scholarly journals Multiplex Real-Time PCR Method To Identify Shiga Toxin Genes stx1 and stx2 and Escherichia coli O157:H7/H− Serotype

2003 ◽  
Vol 69 (10) ◽  
pp. 6327-6333 ◽  
Author(s):  
Karen C. Jinneman ◽  
Ken J. Yoshitomi ◽  
Stephen D. Weagant
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Escherichia Coli O157 ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
E Coli ◽  
Pcr Method ◽  
Shiga Toxin Genes

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.

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.

Potential pitfalls in the quantitative molecular detection ofEscherichia coliO157:H7 in environmental matrices

2006 ◽  
Vol 52 (5) ◽  
pp. 482-488 ◽  
Author(s):  
Rebekka R.E Artz ◽  
Lisa M Avery ◽  
Davey L Jones ◽  
Ken Killham
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Environmental Samples ◽  
Detection Sensitivity ◽  
Escherichia Coli O157 ◽  
Soil System ◽  
E Coli ◽  
Animal Wastes ◽  
Pcr Assays

The detection sensitivity and potential interference factors of a commonly used assay based on real-time polymerase chain reaction (PCR) for Escherichia coli O157:H7 using eae gene-specific primers were assessed. Animal wastes and soil samples were spiked with known replicate quantities of a nontoxigenic strain of E. coli O157:H7 in a viable or dead state and as unprotected DNA. The detection sensitivity and accuracy of real-time PCR for E. coli O157:H7 in animal wastes and soil is low compared to enrichment culturing. Nonviable cells and unprotected DNA were shown to produce positive results in several of the environmental samples tested, leading to potential overestimates of cell numbers due to prolonged detection of nonviable cells. This demonstrates the necessity for the specific calibration of real-time PCR assays in environmental samples. The accuracy of the eae gene–based detection method was further evaluated over time in a soil system against an activity measurement, using the bioluminescent properties of an E. coli O157:H7 Tn5luxCDABE construct. The detection of significant numbers of viable but nonculturable (VBNC) as well as nonviable and possibly physically protected cells as shown over a period of 90 days further complicates the use of real-time PCR assays for quick diagnostics in environmental samples and infers that enrichment culturing is still required for the final verification of samples found positive by real-time PCR methods.Key words: Escherichia coli O157:H7, real-time PCR, animal waste, soil, VBNC.

scholarly journals Molecular analysis of Escherichia coli O157 strains isolated from cattle and pigs by the use of PCR and pulsed-field gel electrophoresis methods

2012 ◽  
Vol 47 (No. 6) ◽  
pp. 149-158 ◽  
Author(s):  
J. Osek ◽  
P. Gallien
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Genetic Relatedness ◽  
Rflp Analysis ◽  
Toxin Gene ◽  
Escherichia Coli O157 ◽  
Chromosomal Dna ◽  
E Coli ◽  
Shiga Toxin Genes ◽  
Porcine Origin

Fourteen Escherichia coli O157 strains isolated from cattle and pigs in Poland and in Germany were investigated, using PCR, for the genetic markers associated with Shiga toxin-producing E. coli (STEC). Only two strains, both of cattle origin, were positive for the fliC (H7) gene and could be classified as O157 : H7. Nine isolates had stx shiga toxin genes, either stx1 (1 strain), stx2 (4 isolates) or both (4 strains). The stx2-carrying samples were further subtyped by PCR for the stx2c, stx2d, and stx2e toxin variants. It was shown that all but one stx2-positive bacteria possessed the stx2c Shiga toxin gene type and one stx2 STEC isolate had the stx2d virulence factor sub-type. The eaeA (intimin) gene was found in 9 strains (8 isolates from cattle and one strain from pigs); all of them harboured the genetic marker characteristic of the gamma intimin variant. The translocated intimin receptor (tir) gene was detected in 7 isolates tested and among them only one tir-positive strain was recovered from pigs. The ehly E. coli enterohemolysin gene was amplified in all but one strains obtained from cattle and only in one isolate of porcine origin. The genetic relatedness of the analysed E. coli O157 strains was examined by restriction fragment length polymorphism (RFLP) of chromosomal DNA digested with XbaI. Two distinct but related RFLP pattern clusters were observed: one with 9 strains (8 isolates of bovine origin and one strain obtained from pigs) and the other one comprises the remaining 5 E. coli isolates (4 of porcine origin and one strain recovered from cattle). The results suggest that pigs, besides cattle, may be a reservoir of E. coli O157 strains potentially pathogenic to humans. Moreover, epidemiologically unrelated isolates of the O157 serogroup, recovered from different animal species, showed a clonal relationship as demonstrated by the RFLP analysis.

