scholarly journals Detection of Shiga toxin-producing Escherichia coli in ground beef using the GeneDisc real-time PCR system

2012 ◽  
Vol 2 ◽  
Author(s):  
Pina M. Fratamico ◽  
Lori K. Bagi
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Ground Beef

Influence of Primer Sequences and DNA Extraction Method on Detection of Non-O157 Shiga Toxin–Producing Escherichia coli in Ground Beef by Real-Time PCR Targeting the eae, stx, and Serogroup-Specific Genes†

2012 ◽  
Vol 75 (11) ◽  
pp. 1939-1950 ◽  
Author(s):  
JAMIE L. WASILENKO ◽  
PINA M. FRATAMICO ◽  
NEELAM NARANG ◽  
GLENN E. TILLMAN ◽  
SCOTT LADELY ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Dna Extraction ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Extraction Method ◽  
Ground Beef ◽  
Dna Extraction Method ◽  
Big Six ◽  
Pcr Targeting

Non-O157 Shiga toxin–producing Escherichia coli (STEC) infections, particularly those caused by the “big six” or “top six” non-O157 serogroups (O26, O45, O103, O111, O121, and O145) can result in severe illness and complications. Because of their significant public health impact and the notable prevalence of STEC in cattle, methods for detection of the big six non-O157 STEC in ground beef have been established. Currently, the U.S. Department of Agriculture, Food Safety and Inspection Service detection methods for screening beef samples for non-O157 STEC target the stx1, stx2, and eae virulence genes, with the 16S rRNA gene as an internal control, in a real-time PCR multiplex assay. Further, the serogroup is determined by PCR targeting genes in the E. coli O-antigen gene clusters of the big six non-O157 serogroups. The method that we previously reported was improved so that additional stx variants, stx1d, stx2e, and stx2g, are detected. Additionally, alignments of the primers targeting the eae gene were used to improve the detection assay so that eae subtypes that could potentially be of clinical significance would also be detected. Therefore, evaluation of alternative real-time PCR assay primers and probes for the stx and eae reactions was carried out in order to increase the stx and eae subtypes detected. Furthermore, a Tris-EDTA DNA extraction method was compared with a previously used procedure that was based on a commercially available reagent. The Tris-EDTA DNA extraction method significantly decreased the cycle threshold values for the stx assay (P < 0.0001) and eae assay (P < 0.0001), thereby increasing the ability to detect the targets. The use of different stx primers and probes increased the subtypes detected to include stx1d, stx2e, and stx2g, and sequence data showed that modification of the eae primer should allow the known eae subtypes to be detected.

scholarly journals Application of real-time PCR for detection of antibiotic resistant pathogens and Shiga-toxin producing Escherichia coli

2015 ◽  
Author(s):  
◽  
Prashant Singh
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Ground Beef ◽  
The United States ◽  
Multiple Drug ◽  
United States Department ◽  
Antibiotic Resistant ◽  
Food Pathogens

Salmonella and Shiga toxin producing Escherichia coli (STEC) are among the most important food pathogens. Increasing use of antibiotics for treatment and as a therapeutic agent on food animals has been proposed as a reason for the emergence of multiple drug resistant (MDR) strains of food pathogens. In this study real-time PCR methods were developed for the detection antibiotic resistant strains of Salmonella, extended-spectrum [beta]-lactam (ESBL) and carbapenem resistant pathogens. The United States Department of Agriculture Food Safety and Inspection Service (USDA-FSIS) declared seven STEC serogroups O157, O26, O45, O103, O111, O121 and O145 as adulterants in ground beef and beef trims. Multiplex real-time PCR melt curve assays with IAC were standardized for the detection of seven STEC serogroups with their virulence genes and Salmonella. The assay was able to detect all STEC strains in 325 g of ground beef and beef trims spiked with 10 CFU.

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 Association of Ct Values from Real-Time PCR with Culture in Microbiological Clearance Samples for Shiga Toxin-Producing Escherichia coli (STEC)

2020 ◽  
Vol 8 (11) ◽  
pp. 1801
Author(s):  
Michael Bording-Jorgensen ◽  
Brendon D. Parsons ◽  
Gillian A.M. Tarr ◽  
Binal Shah-Gandhi ◽  
Colin Lloyd ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Gene Detection ◽  
Culture Positivity ◽  
Hands On ◽  
Culture Independent ◽  
Chromogenic Agar ◽  
Stool Samples

Shiga toxin-producing Escherichia coli (STEC) are associated with acute gastroenteritis worldwide, which induces a high economic burden on both healthcare and individuals. Culture-independent diagnostic tests (CIDT) in frontline microbiology laboratories have been implemented in Alberta since 2019. The objectives of this study were to determine the association between gene detection and culture positivity over time using STEC microbiological clearance samples and also to establish the frequency of specimen submission. Both stx genes’ amplification by real-time PCR was performed with DNA extracted from stool samples using the easyMAG system. Stools were inoculated onto chromogenic agar for culture. An association between gene detection and culture positivity was found to be independent of which stx gene was present. CIDT can provide rapid reporting with less hands-on time and technical expertise. However, culture is still important for surveillance and early cluster detection. In addition, stool submissions could be reduced from daily to every 3–5 days until a sample is negative by culture.

