scholarly journals Simultaneous Detection of Salmonella Strains and Escherichia coli O157:H7 with Fluorogenic PCR and Single-Enrichment-Broth Culture

2000 ◽  
Vol 66 (12) ◽  
pp. 5472-5476 ◽  
Author(s):  
Vijay K. Sharma ◽  
Steve A. Carlson
Keyword(s):  
Escherichia Coli ◽  
Simultaneous Detection ◽  
Broth Culture ◽  
Escherichia Coli O157 ◽  
Specific Detection ◽  
Pcr Assay ◽  
Enrichment Broth ◽  
E Coli ◽  
Serovar Typhimurium ◽  
Low Levels

ABSTRACT A multiplex fluorogenic PCR assay for simultaneous detection of pathogenic Salmonella strains and Escherichia coli O157:H7 was developed and evaluated for use in detecting very low levels of these pathogens in meat and feces. Two sets of primers were used to amplify a junctional segment of virulence genessipB and sipC of Salmonella and an intragenic segment of gene eae of E. coliO157:H7. Fluorogenic reporter probes were included in the PCR assay for automated and specific detection of amplified products. The assay could detect <10 CFU of Salmonella enterica serovar Typhimurium or E. coli O157:H7 per g of meat or feces artificially inoculated with these pathogens and cultured for 6 to 18 h in a single enrichment broth. Detection of amplification products could be completed in ≤4 h after enrichment.

Validation of Low-Volume Enrichment Protocols for Detection of Escherichia coli O157 in Raw Ground Beef Components, Using Commercial Kits

2009 ◽  
Vol 72 (3) ◽  
pp. 669-673 ◽  
Author(s):  
IMTIAZ AHMED ◽  
DENISE HUGHES ◽  
IAN JENSON ◽  
TASS KARALIS
Keyword(s):  
Escherichia Coli ◽  
Cost Savings ◽  
Ground Beef ◽  
Escherichia Coli O157 ◽  
Enrichment Broth ◽  
E Coli ◽  
Cultural Methods ◽  
Beef Products ◽  
Low Levels ◽  
Commercial Kits

Testing of beef destined for use in ground beef products for the presence of Escherichia coli O157:H7 has become an important cornerstone of control and verification activities within many meat supply chains. Validation of the ability of methods to detect low levels of E. coli O157:H7 is critical to confidence in test systems. Many rapid methods have been validated against standard cultural methods for 25-g samples. In this study, a number of previously validated enrichment broths and commercially available test kits were validated for the detection of low numbers of E. coli O157:H7 in 375-g samples of raw ground beef component matrices using 1 liter of enrichment broth (large-sample:low-volume enrichment protocol). Standard AOAC International methods for 25-g samples in 225 ml of enrichment broth, using the same media, incubation conditions, and test kits, were used as reference methods. No significant differences were detected in the ability of any of the tests to detect low levels of E. coli O157:H7 in samples of raw ground beef components when enriched according to standard or large-sample:low-volume enrichment protocols. The use of large-sample:low-volume enrichment protocols provides cost savings for media and logistical benefits when handling and incubating large numbers of samples.

Comparative Evaluation of a Phage Protein Ligand Assay with VIDAS and BAX Methodology for Detection of Escherichia coli O157:H7 Using a Standard Nonproprietary Enrichment Broth

2012 ◽  
Vol 95 (6) ◽  
pp. 1669-1671 ◽  
Author(s):  
Jingzhang Lu ◽  
Mujin Tang ◽  
Huiling Liu ◽  
Lihua Huang ◽  
Zhigang Wan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Foodborne Pathogen ◽  
Raw Milk ◽  
Alternative Methods ◽  
Escherichia Coli O157 ◽  
Pcr Assay ◽  
Enrichment Broth ◽  
E Coli ◽  
Phage Protein ◽  
Positive Results

