scholarly journals Comparison of Methods for DNA Isolation from Food Samples for Detection of Shiga Toxin-Producing Escherichia coli by Real-Time PCR

2003 ◽  
Vol 69 (3) ◽  
pp. 1844-1846 ◽  
Author(s):  
Loree C. Heller ◽  
Carisa R. Davis ◽  
K. Kealy Peak ◽  
David Wingfield ◽  
Andrew C. Cannons ◽  
...  

ABSTRACT In this study, food samples were intentionally contaminated with Escherichia coli O157:H7, and then DNA was isolated by using four commercial kits. The isolated DNA samples were compared by using real-time PCR detection of the Shiga toxin genes. The four kits tested worked similarly.

LWT ◽  
2020 ◽  
Vol 131 ◽  
pp. 109785
Author(s):  
Prashant Singh ◽  
Gabriel Cubillos ◽  
Gabrielle Kirshteyn ◽  
Joseph M. Bosilevac

2003 ◽  
Vol 69 (10) ◽  
pp. 6327-6333 ◽  
Author(s):  
Karen C. Jinneman ◽  
Ken J. Yoshitomi ◽  
Stephen D. Weagant

ABSTRACT A multiplex real-time PCR method to simultaneously detect the stx1 and stx2 genes of Shiga toxin-producing Escherichia coli and a unique conserved single-nucleotide polymorphism in the E. coli O157:H7/H− uidA gene has been developed. There is more than 98.6% sensitivity and 100% specificity for all three gene targets based on a panel of 138 isolates. The PCR efficiencies were ≥1.89, and as few as 6 CFU/reaction could be detected.


2020 ◽  
Vol 8 (11) ◽  
pp. 1801
Author(s):  
Michael Bording-Jorgensen ◽  
Brendon D. Parsons ◽  
Gillian A.M. Tarr ◽  
Binal Shah-Gandhi ◽  
Colin Lloyd ◽  
...  

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.


2013 ◽  
Vol 198 (2) ◽  
pp. 538-540 ◽  
Author(s):  
Jae-Won Byun ◽  
Byeong Yeal Jung ◽  
Ha-Young Kim ◽  
John M. Fairbrother ◽  
Myoung-Heon Lee ◽  
...  

2005 ◽  
Vol 68 (8) ◽  
pp. 1593-1599 ◽  
Author(s):  
MICHAEL A. GRANT

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.


2019 ◽  
Vol 82 (9) ◽  
pp. 1512-1523
Author(s):  
ODBERT A. TRIPLETT ◽  
JIEKUN XUAN ◽  
STEVEN FOLEY ◽  
RAJESH NAYAK ◽  
WILLIAM H. TOLLESON

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.


2015 ◽  
Vol 53 (3) ◽  
pp. 1019-1023 ◽  
Author(s):  
Linda Chui ◽  
Laura Patterson-Fortin ◽  
Julie Kuo ◽  
Vincent Li ◽  
Valerie Boras

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.


2013 ◽  
Vol 76 (5) ◽  
pp. 871-873 ◽  
Author(s):  
HEIKE MARGOT ◽  
NICOLE CERNELA ◽  
CAROL IVERSEN ◽  
CLAUDIO ZWEIFEL ◽  
ROGER STEPHAN

Following the recent outbreak of Shiga toxin–producing Escherichia coli (STEC) O104:H4 infection in Germany, the demand for fast detection of STEC has again increased. Various real-time PCR-based methods enabling detection of Shiga toxin genes (stx) have been developed and can be used for applications in food microbiology. The present study was conducted to evaluate the reliability of seven commercially available real-time PCR systems for detection of stx1 and stx2 subtypes. For this purpose, pure cultures of 18 STEC strains harboring all known stx1 and/or stx2 subtypes were tested. Only one of the seven real-time PCR systems detected all known stx1 and stx2 subtypes. Six systems failed to detect the stx2f subtype. One system missed stx2 subtypes reported in association with severe human disease. Because the presence of certain stx genes (subtypes) is considered an important indicator of STEC virulence, systems differentiating between the stx1 and stx2 gene groups provide added value. Reliable and fast detection of stx genes is of major importance for both diagnostic laboratories and the food industry.


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