scholarly journals Use of Fluorescence Quantitative Polymerase Chain Reaction (PCR) for the Detection of Escherichia coli Adhesion to Pig Intestinal Epithelial Cells

2016 ◽  
Vol 19 (3) ◽  
pp. 619-625 ◽  
Author(s):  
C.H. Dai ◽  
L.N. Gan ◽  
W.U. Qin ◽  
C. Zi ◽  
G.Q. Zhu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Relative Number ◽  
Intestinal Epithelial ◽  
Chain Reaction ◽  
E Coli ◽  
Polymerase Chain

AbstractAn efficient and accurate method to testEscherichia coli(E. coli) adhesion to intestinal epithelial cells will contribute to the study of bacterial pathogenesis and the function of genes that encode receptors related to adhesion. This study used the quantitative real-time polymerase chain reaction (qPCR) method. qPCR primers were designed from thePILINgene ofE. coliF18ab, F18ac, and K88ac, and the pig β-ACTINgene. Total deoxyribonucleic acid (DNA) fromE. coliand intestinal epithelial cells (IPEC-J2 cells) were used as templates for qPCR. The 2−ΔΔCtformula was used to calculate the relative number of bacteria in cultures of different areas. We found that the relative numbers of F18ab, F18ac, and K88ac that adhered to IPEC-J2 cells did not differ significantly in 6-, 12-, and 24-well culture plates. This finding indicated that there was no relationship between the relative adhesion number ofE. coliand the area of cells, so the method of qPCR could accurately test the relative number ofE. coli. This study provided a convenient and reliable testing method for experiments involvingE. coliadhesion, and also provided innovative ideas for similar detection methods.

scholarly journals Plant-Derived Antimicrobials ReduceE. coliO157:H7 Virulence Factors Critical for Colonization in Cattle Gastrointestinal TractIn Vitro

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Sangeetha Ananda Baskaran ◽  
Kumar Venkitanarayanan
Keyword(s):  
Polymerase Chain Reaction ◽  
Epithelial Cells ◽  
Rumen Fluid ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
E Coli ◽  
Intestinal Contents ◽  
Polymerase Chain

This study investigated the effect of subinhibitory concentrations (SIC) of five plant-derived antimicrobials (PDAs), namely, trans cinnamaldehyde, eugenol, carvacrol, thymol, andβ-resorcylic acid, onE. coliO157:H7 (EHEC) attachment and invasion of cultured bovine colonic (CO) and rectoanal junction (RAJ) epithelial cells. In addition, PDAs’ effect on EHEC genes critical for colonization of cattle gastrointestinal tract (CGIT) was determined in bovine rumen fluid (RF) and intestinal contents (BICs). Primary bovine CO and RAJ epithelial cells were established and were separately inoculated with three EHEC strains with or without (control) SIC of each PDA. Following incubation, EHEC that attached and invaded the cells were determined. Furthermore, the expression of EHEC genes critical for colonization in cattle was investigated using real-time, quantitative polymerase chain reaction in RF and BICs. All the PDAs decreased EHEC invasion of CO and RAJ epithelial cells (P<0.05). The PDAs also downregulated (P<0.05) the expression of EHEC genes critical for colonization in CGIT. Results suggest that the PDAs could potentially be used to control EHEC colonization in cattle; however follow-upin vivostudies in cattle are warranted.

scholarly journals Development of an Escherichia coli K12-specific quantitative polymerase chain reaction assay and DNA isolation suited to biofilms associated with iron drinking water pipe corrosion products

2014 ◽  
Vol 12 (4) ◽  
pp. 763-771 ◽  
Author(s):  
Jingrang Lu ◽  
Tammie L. Gerke ◽  
Helen Y. Buse ◽  
Nicholas J. Ashbolt
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Drinking Water ◽  
Polymerase Chain Reaction Assay ◽  
Quantitative Polymerase Chain Reaction ◽  
The Novel ◽  
Water Pipe ◽  
Chain Reaction ◽  
E Coli ◽  
Polymerase Chain