Abstract Escherichia coli O157:H7 is a major foodborne pathogen of concern worldwide. This study was conducted to compare the sensitivity and minimum enrichment time for detection of E. coli O157:H7 by the VIDAS ultraperformance E. coli test (VIDAS ECPT UP) with that of two other commercial detection kits, the ELISA-based VIDAS ECO system and the PCR-based BAX® system. Only VIDAS ECPT UP detected all 18 positive results of bacterial suspensions at the level of 104 CFU/mL E. coli O157:H7 and 106 CFU/mL E. coli as background flora, whereas the BAX system PCR assay detected six positive results and VIDAS ECO detected no positive results. A 6 h enrichment at 42°C is enough for detection of all 18 strains in artificial contaminated raw beef meat, raw milk, and raw chicken, and for detection of most of them in soybean sprout and fresh papaya juice with VIDAS ECPT UP, whereas enrichment of more than 8 h was required for detection of the strains with the VIDAS ECO and PCR-BAX systems. These results indicate that the VIDAS ECPT UP is superior to the other two alternative methods when a standard enrichment broth is used that is different from the broths recommended by the manufacturers.

scholarly journals Effect of Electron Beam Irradiation on Survival of Escherichia coli O157:H7 and Salmonella enterica serovar Thyphimurium in Minced Camel Meat during Refrigerated Storage

2019 ◽  
Author(s):  
A. Amiri ◽  
H. Zandi ◽  
H. Mozaffari Khosravi
Keyword(s):  
Escherichia Coli ◽  
Electron Beam ◽  
Electron Beam Irradiation ◽  
Escherichia Coli O157 ◽  
Refrigerated Storage ◽  
Beam Irradiation ◽  
E Coli ◽  
Serovar Typhimurium ◽  
Camel Meat ◽  
Meat Samples

Background: Electron beam irradiation is one of the effective ways to control foodborne pathogens. We evaluated the effect of electron beam irradiation on survival of Escherichia coli O157:H7 and Salmonella enterica serovar Thyphimurium in minced camel meat during refrigerated storage. Methods: The meat samples were inoculated with E. coli O157:H7 and S. enterica serovar Thyphimurium and then irradiated with doses of 0, 1, 2, 3, and 5 kGy. The samples were stored at 4±1 °C and evaluated microbiologically up to 10 days. Data were analyzed using SPSS software version 18. Results: The microbial loads of minced camel meat samples were significantly reduced (p<0.0001) with increasing the dose of irradiation. The most effective dose was 5 kGy that highly reduced S. enterica serovar Typhimurium, and completely destroyed E. coli O157:H7. However, E. coli O157:H7 was more sensitive to electron beam irradiation than S. enterica serovar Typhimurium. Conclusion: Electron beam irradiation effectively reduced the population of both E. coli O157:H7 and S. enterica serovar Typhimurium in minced camel meat in a dose dependent manner.

Horizontal Transmission of Escherichia coli O157:H7 during Cattle Housing

2004 ◽  
Vol 67 (12) ◽  
pp. 2651-2656 ◽  
Author(s):  
P. McGEE ◽  
L. SCOTT ◽  
J. J. SHERIDAN ◽  
B. EARLEY ◽  
N. LEONARD
Keyword(s):  
Escherichia Coli ◽  
Drinking Water ◽  
Animal Behavior ◽  
Horizontal Transmission ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Holstein Friesian ◽  
Water Feed ◽  
Low Levels ◽  
The Right

Ruminant livestock, particularly cattle, is considered the primary reservoir of Escherichia coli O157:H7. This study examines the transmission of E. coli O157:H7 within groups of cattle during winter housing. Holstein Friesian steers were grouped in six pens of five animals. An animal inoculated with and proven to be shedding a marked strain of E. coli O157: H7 was introduced into each pen. Fecal (rectal swabs) and hide samples (900 cm2 from the right rump) were taken from the 36 animals throughout the study. Water, feed, and gate or partition samples from each pen were also examined. Within 24 h of introducing the inoculated animals into the pens, samples collected from the drinking water, pen barriers, and animal hides were positive for the pathogen. Within 48 h, the hides of 20 (66%) of 30 cohort animals from the six pens were contaminated with E. coli O157:H7. The first positive fecal samples from the noninoculated cohort animals were detected 3 days after the introduction of the inoculated steers. During the 23 days of the study, 15 of 30 cohort animals shed the marked E. coli O157: H7 strain in their feces on at least one occasion. Animal behavior in the pens was monitored during a 12-h period using closed circuit television cameras. The camera footage showed an average of 13 instances of animal grooming in each pen per hour. The study suggests that transmission of E. coli O157:H7 between animals may occur following ingestion of the pathogen at low levels and that animal hide may be an important source of transmission.

