scholarly journals Toward an International Standard for PCR-Based Detection of Foodborne Escherichia coli O157: Validation of the PCR-Based Method in a Multicenter Interlaboratory Trial

2004 ◽  
Vol 87 (4) ◽  
pp. 856-860 ◽  
Author(s):  
Amir Abdulmawjood ◽  
Michael Bülte ◽  
Stefanie Roth ◽  
Hahn Schönenbrücher ◽  
Nigel Cook ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Escherichia Coli O157 ◽  
Diagnostic Specificity ◽  
Chain Reaction ◽  
E Coli ◽  
Colony Forming Units ◽  
Interlaboratory Trial ◽  
Pure Cultures ◽  
Pcr Method ◽  
Polymerase Chain

Abstract The performance of a polymerase chain reaction (PCR) method for detection of Escherichia coli O157, previously validated on DNA extracted from pure cultures, was evaluated on spiked cattle swabs through an interlaboratory trial, including 12 participating laboratories from 11 European countries. Twelve cattle swab samples, spiked at 4 levels (0, 1–10, 10–100, and 100–1000 colony-forming units, in triplicate) with E. coli O157 were prepared centrally in the originating laboratory; the receiving laboratories performed pre-PCR treatment followed by PCR. The results were reported as positive when the correct amplicons were present after gel electrophoresis. The statistical analysis, performed on 10 sets of reported results, determined the diagnostic sensitivity to be 92.2%. The diagnostic specificity was 100%. The accordance (repeatability) was 90.0%, calculated from all positive inoculation levels. The concordance (reproducibility) was 85.0%, calculated from all positive inoculation levels. The concordance odds ratio (degree of interlaboratory variation calculated from all positive inoculation levels) was 1.58, indicating the robustness of the PCR method. Thus, the interlaboratory variation due to personnel, reagents, minor temperature or pH fluctuations and, not least, thermal cyclers, did not affect the performance of the method, which is currently being considered as part of an international PCR standard.

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 Molecular Detection of Plasmid-Mediated Quinolone Resistance in Ciprofloxacin-Resistant Escherichia coli from Urine of Patients attending Garki Hospital, Abuja, Nigeria

2020 ◽  
Vol 1 (4) ◽  
Author(s):  
Moses Oghenaigah Eghieye ◽  
Istifanus Haruna Nkene ◽  
Rejoice Helma Abimiku ◽  
Yakubu Boyi Ngwai ◽  
Ibrahim Yahaya ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Urinary Tract ◽  
Molecular Detection ◽  
Quinolone Resistance ◽  
Chain Reaction ◽  
E Coli ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Tract Infections

Urinary tract infections (UTIs) caused by Escherichia coli (E. coli) is common worldwide; and its successful treatment using antibiotics is limited by acquisition of resistance by the bacteria. This study investigated the occurrence of plasmid-mediated quinolone resistance (PMQR) genes in ciprofloxacin-resistant E. coli from urine of patients with suspected cases of UTIs attending Garki Hospital Abuja (GHA), Nigeria. A total of 8 confirmed ciprofloxacin-resistant E. coli was screened for carriage of PMQR genes using polymerase chain reaction (PCR) method. The occurrences of the PMQR genes detected were in the order: aac-(6′)-Ib-cr (87.5%) > qnrB (50.0%) > qnrS (37.5%) > oqxAB (12.5%) > qnrA(0.0%). qnrB and qnrS did not exist alone, but in combination with other genes; aac-(6′)-Ib-crexisted both alone and in combination with others; the most prevalent patterns of existence were aac-(6′)-Ib-cr alone and aac-(6′)-Ib-cr + qnrB + qnrS at 25.0% each. This study has shown that the ciprofloxacin-resistant E. coli harbored aac-(6′)-Ib-cr, qnrB, qnrS and oqxAB PMQR genes, with aac-(6′)-Ib-cr being the most prevalent. The genes were present either alone or in combination with one another. This has implication for the clinical application of fluoroquinolones to treat UTI in the study location and environs. 

