scholarly journals Development of 11-Plex MOL-PCR Assay for the Rapid Screening of Samples for Shiga Toxin-Producing Escherichia coli

2016 ◽  
Vol 6 ◽  
Author(s):  
Travis A. Woods ◽  
Heather M. Mendez ◽  
Sandy Ortega ◽  
Xiaorong Shi ◽  
David Marx ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Rapid Screening ◽  
Pcr Assay

An Automated Fluorescent PCR Method for Detection of Shiga Toxin-Producing Escherichia coli in Foods

1998 ◽  
Vol 64 (11) ◽  
pp. 4210-4216 ◽  
Author(s):  
Shu Chen ◽  
Renlin Xu ◽  
Arlene Yee ◽  
Kai Yuan Wu ◽  
Chang-Ning Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Raw Milk ◽  
Clinical Samples ◽  
Pcr Assay ◽  
System A ◽  
Pure Cultures ◽  
Pcr Method ◽  
Meat Samples ◽  
Shiga Toxin Genes

ABSTRACT An automated fluorescence-based PCR system (a model AG-9600 AmpliSensor analyzer) was investigated to determine whether it could detect Shiga toxin-producing Escherichia coli (STEC). The AmpliSensor PCR assay involves amplification-mediated disruption of a fluorogenic DNA signal duplex (AmpliSensor) that is homologous to conserved target sequences in a 323-bp amplified fragment of Shiga toxin genes stx 1, stx 2, and stx e. Using the Amplisensor assay, we detected 113 strains of STEC belonging to 50 different serotypes, while 18 strains of non-Shiga-toxin-producing E. coli and 68 strains of other bacteria were not detected. The detection limits of the assay were less than 1 to 5 CFU per PCR mixture when pure cultures of five reference strains were used and 3 CFU per 25 g of food when spiked ground beef samples that were preenriched overnight were used. The performance of the assay was also evaluated by using 53 naturally contaminated meat samples and 48 raw milk samples. Thirty-two STEC-positive samples that were confirmed to be positive by the culture assay were found to be positive when the AmpliSensor assay was used. Nine samples that were found to be positive when the PCR assay was used were culture negative. The system described here is an automated PCR-based system that can be used for detection of all serotypes of STEC in food or clinical samples.

Isolation and detection of Shiga toxin-producing Escherichia coli in clinical stool samples using conventional and molecular methods

2009 ◽  
Vol 58 (7) ◽  
pp. 905-911 ◽  
Author(s):  
Matthew W. Gilmour ◽  
Linda Chui ◽  
Theodore Chiu ◽  
Dobryan M. Tracz ◽  
Kathryn Hagedorn ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Molecular Methods ◽  
Genetic Profile ◽  
Pcr Assay ◽  
Suspension Array ◽  
Stool Samples ◽  
Pcr Targeting

The isolation of Shiga toxin-producing Escherichia coli (STEC) other than serogroup O157 from clinical stool samples is problematic due to the lack of differential phenotypic characteristics from non-pathogenic E. coli. The development of molecular reagents capable of identifying both toxin and serogroup-specific genetic determinants holds promise for a more comprehensive characterization of stool samples and isolation of STEC strains. In this study, 876 stool samples from paediatric patients with gastroenteritis were screened for STEC using a cytotoxicity assay, commercial immunoassay and a conventional PCR targeting Shiga-toxin determinants. In addition, routine culture methods for isolating O157 STEC were also performed. The screening assays identified 45 stools presumptively containing STEC, and using non-differential culture techniques a total of 20 O157 and 22 non-O157 strains were isolated. These included STEC serotypes O157 : H7, O26 : H11, O121 : H19, O26 : NM, O103 : H2, O111 : NM, O115 : H18, O121 : NM, O145 : NM, O177 : NM and O5 : NM. Notably, multiple STEC serotypes were isolated from two clinical stool samples (yielding O157 : H7 and O26 : H11, or O157 : H7 and O103 : H2 isolates). These data were compared to molecular serogroup profiles determined directly from the stool enrichment cultures using a LUX real-time PCR assay targeting the O157 fimbrial gene lpfA, a microsphere suspension array targeting allelic variants of espZ and a gnd-based molecular O-antigen serogrouping method. The genetic profile of individual stool cultures indicated that the espZ microsphere array and lpfA real-time PCR assay could accurately predict the presence and provide preliminary typing for the STEC strains present in clinical samples. The gnd-based molecular serogrouping method provided additional corroborative evidence of serogroup identities. This toolbox of molecular methods provided robust detection capabilities for STEC in clinical stool samples, including co-infection of multiple serogroups.

