A Multiplex Polymerase Chain Reaction Assay for Rapid Detection and Identification of Escherichia coli O157:H7 in Foods and Bovine Feces†

2000 ◽  
Vol 63 (8) ◽  
pp. 1032-1037 ◽  
Author(s):  
PINA M. FRATAMICO ◽  
LORI K. BAGI ◽  
TIZIANA PEPE
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Multiplex Polymerase Chain Reaction ◽  
Ground Beef ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
E Coli ◽  
Polymerase Chain ◽  
Bovine Feces

A multiplex polymerase chain reaction (PCR) assay was designed to simplify detection of Escherichia coli O157:H7 and to identify the H serogroup and the type of Shiga toxin produced by this bacterium. Primers for a plasmid-encoded hemolysin gene (hly933), and chromosomal flagella (fliCh7; flagellar structural gene of H7 serogroup), Shiga toxins (stx1, stx2), and attaching and effacing (eaeA) genes were used in a multiplex PCR for coamplification of the corresponding DNA sequences from enterohemorrhagic E. coli (EHEC) O157:H7. Enrichment cultures of ground beef, blue cheese, mussels, alfalfa sprouts, and bovine feces, artificially inoculated with various levels of E. coli O157:H7 strain 933, were subjected to a simple DNA extraction step prior to the PCR, and the resulting amplification products were analyzed by agarose gel electrophoresis. Sensitivity of the assay was ≤1 CFU/g of food or bovine feces (initial inoculum level), and results could be obtained within 24 h. Similar detection levels were obtained with ground beef samples that underwent enrichment culturing immediately after inoculation and samples that were frozen or refrigerated prior to enrichment. The multiplex PCR facilitates detection of E. coli O157:H7 and can reduce the time required for confirmation of isolates by up to 3 to 4 days.

Evaluation of an Enzyme-Linked Immunosorbent Assay, Direct Immunofluorescent Filter Technique, and Multiplex Polymerase Chain Reaction for Detection of Escherichia coli O157:H7 Seeded in Beef Carcass Wash Water†

1998 ◽  
Vol 61 (8) ◽  
pp. 934-938 ◽  
Author(s):  
PINA M. FRATAMICO ◽  
TERENCE P. STROBAUGH
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Multiplex Pcr ◽  
Multiplex Polymerase Chain Reaction ◽  
Wash Water ◽  
Enzyme Linked Immunosorbent Assay ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
E Coli ◽  
Polymerase Chain

In commercial beef processing, carcasses are customarily washed with water to remove physical and microbial contamination. Assaying the water that is shed from the carcasses after washing is a convenient method to determine whether the carcass is contaminated with Escherichia coli O157:H7 or other bacterial pathogens. E. coli O157:H7 was inoculated into carcass wash water at various levels and the bacteria were then concentrated by filtration. After collection of bacteria in the filter units, the nylon membranes were cut out and placed in tubes containing growth medium, and the tubes were mixed vigorously to dislodge the bacteria from the membranes. Prior to enrichment, samples were removed for testing by a multiplex polymerase chain reaction (PCR) and a direct immunofluorescent filter technique (DIFT). The remaining samples were subjected to 4-h enrichment culturing at 37°C, after which aliquots were removed for testing by multiplex PCR, DIFT, and an enzyme-linked immunosorbent assay (ELISA). Following 4-h enrichment culturing, E. coli O157:H7 was detected in wash water samples initially inoculated with ca. 100, 0.1, and 1 CFU/ml by ELISA, DIFT, and multiplex PCR, respectively. Testing of the wash water using the ELISA and the DIFT can be accomplished in less than 8 h. On the basis of these results, assaying carcass wash water by ELISA, DIFT, or multiplex PCR can be useful for detection of E. coli O157:H7 beef carcass contamination and can potentially be employed to identify carcasses for further processing to inactivate the organism.

A rapid (one day), sensitive real-time polymerase chain reaction assay for detecting Escherichia coli O157:H7 in ground beef

2006 ◽  
Vol 52 (10) ◽  
pp. 992-998 ◽  
Author(s):  
Jane Holicka ◽  
Rebecca A Guy ◽  
Anita Kapoor ◽  
David Shepherd ◽  
Paul A Horgen
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Polymerase Chain Reaction Assay ◽  
Ground Beef ◽  
Automatic Process ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
E Coli ◽  
Polymerase Chain

The purpose of this study was to apply our rapid, integrated double enrichment 5′ nuclease real-time polymerase chain reaction assay for the detection of Escherichia coli O157:H7 and evaluate its efficacy. The assay targeted ground beef, an important vehicle in disease epidemiology. The assay reliably determined in 8 h the presence of E. coli O157:H7 in ground beef at the level of 1 colony-forming unit (CFU)/g. The sensitivity and specificity of the assay were compared with that of standard enrichment diagnostic techniques. A correlation of 100% in detection was achieved to the limit of 1 CFU/g. This assay can be used as a rapid, automatic process for identification of E. coli O157:H7 in ground beef or can be integrated with standard culture procedures, resulting in considerable cost and time savings.Key words: real-time PCR, E. coli O157:H7, ground beef, molecular diagnostics, rapid O157:H7 assay.

scholarly journals Multiplex Polymerase Chain Reaction Assay for Identification of Enterotoxigenic Escherichia Coli Strains

2001 ◽  
Vol 13 (4) ◽  
pp. 308-311 ◽  
Author(s):  
Jacek Osek
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Multiplex Polymerase Chain Reaction ◽  
Direct Determination ◽  
Enteric Bacteria ◽  
Enterotoxigenic Escherichia Coli ◽  
Chain Reaction ◽  
Gram Negative ◽  
E Coli ◽  
Polymerase Chain

A multiplex polymerase chain reaction (PCR) system was developed for identification of enterotoxigenic Escherichia coli (ETEC) strains and to differentiate them from other gram negative enteric bacteria. This test simultaneously amplifies heat-labile (LTI) and heat-stable (STI and STII) toxin sequences and the E. coli-specific universal stress protein ( uspA). The specificity of the method was validated by single PCR tests performed with the reference E. coli and non- E. coli strains and with bacteria isolated from pig feces. The multiplex PCR allowed the rapid and specific identification of enterotoxin-positive E. coli and may be used as a method for direct determination of ETEC and to differentiate them from other E. coli and gram-negative enteric isolates.