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.

Description of a Novel Intimin Variant (Type ζ) in the Bovine O84:NM Verotoxin-Producing Escherichia coli Strain 537/89 and the Diagnostic Value of Intimin Typing

2003 ◽  
Vol 228 (4) ◽  
pp. 370-376 ◽  
Author(s):  
Joerg Jores ◽  
Karen Zehmke ◽  
Juergen Eichberg ◽  
Leonid Rumer ◽  
Lothar H. Wieler
Keyword(s):  
Escherichia Coli ◽  
Diagnostic Value ◽  
Coli Strain ◽  
Chain Reaction ◽  
E Coli ◽  
Pcr Method ◽  
Polymerase Chain ◽  
Enterocyte Effacement ◽  
Phylogenetic Lineages ◽  
Adhesion Factor

Infections with verotoxin-producing Escherichia coli (VTEC) has resulted in increasing numbers of human illnesses annually. These illnesses usually result from the ability of VTEC to cause the attaching and effacing lesions (AE lesion). The AE phenotype is encoded by the locus of enterocyte effacement (LEE) pathogenicity island. A key adhesion factor involved is the outer membrane protein intimin, encoded by the eae gene within the LEE. Intimin types α, β, γ, δ, and ε have been described previously. Each intimin represents distinct phylogenetic lineages of LEE-positive strains. A new intimin type ζ was identified in a VTEC strain of the serotype O84:NM (nonmotile) that was isolated from a calf with diarrhea. ζ intimin showed the highest similarity (88%) of its amino acid sequence to the α intimin. For diagnostic purposes, we established a polymerase chain reaction (PCR) method for diagnosis of the key virulence traits of VTEC (i.e., verotoxins and intimins). This method also distinguishes between the toxins (VT1 and VT2) and the six intimin types. By applying the PCR method, intimin ζ in strains of other VTEC serotypes O84:H2, O92:NM, O119:H25, and O150:NM was identified. Because the intimin types represent distinctive phylogenetic E. coli lineages, application of the intimin subtyping PCR offers significant benefits. These include improving diagnosis of VTEC infection and increasing the understanding of evolution of attaching and effacing VTEC and other LEE-positive bacteria.

scholarly journals Multicenter Validation of PCR-Based Method for Detection of Salmonella in Chicken and Pig Samples

2004 ◽  
Vol 87 (4) ◽  
pp. 861-866 ◽  
Author(s):  
Burkhard Malorny ◽  
Nigel Cook ◽  
Martin D'Agostino ◽  
Dario De Medici ◽  
Luciana Croci ◽  
...  
Keyword(s):  
Experimental Protocol ◽  
Diagnostic Specificity ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Colony Forming Units ◽  
Interlaboratory Trial ◽  
Peptone Water ◽  
Polymerase Chain ◽  
Specific Pcr Assay

Abstract As part of a standardization project, an interlaboratory trial including 15 laboratories from 13 European countries was conducted to evaluate the performance of a noproprietary polymerase chain reaction (PCR)-based method for the detection of Salmonella on artificially contaminated chicken rinse and pig swab samples. The 3 levels were 1–10, 10–100, and 100–1000 colony-forming units (CFU)/100 mL. Sample preparations, including inoculation and pre-enrichment in buffered peptone water (BPW), were performed centrally in a German laboratory; the pre-PCR sample preparation (by a resin-based method) and PCR assay (gel electrophoresis detection) were performed by the receiving laboratories. Aliquots of BPW enrichment cultures were sent to the participants, who analyzed them using a thermal lysis procedure followed by a validated Salmonella-specific PCR assay. The results were reported as negative or positive. Outlier results caused, for example, by gross departures from the experimental protocol, were omitted from the analysis. For both the chicken rinse and the pig swab samples, the diagnostic sensitivity was 100%, with 100% accordance (repeatability) and concordance (reproducibility). The diagnostic specificity was 80.1% (with 85.7% accordance and 67.5% concordance) for chicken rinse, and 91.7% (with 100% accordance and 83.3% concordance) for pig swab. Thus, the interlaboratory variation due to personnel, reagents, thermal cyclers, etc., did not affect the performance of the method, which will be proposed as part of a developing international PCR standard.