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.

Survey of Intact and Nonintact Raw Pork Collected at Retail Stores in the Mid-Atlantic Region of the United States for the Seven Regulated Serogroups of Shiga Toxin–Producing Escherichia coli

2019 ◽  
Vol 82 (11) ◽  
pp. 1844-1850
Author(s):  
YANGJIN JUNG ◽  
ANNA C. S. PORTO-FETT ◽  
BRADLEY A. SHOYER ◽  
LAURA E. SHANE ◽  
ELIZABETH HENRY ◽  
...  
Keyword(s):  
United States ◽  
Escherichia Coli ◽  
Shiga Toxin ◽  
Virulence Genes ◽  
The United States ◽  
Retail Stores ◽  
Pcr Assay ◽  
Atlantic Region ◽  
O Antigens ◽  
Pork Samples

ABSTRACT A total of 514 raw pork samples (395 ground or nonintact and 119 intact samples) were purchased at retail stores in Pennsylvania, Delaware, and New Jersey between July and December 2017. All raw pork samples were screened for serogroup O26, O45, O103, O111, O121, O145, or O157:H7 cells of Shiga toxin–producing Escherichia coli (STEC-7) using standard microbiological and molecular methods. In short, 21 (5.3%) of the 395 ground or nonintact pork samples and 3 (3.4%) of the 119 intact pork samples tested positive via the BAX system real-time PCR assay for the stx and eae virulence genes and for the somatic O antigens for at least one of the STEC-7 serogroups. However, none of these 24 presumptive-positive pork samples subsequently yielded a viable isolate of STEC displaying a STEC-7 serogroup-specific surface antigen in combination with the stx and eae genes. These data suggest that cells of STEC serogroups O26, O45, O103, O111, O121, O145, or O157:H7 are not common in retail raw pork samples in the mid-Atlantic region of the United States.

scholarly journals Development of a real-time PCR assay and comparison to CHROMagarTM STEC to screen for Shiga toxin-producing Escherichia coli in stool, Cape Town, South Africa

2017 ◽  
Vol 6 (1) ◽  
Author(s):  
John B. Kalule ◽  
Karen H. Keddy ◽  
Anthony Smith ◽  
Mark P. Nicol ◽  
Lourens Robberts
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Predictive Value ◽  
Limit Of Detection ◽  
Reference Standard ◽  
Pcr Assay ◽  
Diagnostic Technology ◽  
Sensitivity Specificity

Introduction: Shiga toxin-producing Escherichia coli (STEC) is an emerging infectious pathogen which could lead to haemolytic uremic syndrome. Even though previous studies have compared the performance of CHROMagarTMSTEC to real-time polymerase chain reaction (PCR) in Europe, no study has been done to assess its performance on African isolates.Objectives: This project aimed to validate and test an in-house-developed duplex real-time PCR and use it as a reference standard to determine the performance of CHROMagarTMSTEC on African isolates from diarrhoeic stool samples.Methods: This study evaluated STEC diagnostic technology on African isolates. An in-house-developed duplex real-time PCR assay for detection of stx1 and stx2 was validated and tested on diarrhoeic stool samples and then used as a reference standard to assess the performance of CHROMagarTMSTEC. Real-time PCR was used to screen for stx in tryptic soy broth and the suspected STEC isolates, while conventional PCR was used to detect the other virulence genes possessed by the isolates.Results: The real-time PCR limit of detection was 5.3 target copies/μL of broth. The mean melting temperature on melt-curve analysis for detection of stx1 was 58.2 °C and for stx2 was 65.3 °C. Of 226 specimens screened, real-time PCR detected stx in 14 specimens (6.2%, 95% confidence interval = 3.43% – 10.18%). The sensitivity, specificity, negative predictive value and positive predictive value of the CHROMagarTMSTEC were 33.3%, 77.4%, 95.3% and 11.3%.Conclusions: The in-house developed real-time PCR assay is a sensitive and specific option for laboratory detection of STEC as compared to CHROMagarTMSTEC in this setting.

