Direct detection of Escherichia coli O157 and its major virulence factor genes in animal faeces at slaughter using multiplex polymerase chain reaction (PCR)

2011 ◽  
Vol 5 (14) ◽  
Author(s):  
Khandaghi Jalil
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Virulence Factor ◽  
Multiplex Polymerase Chain Reaction ◽  
Direct Detection ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
Major Virulence Factor ◽  
Polymerase Chain ◽  
Virulence Factor Genes

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.

Molecular Analysis of Uropathogenic E.coli Isolates from Urinary Tract Infections

2019 ◽  
Vol 19 (3) ◽  
pp. 322-326 ◽  
Author(s):  
Hassan Valadbeigi ◽  
Elham Esmaeeli ◽  
Sobhan Ghafourian ◽  
Abbas Maleki ◽  
Nourkhoda Sadeghifard
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Multiplex Polymerase Chain Reaction ◽  
Virulence Genes ◽  
Uropathogenic Escherichia Coli ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Tract Infections

Introduction: The aim of the current study was to investigate the prevalence of virulence genes in uropathogenic Escherichia coli (UPEC) isolates in Ilam. Materials and Methods: For this purpose, a total of 80 UPEC isolates were collected for patients with UTIs during a 6 months period. The multiplex polymerase chain reaction (multiplex PCR) was used to detect the papEF, fimH, iucD, hlyA, fyuA, and ompT genes. Results: The prevalence of fimH, papEF, iucD, fyuA, hlyA, hlyA, and ompT genes were 87.5%, 47.5%, 60%, 67.5%, 27.5%, 47.5% and 71.2%, respectively. Among all of the isolates, 27 profiles were obtained. Conclusion: Our findings demonstrated that the most prevalence was found for fimH, and different distribution of virulence genes suggested different ability of pathogenicity.

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.

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