Evaluation of a Polymerase Chain Reaction–Based System for Detection of Salmonella Enteritidis, Escherichia coli O157:H7, Listeria spp., and Listeria monocytogenes on Fresh Fruits and Vegetables†

2001 ◽  
Vol 64 (6) ◽  
pp. 788-795 ◽  
Author(s):  
ADRIENNE E. H. SHEARER ◽  
CHRISTINE M. STRAPP ◽  
ROLF D. JOERGER
Keyword(s):  
Escherichia Coli ◽  
Polymerase Chain Reaction ◽  
Listeria Monocytogenes ◽  
Salmonella Enteritidis ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
Culture Methods ◽  
E Coli ◽  
Polymerase Chain ◽  
Alfalfa Sprouts

A polymerase chain reaction (PCR)-based detection system, BAX, was evaluated for its sensitivity in detecting Salmonella Enteritidis, Escherichia coli O157:H7, Listeria sp., and Listeria monocytogenes on fresh produce. Fifteen different types of produce (alfalfa sprouts, green peppers, parsley, white cabbage, radishes, onions, carrots, mushrooms, leaf lettuce, tomatoes, strawberries, cantaloupe, mango, apples, and oranges) were inoculated, in separate studies, with Salmonella Enteritidis, E. coli O157:H7, and L. monocytogenes down to the predicted level of 1 CFU per 25-g sample. Detection by BAX was compared to recovery of the inoculated bacteria by culture methods according to the Food and Drug Administration's (FDA) Bacteriological Analytical Manual (BAM). BAX was essentially as sensitive as the culture-based method in detecting Salmonella Enteritidis and L. monocytogenes and more sensitive than the culture-based method for the detection of E. coli O157:H7 on green pepper, carrot, radish, and sprout samples. Detection of the pathogenic bacteria in samples spiked with a predicted number of less than 10 CFU was possible for most produce samples, but both methods failed to detect L. monocytogenes on carrot samples and one of two mushroom and onion samples spiked with less than 100 CFU. Both BAX and the culture method were also unable to consistently recover low numbers of E. coli O157:H7 from alfalfa sprouts. The PCR method allowed detection of Salmonella Enteritidis, E. coli O157:H7, and L. monocytogenes at least 2 days earlier than the conventional culture methods.

Survey of Retail Alfalfa Sprouts and Mushrooms for the Presence of Escherichia coli O157:H7, Salmonella, and Listeria with BAX, and Evaluation of this Polymerase Chain Reaction–Based System with Experimentally Contaminated Samples†

2003 ◽  
Vol 66 (2) ◽  
pp. 182-187 ◽  
Author(s):  
CHRISTINE M. STRAPP ◽  
ADRIENNE E. H. SHEARER ◽  
ROLF D. JOERGER
Keyword(s):  
Escherichia Coli ◽  
Detection Rate ◽  
Salmonella Enteritidis ◽  
Detection System ◽  
Escherichia Coli O157 ◽  
Chain Reaction ◽  
E Coli ◽  
Polymerase Chain ◽  
Alfalfa Sprouts ◽  
Alfalfa Sprout