scholarly journals Evaluation of multiplex SYBR green real-time PCR assay for detection of pathogenic Escherichia coli

2020 ◽  
Vol 9 (2) ◽  
pp. 448
Author(s):  
Ema Komalasari ◽  
Winiati P. Rahayu ◽  
Siti Nurjanah
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Real Time Pcr ◽  
Sybr Green ◽  
Rt Pcr ◽  
E Coli ◽  
Melting Curves ◽  
Pathogenic Escherichia Coli ◽  
Wide Range ◽  
Pcr Method

Pathogenic Escherichia coli (E. coli) has been implicated in a wide range of disease causing infections. It is essential to generate a method for detecting and differentiating each pathotype of E. coli which is more quickly and efficiently by using less reagent. This study aimed to evaluate a SYBR Green multiplex real-time PCR method for detecting four types of pathogenic E. coli. Two of multiplex real-time PCR system, 6-plex and 3-plex, were set to detect six different virulence factors from ETEC, EPEC, EHEC, and EIEC and evaluate the melting curves and specificity compared to simplex method. The results showed that 3-plex rt-PCR method gave more reliable melting curves than 6-plex. The 3-plex rt-PCR also provided similar melting value (Tm) to simplex system. The results of this specificity assay supported the selection of 3-plex rt-PCR conditions for detection of pathogenic E. coli.

scholarly journals High-resolution melting real-time PCR assays for detection of Escherichia coli O26 and O111 strains possessing Shiga toxin genes

LWT ◽  
2020 ◽  
Vol 131 ◽  
pp. 109785
Author(s):  
Prashant Singh ◽  
Gabriel Cubillos ◽  
Gabrielle Kirshteyn ◽  
Joseph M. Bosilevac
Keyword(s):  
Escherichia Coli ◽  
High Resolution ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Toxin Genes ◽  
Pcr Assays ◽  
Shiga Toxin Genes

Recirculating Immunomagnetic Separation and Optimal Enrichment Conditions for Enhanced Detection and Recovery of Low Levels of Escherichia coli O157:H7 from Fresh Leafy Produce and Surface Water

2007 ◽  
Vol 70 (12) ◽  
pp. 2717-2724 ◽  
Author(s):  
SUNEE HIMATHONGKHAM ◽  
MARY LEE DODD ◽  
JENNY K. YEE ◽  
DAVID K. LAU ◽  
RAYMOND G. BRYANT ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Surface Water ◽  
Real Time ◽  
Real Time Pcr ◽  
Immunomagnetic Separation ◽  
Escherichia Coli O157 ◽  
Simple Method ◽  
E Coli ◽  
Low Levels ◽  
Spinach Leaves