Real-time PCR for differentiation of F18 variants among enterotoxigenic and Shiga toxin-producing Escherichia coli from piglets with diarrhoea and oedema disease

2013 ◽  
Vol 198 (2) ◽  
pp. 538-540 ◽  
Author(s):  
Jae-Won Byun ◽  
Byeong Yeal Jung ◽  
Ha-Young Kim ◽  
John M. Fairbrother ◽  
Myoung-Heon Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Oedema Disease

Comparison of a New Enrichment Procedure for Shiga Toxin–Producing Escherichia coli with Five Standard Methods

2005 ◽  
Vol 68 (8) ◽  
pp. 1593-1599 ◽  
Author(s):  
MICHAEL A. GRANT
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Target Cells ◽  
Macconkey Agar ◽  
Standard Methods ◽  
E Coli ◽  
Canadian Health ◽  
Health Products

A new procedure for enrichment of Escherichia coli O157:H7 and other Shiga toxin–producing E. coli was compared to five standard methods: the British Public Health Laboratory Service, International Standard Method, U.S. Department of Agriculture, Canadian Health Products and Food Branch, and U.S. Food and Drug Administration. The new procedure was comparable to the standard methods in its ability to detect target cells inoculated into foods at approximately 1 CFU g−1. Comparisons were also made of the ability of the six enrichment procedures to detect E. coli O157:H7 against a large background of competitor microorganisms. In these experiments the new procedure yielded more target cells than the other five enrichments by two to three orders of magnitude as determined by enumeration on sorbitol MacConkey agar with tellurite and cefixime and Rainbow agar with tellurite and novobiocin and by verification of presumptive colonies by real-time PCR. For example, the population of enterohemorrhagic E. coli strain 6341 recovered on sorbitol MacConkey agar with tellurite and cefixime after enrichment with the experimental method was 2.42 × 108 CFU ml−1 and 1.80 × 106 CFU ml−1 after enrichment with the Canadian Health Products and Food Branch method, the second most effective in this experiment. In addition, broth cultures resulting from each of the six enrichment procedures were used to prepare templates for real-time PCR detection of stx1/stx2. Resulting threshold cycle (Ct) values after the experimental enrichment were similar to positive control values, whereas the five standard methods produced delayed Ct values or were not detected.

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.

Immunomagnetic Capture of Big Six Shiga Toxin–Producing Escherichia coli Strains in Apple Juice with Detection by Multiplex Real-Time PCR Eliminates Interference from the Food Matrix

2019 ◽  
Vol 82 (9) ◽  
pp. 1512-1523
Author(s):  
ODBERT A. TRIPLETT ◽  
JIEKUN XUAN ◽  
STEVEN FOLEY ◽  
RAJESH NAYAK ◽  
WILLIAM H. TOLLESON
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Internal Control ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Apple Juice ◽  
Dna Amplification ◽  
Probability Of Detection ◽  
Solid Agar ◽  
Big Six

ABSTRACT Having reliable methods for detecting Shiga toxin–producing Escherichia coli (STEC) in foods is an important food safety goal. The majority of STEC outbreaks have involved either the O157:H7 serotype or one of six non-O157 serogroups, O26, O45, O103, O111, O121, and O145, termed “The Big Six.” We have compared detection by PCR of the Shiga toxin genes stx1a and stx2a from STEC bacteria isolated from unclarified apple juice by simple centrifugation with the use of an immunocapture technique to minimize contaminants (such as pectin and polyphenols that may copurify with DNA) that may interfere with DNA amplification efficiencies and limit sensitivity. An internal control for successful immunocapture, DNA extraction, and PCR amplification was generated by introducing the pmRaspberry plasmid into an stx null strain, yielding an E. coli O45 pmRaspberry derivative that can be added to food samples directly. Using serial dilutions of a representative Big Six STEC in apple juice, our immunocapture method resulted in a 50% probability of detection value of 3.34, 2.25, and 4.25 CFU for detection by multiplex real-time PCR, growth on solid agar, and multiplex endpoint PCR, respectively. The time to result was 6.5 h, 9.5 h, and 1.5 days for immunocapture of Big Six STECs and detection by multiplex real-time PCR, endpoint PCR, and growth on solid agar, respectively. A set of 52 Big Six STEC isolates and 30 non–Big Six STEC strains was used to establish the inclusivity and exclusivity of the method. Finally, the ability to detect Big Six STEC contamination reliably was confirmed at 4.5 and 45 CFU/25-mL portions of refrigerated apple juice.

scholarly journals Evaluation of Enzyme Immunoassays and Real-Time PCR for Detecting Shiga Toxin-Producing Escherichia coli in Southern Alberta, Canada

2015 ◽  
Vol 53 (3) ◽  
pp. 1019-1023 ◽  
Author(s):  
Linda Chui ◽  
Laura Patterson-Fortin ◽  
Julie Kuo ◽  
Vincent Li ◽  
Valerie Boras
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Content Type ◽  
Enzyme Immunoassays ◽  
Southern Alberta

Two immunoassays (Shiga Toxin Chek and Shiga Toxin Quik Chek) and real-time PCR were used to detect Shiga toxin-producingEscherichia coli. For enriched culture, the sensitivity and specificity of the three methods ranged from 80.0% to 98.2% and 98.0% to 100.0%, respectively. STEC isolates were identified in 2.6% of the 784 samples.

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