Promising Nucleic Acid Lateral Flow Assay Plus PCR for Shiga Toxin–Producing Escherichia coli

2015 ◽  
Vol 78 (8) ◽  
pp. 1560-1568 ◽  
Author(s):  
YOSHITAKA TERAO ◽  
KANA TAKESHITA ◽  
YASUTAKA NISHIYAMA ◽  
NAOKI MORISHITA ◽  
TAKASHI MATSUMOTO ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Detection Limit ◽  
Nucleic Acid ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Pcr Assay ◽  
E Coli ◽  
Lower Detection Limit ◽  
Lower Detection

Shiga toxin (Stx)–producing Escherichia coli (STEC) is a frequent cause of foodborne infections, and methods for rapid and reliable detection of STEC are needed. A nucleic acid lateral flow assay (NALFA) plus PCR was evaluated for detecting STEC after enrichment. When cell suspensions of 45 STEC strains, 14 non-STEC strains, and 13 non–E. coli strains were tested with the NALFA plus PCR, all of the STEC strains yielded positive results, and all of the non-STEC and non–E. coli strains yielded negative results. The lower detection limit for the STEC strains ranged from 0.1 to 1 pg of genomic DNA (about 20 to 200 CFU) per test, and the NALFA plus PCR was able to detect Stx1- and Stx2-producing E. coli strains with similar sensitivities. The ability of the NALFA plus PCR to detect STEC in enrichment cultures of radish sprouts, tomato, raw ground beef, and beef liver inoculated with 10-fold serially diluted STEC cultures was comparable to that of a real-time PCR assay (at a level of 100 to 100,000 CFU/ml in enrichment culture). The bacterial inoculation test in raw ground beef revealed that the lower detection limit of the NALFA plus PCR was also comparable to that obtained with a real-time PCR assay that followed the U.S. Department of Agriculture guidelines. Although further evaluation is required, these results suggest that the NALFA plus PCR is a specific and sensitive method for detecting STEC in a food manufacturing plant.

scholarly journals Identification, Shiga toxin subtypes and prevalence of minor serogroups of Shiga toxin-producing Escherichia coli in feedlot cattle feces

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kaylen M. Capps ◽  
Justin B. Ludwig ◽  
Pragathi B. Shridhar ◽  
Xiaorong Shi ◽  
Elisabeth Roberts ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Foodborne Pathogens ◽  
Shiga Toxin ◽  
Feedlot Cattle ◽  
Pcr Assay ◽  
Cattle Feces ◽  
Virulence Potential ◽  
Multiplex Pcr Assay ◽  
Human Infections

AbstractShiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that cause illnesses in humans ranging from mild to hemorrhagic enteritis with complications of hemolytic uremic syndrome and even death. Cattle are a major reservoir of STEC, which reside in the hindgut and are shed in the feces, a major source of food and water contaminations. Seven serogroups, O26, O45, O103, O111, O121, O145 and O157, called ‘top-7’, are responsible for the majority of human STEC infections in North America. Additionally, 151 serogroups of E. coli are known to carry Shiga toxin genes (stx). Not much is known about fecal shedding and prevalence and virulence potential of STEC other than the top-7. Our primary objectives were to identify serogroups of STEC strains, other than the top-7, isolated from cattle feces and subtype stx genes to assess their virulence potential. Additional objective was to develop and validate a novel multiplex PCR assay to detect and determine prevalence of six serogroups, O2, O74, O109, O131, O168, and O171, in cattle feces. A total of 351 strains, positive for stx gene and negative for the top-7 serogroups, isolated from feedlot cattle feces were used in the study. Of the 351 strains, 291 belonged to 16 serogroups and 60 could not be serogrouped. Among the 351 strains, 63 (17.9%) carried stx1 gene and 300 (82.1%) carried stx2, including 12 strains positive for both. The majority of the stx1 and stx2 were of stx1a (47/63; 74.6%) and stx2a subtypes (234/300; 78%), respectively, which are often associated with human infections. A novel multiplex PCR assay developed and validated to detect six serogroups, O2, O74, O109, O131, O168, and O171, which accounted for 86.9% of the STEC strains identified, was utilized to determine their prevalence in fecal samples (n = 576) collected from a commercial feedlot. Four serogroups, O2, O109, O168, and O171 were identified as the dominant serogroups prevalent in cattle feces. In conclusion, cattle shed in the feces a number of STEC serogroups, other than the top-7, and the majority of the strains isolated possessed stx2, particularly of the subtype 2a, suggesting their potential risk to cause human infections.

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