Detection of Escherichia coli O157, Salmonella enterica Serovar Typhimurium, and Staphylococcal Enterotoxin B in a Single Sample Using Enzymatic Bio-Nanotransduction

2007 ◽  
Vol 70 (4) ◽  
pp. 841-850 ◽  
Author(s):  
JOSH R. BRANEN ◽  
MARTHA J. HASS ◽  
ERIN R. DOUTHIT ◽  
WUSI C. MAKI ◽  
A. LARRY BRANEN
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Staphylococcal Enterotoxin B ◽  
Escherichia Coli O157 ◽  
Dna Templates ◽  
E Coli ◽  
Serovar Typhimurium ◽  
Biological Recognition ◽  
Enterotoxin B

Enzymatic bio-nanotransduction is a biological detection scheme based on the production of nucleic acid nano-signals (RNA) in response to specific biological recognition events. In this study, we applied an enzymatic bio-nanotransduction system to the detection of important food-related pathogens and a toxin. Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and staphylococcal enterotoxin B (SEB) were chosen because of the implications of these targets to food safety. Primary antibodies to each of the targets were used to functionalize magnetic beads and produce biological recognition elements (antibodies) conjugated to nano-signal–producing DNA templates. Immunomagnetic capture that was followed by in vitro transcription of DNA templates bound to target molecules produced RNA nano-signals specific for every target in the sample. Discrimination of RNA nano-signals with a standard enzyme-linked oligonucleotide fluorescence assay provided a correlation between nano-signal profiles and target concentrations. The estimated limit of detection was 2.4 × 103 CFU/ml for E. coli O157:H7, 1.9 × 104 CFU/ml for S. enterica serovar Typhimurium, and 0.11 ng/ml for SEB with multianalyte detection in buffer. Low levels of one target were also detected in the presence of interference from high levels of the other targets. Finally, targets were detected in milk, and detection was improved for E. coli O157 by heat treatment of the milk.

Distribution Patterns of Escherichia coli O157:H7 in Ground Beef Produced by a Laboratory-Scale Grinder†

2002 ◽  
Vol 65 (12) ◽  
pp. 1894-1902 ◽  
Author(s):  
ROLANDO A. FLORES ◽  
MARK L. TAMPLIN
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Distribution Patterns ◽  
Small Scale ◽  
Escherichia Coli O157 ◽  
Inoculum Level ◽  
E Coli ◽  
Specific Distribution ◽  
Low Levels ◽  
G Prior

This study determined the distribution patterns of Escherichia coli O157:H7 in ground beef when a contaminated beef trim was introduced into a batch of uncontaminated beef trims prior to grinding in a small-scale laboratory grinder. A beef trim (15.3 ± 2 g) was inoculated with a rifampicin-resistant strain of E. coli O157:H7 (E. coli O157:H7rif) and introduced into a stream of noncontaminated beef (322 ± 33 g) prior to grinding. Seven inoculum levels (6, 5, and 4 total log CFU [high]; and 3, 2, 1, and 0 total log CFU [low]) were studied in triplicate. E. coli O157:H7rif was not detected in 3.1 to 43% of the ground beef inoculated with the high levels or in 3.4 to 96.9% of the ground beef inoculated with the low levels. For all inoculum levels studied, the five ground beef fractions (each 7.8 ± 0.6 g) with the highest pathogen levels accounted for 59 to 100% of the total pathogens detected. For all inoculum levels, there was a linear relationship between the quantity of ground beef containing E. coli O157:H7rif and the inoculum level. The quantity of E. coli O157:H7rif in the beef remaining in the grinder was proportional to the inoculum level and was related to the location in the grinder. Different components of the grinder accumulated E. coli O157:H7rif in different quantities, with the most significant accumulation being in the nut (collar) that attaches the die to the blade. This study determined specific distribution patterns of E. coli O157:H7rif after the grinding of a contaminated beef trim along with uncontaminated trims, and the results indicate that the grinding operation should be regarded as a means of distribution of microbial contamination in risk analyses of ground beef operations.