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.

scholarly journals High Frequency of Diarrheagenic Escherichia coli in HIV-Infected Patients and Patients with Thalassemia in Kerman, Iran

2015 ◽  
Vol 16 (4) ◽  
pp. 353-358 ◽  
Author(s):  
Hesam Alizade ◽  
Hamid Sharifi ◽  
Zahedeh Naderi ◽  
Reza Ghanbarpour ◽  
Mehdi Bamorovat ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Statistical Analysis ◽  
High Frequency ◽  
Multiplex Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Fecal Samples ◽  
E Coli ◽  
Diarrheagenic Escherichia Coli ◽  
Polymerase Chain

This study was conducted on patients with thalassemia and HIV-infected patients to determine the frequency of diarrheagenic Escherichia coli in Kerman, Iran. We analyzed 68 and 49 E coli isolates isolated from healthy fecal samples of patients with thalassemia and HIV-infected patients, respectively. The E coli isolates were studied using a multiplex polymerase chain reaction to identify the enterotoxigenic E coli (ETEC), enterohemorrhagic E coli (EHEC), and enteropathogenic E coli (EPEC) groups. Statistical analysis was carried out to determine the correlation of diarrheagenic E coli between HIV-infected patients and patients with thalassemia using Stata 11.2 software. The frequency of having at least 1 diarrheagenic E coli was more common in patients with thalassemia (67.64%) than in HIV-infected patients (57.14%; P = .25), including ETEC (67.64% versus 57.14%), EHEC (33.82% versus 26.53%), and EPEC (19.11% versus 16.32%). The results of this study indicate that ETEC, EHEC, and EPEC pathotypes are widespread among diarrheagenic E coli isolates in patients with thalassemia and HIV-infected patients.

scholarly journals Verotoxins in commensal Escherichia coli in cattle: the effect of injectable subcutaneous oxytetracycline in addition to in-feed chlortetracycline on prevalence

2004 ◽  
Vol 132 (1) ◽  
pp. 77-85 ◽  
Author(s):  
A. M. O'CONNOR ◽  
K. A. ZIEBELL ◽  
C. POPPE ◽  
S. A. McEWEN
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Observational Study ◽  
Multiplex Pcr ◽  
Virulence Genes ◽  
Chain Reaction ◽  
E Coli ◽  
Polymerase Chain

Using a self-paired observational study, the association between therapeutic oxytetracycline use and the prevalence of virulence genes in commensal Escherichia coli (E. coli) from cattle was examined. Faeces were collected from 39 yearling bulls prior to and after treatment with oxytetracycline and from 44 untreated animals. Between samplings all animals received in-feed chlortetracycline for 16 days. Five E. coli were isolated from each sample and tested by a polymerase chain reaction (PCR) capable of detecting all verotoxin (vt) genes. Positive isolates were further tested with a multiplex PCR to detect vt1, vt2, eaeA and hlyA. For vt, 23 animals were positive at both samplings, 26 negative at both samplings, 22 negative animals became positive and 12 positive animals became negative. Sixty-eight per cent of the discordant pairs changed from vt-negative to vt-positive (95% CI 48–80) suggesting pressure toward becoming vt-positive perhaps due to the transfer of genes due to mixing of cattle in the months between samplings or an effect of chlortetracycline.

Detection of Viable Shiga Toxin–Producing Escherichia coli by Quantitative Competitive Polymerase Chain Reaction

2003 ◽  
Vol 66 (7) ◽  
pp. 1277-1282 ◽  
Author(s):  
W. LI ◽  
M. A. DRAKE
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Cell Viability ◽  
Shiga Toxin ◽  
Ground Beef ◽  
Multiple Time ◽  
Chain Reaction ◽  
Competitive Polymerase Chain Reaction ◽  
E Coli ◽  
Polymerase Chain

With the use of Escherichia coli O157:H7 as a model, a procedure for the quantitative detection of viable Shiga toxin–producing E. coli (STEC) in broth and cooked ground beef enrichments with multiple–time point quantitative competitive polymerase chain reaction (QC-PCR) was developed. The A subunit (a 401-bp fragment) of the stx2 gene was chosen as a target sequence. Immunomagnetic separation (IMS) was used to isolate and concentrate cells from ground beef enrichments. Cell viability was confirmed on the basis of the quantitative increase in the signal of target bands from QC-PCR across multiple time points. The application of IMS increased detection limits relative to those for QC-PCR without IMS. E. coli O157:H7 inoculated at 0.20 CFU/g of cooked ground beef (25 g of ground beef plus 225 ml of Bacto modified EC medium plus novobiocin) was detected and confirmed to be viable in <15 h. A DNA-based molecular approach can be used to determine cell viability.

Sign in / Sign up

Export Citation Format

Share Document