A quantitative polymerase chain reaction assay (115 bp amplicon) specific to Escherichia coli K12 with an ABITM internal control was developed based on sequence data encoding the rfb gene cluster. Assay specificity was evaluated using three E. coli K12 strains (ATCC W3110, MG1655 & DH1), 24 non-K12 E. coli and 23 bacterial genera. The biofilm detection limit was 103 colony-forming units (CFU) E. coli K12 mL−1, but required a modified protocol, which included a bio-blocker Pseudomonas aeruginosa with ethylenediaminetetraacetic acid buffered to pH 5 prior to cell lysis/DNA extraction. The novel protocol yielded the same sensitivity for drinking water biofilms associated with Fe3O4 (magnetite)-coated SiO2 (quartz) grains and biofilm-surface iron corrosion products from a drinking water distribution system. The novel DNA extraction protocol and specific E. coli K12 assay are sensitive and robust enough for detection and quantification within iron drinking water pipe biofilms, and are particularly well suited for studying enteric bacterial interactions within biofilms.

scholarly journals Occurrence of toxigenic Escherichia coli in raw milk cheese in Brazil

2007 ◽  
Vol 59 (2) ◽  
pp. 508-512 ◽  
Author(s):  
B.R. Paneto ◽  
R.P. Schocken-Iturrino ◽  
C. Macedo ◽  
E. Santo ◽  
J.M. Marin
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Antimicrobial Agents ◽  
Raw Milk ◽  
Diffusion Method ◽  
Chain Reaction ◽  
Disk Diffusion Method ◽  
E Coli ◽  
Polymerase Chain ◽  
Raw Milk Cheese

The occurrence of toxigenic Escherichia coli in raw milk cheese was surveyed in Middle Western Brazil. Fifty samples of cheese from different supermarkets were analyzed for E.coli. The isolates were serotyped and screened for the presence of verotoxigenic E. coli (VTEC) and enterotoxigenic E. coli (ETEC) by Polymerase Chain Reaction (PCR). The susceptibility to thirteen antimicrobial agents was evaluated by the disk diffusion method. E.coli were recovered from 48 (96.0%) of the samples. The serogroups identified were O125 (6.0%), O111 (4.0%), O55 (2.0%) and O119 (2.0%). Three (6.0%) and 1(2.0%) of the E.coli isolates were VTEC and ETEC, respectively. Most frequent resistance was observed to the following antimicrobials: cephalothin (60.0%), nalidixic acid (40.0%), doxycyclin (33.0%), tetracycline (31.0%) and ampicillin (29.0%).

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.

scholarly journals Eliminating false positives in a qPCR assay for the detection of the uidA gene in Escherichia coli

2013 ◽  
Vol 11 (3) ◽  
pp. 382-386 ◽  
Author(s):  
Richard Kibbee ◽  
Natalie Linklater ◽  
Banu Örmeci
Keyword(s):  
Escherichia Coli ◽  
Quantitative Polymerase Chain Reaction ◽  
Qpcr Assay ◽  
Uida Gene ◽  
Chain Reaction ◽  
Taq Polymerase ◽  
E Coli ◽  
Gene Copies ◽  
Polymerase Chain ◽  
Low Levels

Due to contaminant Escherichia coli DNA present in recombinant Taq polymerase reagents, it is not possible to reliably detect low levels of E. coli in samples using the quantitative polymerase chain reaction (qPCR) assay. Native Taq polymerase was successfully used in this study to detect five uidA gene copies (5 fg of genomic DNA) of the uidA gene.

Immunogenicity Evaluation of Chimeric Subunit Vaccine Comprising Adhesion Coli Surface Antigens from Enterotoxigenic Escherichia coli

2019 ◽  
Vol 29 (1-6) ◽  
pp. 91-100
Author(s):  
Dorna Khoobbakht ◽  
Shohreh Zare Karizi ◽  
Mohammad Javad  Motamedi ◽  
Rouhollah Kazemi ◽  
Pooneh Roghanian ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Subunit Vaccine ◽  
Fusion Gene ◽  
Intestinal Epithelial Cells ◽  
High Titer ◽  
Chimeric Protein ◽  
Amino Acid Sequences ◽  
Intestinal Epithelial ◽  
E Coli

