different foods (soft cheese, coalfish, and smoked ham) were analyzed, chosen from the 15 food groups recommended by the AOAC Research Institute for detection of L. monocytogenes. From each food, 20 samples were inoculated with a low level (1–10 CFU/25 g) and 20 samples with a high level (10–50 CFU/25 g) of L. monocytogenes. Additionally, five nonspiked samples were prepared from each food. Depending on the matrix, the food samples were examined with the test kits and compared with the cultural methods according to the U.S. Food and Drug Administration's Bacteriological Analytical Manual or the U.S. Department of Agriculture/Food Safety and Inspection Service Microbiology Laboratory Guidebook.

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.

scholarly journals Reveal 8-Hour Test System for Detection of Escherichia coli O157:H7 in Raw Ground Beef, Raw Beef Cubes, and Iceberg Lettuce Rinse: Collaborative Study

2001 ◽  
Vol 84 (3) ◽  
pp. 719-736 ◽  
Author(s):  
Charles B Bird ◽  
Rebecca J Hoerner ◽  
Lawrence Restaino ◽  
G Anderson ◽  
W Birbari ◽  
...  
Keyword(s):  
Collaborative Study ◽  
Ground Beef ◽  
Culture Method ◽  
The United States ◽  
Test System ◽  
Private Industry ◽  
E Coli ◽  
Inspection Service ◽  
High Level ◽  
The U.S

Abstract Five different food types were analyzed by the Reveal for E. coli O157:H7 8-Hour Test System (Reveal 8) and either the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) culture method or the U.S. Department of Agriculture Food Safety Inspection Service (FSIS) culture method for the presence of E. coli O157:H7. A total of 27 laboratories representing academia and private industry in the United States and Canada participated. Food types were inoculated with E. coli O157:H7 at 2 different levels: a high level where predominantly positive results were expected, and a low level where fractional recovery was anticipated. During this study, 1110 samples and controls were analyzed by both the Reveal 8 and by BAM or FSIS by each of the collaborators (2220 samples in total). For each set of samples, 740 were artificially inoculated with E. coli O157:H7, and 370 were uninoculated controls. The Reveal 8 detected 528 presumptive positives of which 487 were confirmed positive by the BAM culture method. In comparison, BAM and FSIS detected 489 of the 740 artificially contaminated samples as positive. In an additional in-house study performed only on chilled and frozen raw ground beef, 240 artificially inoculated samples were analyzed by both the Reveal 8 and by FSIS. The Reveal 8 detected and confirmed 104 samples as positive compared to 79 confirmed positive by FSIS.

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.

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.

Detection of E. coli O157:H7 in raw ground beef by Pathatrix™ immunomagnetic-separation, real-time PCR and cultural methods

2011 ◽  
Vol 148 (2) ◽  
pp. 87-92 ◽  
Author(s):  
Willis M. Fedio ◽  
Karen C. Jinneman ◽  
Ken J. Yoshitomi ◽  
Ruben Zapata ◽  
Chitra N. Wendakoon ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Ground Beef ◽  
Immunomagnetic Separation ◽  
E Coli ◽  
Cultural Methods

Validation of the Applied Food Diagnostics, Inc. Simultaneous Multiplex Real Time PCR (SIMUL-qPCR) Listeria species and monocytogenes Assay in Selected Foods and Environmental Surfaces: AOAC Performance Tested MethodSM 062001

2021 ◽  
Author(s):  
Thomas Lonczynski ◽  
Laura Cowin
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Reliable Method ◽  
Sample Volume ◽  
Food Samples ◽  
Listeria Species ◽  
Environmental Surfaces ◽  
The Matrix ◽  
Environmental Surface ◽  
One Year

Abstract Background The Simultaneous Multiplex Real Time PCR (SIMUL-qPCR) Listeria species and monocytogenes Assay is a quick, reliable method for detecting Listeria species and monocytogenes in environmental and food samples. The assay multiplexes several targets in one run to properly identify Listeria species and monocytogenes. The assay uses proprietary media, Listeria Recovery and Enrichment Broth (LREB), for enrichment purposes. LREB was specifically formulated to improve the recovery and growth of Listeria while inhibiting competing background flora. Objective This report details the method validation study to validate frankfurters, ready-to-eat sliced turkey, soft fresh raw cheese, chicken salad, ice cream, cooked eggs, pasteurized milk, and frozen/cooked shrimp, as well as environmental surface sponges and swabs for stainless steel, plastic, rubber, ceramic tile, and sealed concrete. Methods Matrix studies, inclusivity/exclusivity, product consistency/stability, and robustness testing were conducted to assess the method’s performance. Results There were no statistically significant differences found between the candidate and reference methods in the matrix studies. Inclusivity/exclusivity testing showed that the assay was able to detect both Listeria species and monocytogenes strains while excluding the non-Listeria isolates. Small variations in critical test parameters (enrichment time, extraction reagent volume and extracted sample volume) did not adversely affect the assay’s performance, and stability testing indicated consistent results for at least one year. Conclusions The data presented in this report show that this a reliable method for detecting Listeria species and monocytogenes. Highlights This assay allows for one sample to be tested for both Listeria species and monocytogenes with one PCR test.