BAX, a polymerase chain reaction (PCR)–based pathogen detection system, was used to survey retail sprouts and mushrooms for contamination with Escherichia coli O157:H7, Salmonella, Listeria spp., and Listeria monocytogenes. No Salmonella or E. coli O157:H7 was detected in the 202 mushroom and 206 alfalfa sprout samples screened. L. monocytogenes was detected in one sprout sample, and seven additional sprout samples tested positive for the genus Listeria. BAX also detected Listeria species in 17 of the mushroom samples. Only 6 of 850 PCR assays (0.7%) failed to amplify control DNA, and therefore reagent failures and the inhibition of PCR by plant compounds were rare. The sensitivity of the detection system was evaluated by assaying samples inoculated with 10 CFU of each of the pathogens. One hundred seventy-two alfalfa sprout samples were inoculated with E. coli O157:H7, and two sets of 130 samples were experimentally contaminated with Salmonella Enteritidis and L. monocytogenes. The frequency of detection depended on the protocols used for inoculation and culturing. Inoculation of samples with approximately 10 CFU from frozen stocks yielded detection rates of 87.5 and 94.5% for L. monocytogenes and Salmonella Enteritidis, respectively, in mushrooms. The corresponding rates for alfalfa sprouts were 94.5 and 76.3%. The E. coli O157:H7 detection rate was 100% for mushrooms but only 48.6% for sprouts when standard BAX culture protocols were used. The substitution of an overnight incubation in modified E. coli medium for the 3-h brain heart infusion incubation increased the rate of E. coli O157:H7 detection to 75% for experimentally contaminated sprouts. The detection rate was 100% when E. coli O157:H7 cells from a fresh overnight culture were used for the inoculation. Test sensitivity is therefore influenced by the type of produce involved and is probably related to the growth of pathogens in the resuscitation and enrichment media.

scholarly journals EFFECTIVENESS OF MENIRAN (PHYLLANTHUS NIRURI LINN) AS ANTIBACTERIAL FOR RESISTANCE ANTIBIOTICS PREVENTION OF ENTEROTOXIN ESCHERICHIA COLI

2018 ◽  
Vol 7 (2) ◽  
pp. 35
Author(s):  
Sri Hidanah ◽  
Emy Koestanti Sabdoningrum ◽  
Retno Sri Wahyuni ◽  
Arini Rahmi Dewi ◽  
Erma - Safitri
Keyword(s):  
Escherichia Coli ◽  
The Body ◽  
Control Group ◽  
Anova Analysis ◽  
Chain Reaction ◽  
Isolation And Identification ◽  
Phyllanthus Niruri ◽  
E Coli ◽  
Pcr Method ◽  
Polymerase Chain

ABSTRACT               Escherichia coli (E. coli) can be isolated from the environment both inside and outside the hospes body. There were 89 serotypes in which 21% showed resistance to various antibiotics, such as E. coli enterotoxin. Alternative efforts were needed as a substitute for antibiotics, one of them through the use of medicinal plants, such as meniran (Phyllanthus niruri Linn).  Meniran plant is an immunomodulator that serves to repair the immune system of the body. The research was done through several stages: isolation and identification of  E. coli enterotoxin from several broiler farms in East Java using the polymerase chain reaction (PCR) method, E. coli resistance test against some antibiotics, making meniran extract and activation test against E. coli enterotoxin The study was divided into five treatments: T0+ (group of chickens were infected by E. coli enterotoxin), T0- (control group, not infected), T1 (infected by E. coli enterotoksin + 20% meniran extract), T2 (infected by E. coli enterotoksin + 25% extract meniran), T3 (infected by E. coli enterotoxin + 30% extract meniran). Data were analyzed by ANOVA (Analysis of Variance). The results were showed that all of  E. coli DNA isolates which tested by the PCR method was showed positive reactions at 600 bp. In the next stage, that E. coli enterotoxin are resistance to some antibiotics, such as Amoxicillin, Amphicillin, Erythromycin, Cephalosporins, Tetracycline, Cloxacillin and Gentamicin. Furthermore, 30% Phyllanthus niruri linn extract  effective as an antibacterial for the prevention of antibiotic resistance from E. coli enterotoxin. 

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