Enterotoxigenic <i>Escherichia coli</i> (ETEC) is the most common agent of diarrhea morbidity in developing countries. ETEC adheres to host intestinal epithelial cells via various colonization factors. The CooD and CotD proteins play a significant role in bacteria binding to the intestinal epithelial cells as adhesin tip subunits of CS1 and CS2 pili. The purpose here was to design a new construction containing <i>cooD</i> and <i>cotD</i> genes and use several types of bioinformatics software to predict the structural and immunological properties of the designed antigen. The fusion gene was synthesized with codon bias of <i>E. coli</i> in order to increase the expression level of the protein. The amino acid sequences, protein structure, and immunogenicity properties of potential antigens were analyzed in silico. The chimeric protein was expressed in <i>E. coli</i>BL21 (DE3). The antigenicity of the recombinant proteins was verified by Western blotting and ELISA. In order to assess the induced immunity, the immunized mice were challenged with wild-type ETEC by an intraperitoneal route. Immunological analyses showed the production of a high titer of IgG serum with no sign of serum-mucosal IgA antibody response. The result of the challenge assay showed that 30% of immunized mice survived. The results of this study showed that CooD-CotD recombinant protein can stimulate immunity against ETEC. The designed chimera could be a prototype for the subunit vaccine, which is worthy of further consideration.

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 Probiotic Escherichia coli Nissle 1917 and Commensal E. coli K12 Differentially Affect the Inflammasome in Intestinal Epithelial Cells

Digestion ◽  
2014 ◽  
Vol 89 (2) ◽  
pp. 110-118 ◽  
Author(s):  
Helen M. Becker ◽  
Aretussa Apladas ◽  
Michael Scharl ◽  
Michael Fried ◽  
Gerhard Rogler
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
E Coli

scholarly journals Escherichia coli O157:H7 Strains That Persist in Feedlot Cattle Are Genetically Related and Demonstrate an Enhanced Ability To Adhere to Intestinal Epithelial Cells

2009 ◽  
Vol 75 (18) ◽  
pp. 5927-5937 ◽  
Author(s):  
Brandon A. Carlson ◽  
Kendra K. Nightingale ◽  
Gary L. Mason ◽  
John R. Ruby ◽  
W. Travis Choat ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Multiplex Pcr ◽  
Intestinal Epithelial Cells ◽  
Feedlot Cattle ◽  
Pfge Type ◽  
Sample Population ◽  
Intestinal Epithelial ◽  
E Coli ◽  
Multiplex Pcr Assay

ABSTRACT A longitudinal study was conducted to investigate the nature of Escherichia coli O157:H7 colonization of feedlot cattle over the final 100 to 110 days of finishing. Rectal fecal grab samples were collected from an initial sample population of 788 steers every 20 to 22 days and microbiologically analyzed to detect E. coli O157:H7. The identities of presumptive colonies were confirmed using a multiplex PCR assay that screened for gene fragments unique to E. coli O157:H7 (rfbE and fliC h7) and other key virulence genes (eae, stx 1, and stx 2). Animals were classified as having persistent shedding (PS), transient shedding (TS), or nonshedding (NS) status if they consecutively shed the same E. coli O157:H7 genotype (based on the multiplex PCR profile), exhibited variable E. coli O157 shedding, or never shed morphologically typical E. coli O157, respectively. Overall, 1.0% and 1.4% of steers were classified as PS and NS animals, respectively. Characterization of 132 E. coli O157:H7 isolates from PS and TS animals by pulsed-field gel electrophoresis (PFGE) typing yielded 32 unique PFGE types. One predominant PFGE type accounted for 53% of all isolates characterized and persisted in cattle throughout the study. Isolates belonging to this predominant and persistent PFGE type demonstrated an enhanced (P < 0.0001) ability to adhere to Caco-2 human intestinal epithelial cells compared to isolates belonging to less common PFGE types but exhibited equal virulence expression. Interestingly, the attachment efficacy decreased as the genetic divergence from the predominant and persistent subtype increased. Our data support the hypothesis that certain E. coli O157:H7 strains persist in feedlot cattle, which may be partially explained by an enhanced ability to colonize the intestinal epithelium.

Sign in / Sign up

Export Citation Format

Share Document