Survival of Escherichia coli O157:H7 during the Manufacture of Pepperoni†‡

1998 ◽  
Vol 61 (2) ◽  
pp. 146-151 ◽  
Author(s):  
DENISE C. R. RIORDAN ◽  
GERALDINE DUFFY ◽  
JAMES J. SHERIDAN ◽  
B. SHAWN EBLEN ◽  
RICHARD C. WHITING ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sodium Nitrite ◽  
Titratable Acidity ◽  
Escherichia Coli O157 ◽  
Growth And Survival ◽  
Commercial Formulation ◽  
E Coli ◽  
Log10 Unit ◽  
Inspection Service ◽  
The U.S

This study investigated the growth and survival of Escherichia coli O157:H7 during the manufacture of pepperoni to determine whether a 5-log10-unit decline in numbers, as recommended by the U.S. Food Safety and Inspection Service (FSIS), could be achieved. A range of pepperoni formulations with variations in salt (2.5 to 4.8%) and sodium nitrite (100 to 400 ppm) levels, and with pH (4.4 to 5.6) adjusted by manipulation of dextrose concentrations were prepared. The batters produced were inoculated with E. coli O157:H7 380-94 at a level of approximately 6.70 log10 CFU/g; changes in pathogen numbers, pH, titratable acidity, and sodium nitrite concentrations were monitored during fermentation and drying. With the standard commercial formulation (i.e., 2.5% salt, 100 ppm sodium nitrite, pH 4.8) E. coli O157:H7 numbers declined by approximately 0.41 log10 CFU/g during fermentation and a further 0.43 log10 CFU/g during subsequent drying (7 days). A regression equation was fitted to the data which showed significantly (P < 0.001) greater reductions in pathogen numbers in samples with increased salt and sodium nitrite contents and lowered pH. However declines were in all cases less than the target reduction of 5 log10 CFU/g.

scholarly journals Detection of food hazards in foods: comparison of real time polymerase chain reaction and cultural methods

2016 ◽  
Vol 5 (1) ◽  
Author(s):  
Paolo Bonilauri ◽  
Lia Bardasi ◽  
Roberto Leonelli ◽  
Mattia Ramini ◽  
Andrea Luppi ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Reference Method ◽  
Food Samples ◽  
Alternative Methods ◽  
Animal Origin ◽  
Screening Methods ◽  
Molecular Screening ◽  
Cultural Methods ◽  
Pcr Method

Foodstuffs should not contain microorganisms or their toxins or metabolites in quantities suggesting an unacceptable risk for human health. The detection of food hazards in foods is performed by several tests that produce results dependent on the analytical method used: an analytical reference method, defined as standard, is associated with each microbiological criterion laid down in Regulation 2073/2005, but, analytical methods other than the reference ones, in particular more rapid methods, could be used. Combined screening methods performed by real time PCR are currently validated as alternative methods according to the ISO 16140:2003 and certified by the Association Française de Normalisation. However, the positive results obtained with these alternative methods, the investigated molecular relations that resulted positive have to be confirmed with cultural methods using the same enrichment media in which the molecular screening was performed. Since it is necessary to assess if these testing schemes provide equivalent guarantees of food safety, the aim of this retrospective study is to analyze the data collected, from 2012 to 2014, by Emilia Romagna Region in the field of <em>Piano Regionale Alimenti</em> (Food Regional Plan), during official controls monitoring food samples, of animal and other than animal origin. Records performed by combined methods of molecular screening of <em>Salmonella</em> spp., <em>Listeria monocytogenes</em> and thermophilic <em>Campylobacter</em> and cultural confirmation results were gathered together and the results were compared in order to assess the sensitivity of the methods. A total of 10.604 food samples were considered in this study: the comparison of the data revealed that the RT-PCR method detected <em>Salmonella</em>, <em>L. monocytogenes</em>, and thermophilic <em>Campylobacter</em> in 2.18, 3.85 and 3.73% of the samples, respectively, whereas by using cultural method these pathogens were isolated in 0.43, 1.57 and 1.57 % of samples, respectively. In spite of the use of the same enrichment broth, the real time PCR method disclosed a percentage of positive samples that were negative to cultural examination ranging between 20 and 43%, with a PCR/culture ratio between 2.37 to 5.00. In conclusion, the results of this study pose a doubt about the sensitivity of the official cultural methods regarding the isolation of the three investigated foodborne pathogens. Moreover this study may be a useful tool for Veterinary Authorities to assess appropriate sampling plans to control the risk relating to the consumption of contaminated foods.

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