A gold nanoparticles-assisted multiplex PCR assay for simultaneous detection of Salmonella typhimurium, Listeria monocytogenes and Escherichia coli O157:H7

2020 ◽  
Vol 12 (2) ◽  
pp. 212-217 ◽  
Author(s):  
Juan Du ◽  
Shujing Wu ◽  
Liyuan Niu ◽  
Junguang Li ◽  
Dianbo Zhao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gold Nanoparticles ◽  
Listeria Monocytogenes ◽  
Salmonella Typhimurium ◽  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Escherichia Coli O157 ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Pcr Product

Unfunctionalized flower-shaped AuNPs is used as colorimetric sensor for PCR product detection by naked eyes.

Polymerase Chain Reaction Assay for Detection of Escherichia coli O157:H7 and Escherichia coli O157:H−

2002 ◽  
Vol 65 (1) ◽  
pp. 5-11 ◽  
Author(s):  
TAKAHISA MIYAMOTO ◽  
NATSUKO ICHIOKA ◽  
CHIE SASAKI ◽  
HIROSHI KOBAYASHI ◽  
KEN-ICHI HONJOH ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Dna Sequence ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
Pcr Assay ◽  
E Coli ◽  
Pcr Product ◽  
Genomic Dnas ◽  
Polymerase Chain

The DNA band patterns generated by polymerase chain reaction (PCR) using the du2 primer and template DNAs from various strains of Escherichia coli and non–E. coli bacteria were compared. Among three to five prominent bands produced, the three bands at about 1.8, 2.7, and 5.0 kb were detected in all of the E. coli O157 strains tested. Some nonpathogenic E. coli and all pathogenic E. coli except E. coli O157 showed bands at 1.8 and 5.0 kb. It seems that the band at 2.7 kb is specific to E. coli O157. Sequence analysis of the 2.7-kb PCR product revealed the presence of a DNA sequence specific to E. coli O157:H− and E. coli O157:H7. Since the DNA sequence from base 15 to base 1008 of the PCR product seems to be specific to E. coli O157, a PCR assay was carried out with various bacterial genomic DNAs and O157-FHC1 and O157-FHC2 primers that amplified the region between base 23 and base 994 of the 2.7-kb PCR product. A single band at 970 bp was clearly detected in all of the strains of E. coli O157:H− and E. coli O157:H7 tested. However, no band was amplified from template DNAs from other bacteria, including both nonpathogenic and pathogenic E. coli except E. coli O157. All raw meats inoculated with E. coli O157:H7 at 3 × 100 to 3.5 × 102 CFU/25 g were positive both for our PCR assay after cultivation in mEC-N broth at 42°C for 18 h and for the conventional cultural method.

Behavior of Escherichia coli O157:H7 during the Manufacture and Aging of Gouda and Stirred-Curd Cheddar Cheeses Manufactured from Raw Milk

2010 ◽  
Vol 73 (12) ◽  
pp. 2217-2224 ◽  
Author(s):  
DENNIS J. D'AMICO ◽  
MARC J. DRUART ◽  
CATHERINE W. DONNELLY
Keyword(s):  
Escherichia Coli ◽  
Population Level ◽  
Raw Milk ◽  
Escherichia Coli O157 ◽  
Milk Whey ◽  
Selective Enrichment ◽  
Point Counts ◽  
E Coli ◽  
Unpasteurized Milk ◽  
Low Levels