The objective of this study was to develop a rapid, simple method for enhanced detection and isolation of low levels of Escherichia coli O157:H7 from leafy produce and surface water using recirculating immunomagnetic separation (RIMS) coupled with real-time PCR and a standard culture method. The optimal enrichment conditions for the method also were determined. Analysis of real-time PCR data (CT values) suggested that incubation of lettuce and spinach leaves rather than rinsates provides better enrichment of E. coli O157:H7. Enrichment of lettuce or spinach leaves at 42°C for 5 h provided better detection than enrichment at 37°C. Extended incubation of surface water for 20 h at 42°C did not improve the detection. The optimized enrichment conditions were also employed with modified Moore swabs, which were used to sample flowing water sites. Positive isolation rates and real-time PCR results indicated an increased recovery of E. coli O157:H7 from all samples following the application of RIMS. Under these conditions, the method provided detection and/or isolation of E. coli O157:H7 at levels as low as 0.07 CFU/g of lettuce, 0.1 CFU/g of spinach, 6 CFU/100 ml of surface water, and 9 CFU per modified Moore swab. During a 6-month field study, modified Moore swabs yielded high isolation rates when deployed in natural watershed sites. The method used in this study was effective for monitoring E. coli O157:H7 in the farm environment, during postharvest processing, and in foodborne outbreak investigations.

scholarly journals Contribution and Interaction of Shiga Toxin Genes to Escherichia coli O157:H7 Virulence

Toxins ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 607 ◽  
Author(s):  
Gillian A.M. Tarr ◽  
Taryn Stokowski ◽  
Smriti Shringi ◽  
Phillip I. Tarr ◽  
Stephen B. Freedman ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Population Level ◽  
Escherichia Coli O157 ◽  
Additive Interaction ◽  
Shiga Toxins ◽  
E Coli ◽  
Uremic Syndrome ◽  
Shiga Toxin Genes

Escherichia coli O157:H7 is the predominant cause of diarrhea-associated hemolytic uremic syndrome (HUS) worldwide. Its cardinal virulence traits are Shiga toxins, which are encoded by stx genes, the most common of which are stx1a, stx2a, and stx2c. The toxins these genes encode differ in their in vitro and experimental phenotypes, but the human population-level impact of these differences is poorly understood. Using Shiga toxin-encoding bacteriophage insertion typing and real-time polymerase chain reaction, we genotyped isolates from 936 E. coli O157:H7 cases and verified HUS status via chart review. We compared the HUS risk between isolates with stx2a and those with stx2a and another gene and estimated additive interaction of the stx genes. Adjusted for age and symptoms, the HUS incidence of E. coli O157:H7 containing stx2a alone was 4.4% greater (95% confidence interval (CI) −0.3%, 9.1%) than when it occurred with stx1a. When stx1a and stx2a occur together, the risk of HUS was 27.1% lower (95% CI −87.8%, −2.3%) than would be expected if interaction were not present. At the population level, temporal or geographic shifts toward these genotypes should be monitored, and stx genotype may be an important consideration in clinically predicting HUS among E. coli O157:H7 cases.

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.

Escherichia coli O157:H7 in Retail Ground Beef in Seattle: Results of a One-Year Prospective Study

1999 ◽  
Vol 62 (2) ◽  
pp. 133-139 ◽  
Author(s):  
PHILLIP I. TARR ◽  
NHIEM THANH TRAN ◽  
RICHARD A. WILSON
Keyword(s):  
Escherichia Coli ◽  
Prospective Study ◽  
Shiga Toxin ◽  
Enrichment Culture ◽  
Ground Beef ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Toxin Genes ◽  
One Year ◽  
Shiga Toxin Genes

Escherichia coli O157:H7 was sought systematically in 1,400 samples of retail ground beef in Seattle in a 1-year prospective study. Sorbitol-nonfermenting, lactose-fermenting, indole-positive colonies isolated after enrichment culture were probed for the presence of Shiga toxin genes. Totals of 67,040 sorbitol-nonfermenting and 66,705 sorbitol-fermenting colonies were characterized, but E. coli O157:H7 was not identified. The sensitivity of this technique was usually sufficient to detect E. coli O157:H7 at a concentration below 1 CFU/g of meat. These data demonstrate that retail ground beef in Seattle is neither frequently nor heavily contaminated with E. coli O157:H7.

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