This study was conducted to examine the fate of Escherichia coli O157:H7 during the manufacture and aging of Gouda and stirred-curd Cheddar cheeses made from raw milk. Cheeses were manufactured from unpasteurized milk experimentally contaminated with one of three strains of E. coli O157:H7 at an approximate population level of 20 CFU/ml. Samples of milk, whey, curd, and cheese were collected for enumeration of bacteria throughout the manufacturing and aging process. Overall, bacterial counts in both cheese types increased almost 10-fold from initial inoculation levels in milk to approximately 145 CFU/g found in cheeses on day 1. From this point, counts dropped significantly over 60 days to mean levels of 25 and 5 CFU/g in Cheddar and Gouda, respectively. Levels of E. coli O157:H7 fell and stayed below 5 CFU/g after an average of 94 and 108 days in Gouda and Cheddar, respectively, yet remained detectable after selective enrichment for more than 270 days in both cheese types. Changes in pathogen levels observed throughout manufacture and aging did not significantly differ by cheese type. In agreement with results of previous studies, our results suggest that the 60-day aging requirement alone is insufficient to completely eliminate levels of viable E. coli O157:H7 in Gouda or stirred-curd Cheddar cheese manufactured from raw milk contaminated with low levels of this pathogen.

Comparison of Methods for Detection and Isolation of Cold- and Freeze-Stressed Escherichia coli O157:H7 in Raw Ground Beef†

2007 ◽  
Vol 70 (7) ◽  
pp. 1663-1669 ◽  
Author(s):  
PINA M. FRATAMICO ◽  
LORI K. BAGI
Keyword(s):  
Escherichia Coli ◽  
Ground Beef ◽  
Escherichia Coli O157 ◽  
Pcr Assay ◽  
Initial Inoculum ◽  
E Coli ◽  
Multiplex Pcr Assay ◽  
Treatment Type ◽  
Agar Media ◽  
Main Effects

A comparison was made of the relative efficiencies of three enrichment media, RapidChek Escherichia coli O157:H7 enrichment broth (REB), R&F broth (RFB), and modified E. coli broth containing novobiocin (mEC+n), and four selective plating media for detection of cold- and freeze-stressed E. coli O157:H7 in raw ground beef. Ground beef (25 g) was inoculated with E. coli O157:H7 at ≤0.5 and ≤2 CFU/g, and samples were then enriched immediately or were stored at 4°C for 72 h or at −20°C for 2 weeks and then enriched. After 8 or 20 h of enrichment, the cultures were plated onto R&F E. coli O157: H7 chromogenic plating medium, cefixime-tellurite sorbitol MacConkey agar, CHROMagar O157, and Rainbow agar O157 and tested using the RapidChek E. coli O157 lateral flow immunoassay and a multiplex PCR assay targeting the E. coli O157: H7 eae, stx1, and stx2 genes. Recovery of E. coli O157:H7 on the four agar media was 4.0 to 7.9 log CFU/ml with the REB enrichment, 1.4 to 7.4 log CFU/ml with RFB, 1.7 to 6.7 log CFU/ml with mEC+n incubated at 42°C, and 1.3 to 3.3 log CFU/ml from mEC+n incubated at 35°C. The percentages of positive ground beef samples containing nonstressed, cold-stressed, and freeze-stressed E. coli O157:H7 as obtained by plating, the immunoassay, and the PCR assay were 97, 88, and 97%, respectively, with REB, 92, 81, and 78%, respectively, with RFB, 97, 58, and 53%, respectively, with mEC+n incubated at 42°C, and 22, 31, and 25%, respectively, with mEC+n incubated at 35°C. Logistic regression analyses of the data indicated significant main effects of treatment, type of medium, enrichment time, inoculum concentration, and detection method. In particular, a positive result was 1.1 times more likely to occur after 20 h of enrichment than after 8 h, 25 times more likely with RFB and REB than with mEC+nat35°C, 3.7 times more likely with an initial inoculum of ≤2.0 CFU/g than with ≤0.5 CFU/g, 2.5 to 3 times more likely using freeze-stressed or nonstressed bacteria than with cold-stressed bacteria, and 2.5 times more likely by plating than by the immunoassay or the PCR assay. REB had better overall performance for enrichment of cold- and freeze-stressed E. coli O157:H7 present in ground beef than did the other media examined.

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