scholarly journals Application of Multiplex PCR for Detection of Non-O157 Verocytotoxin-Producing Escherichia coli in Bloody Stools: Identification of Serogroups O26 and O111

1998 ◽  
Vol 36 (11) ◽  
pp. 3375-3377 ◽  
Author(s):  
M. Louie ◽  
S. Read ◽  
A. E. Simor ◽  
J. Holland ◽  
L. Louie ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Bloody Stool ◽  
Pcr Assay ◽  
E Coli ◽  
Rapid Assays ◽  
Multiplex Pcr Assay ◽  
Stool Specimens ◽  
Bloody Stools ◽  
Etiologic Diagnosis

Primers were designed to amplify sequences of verocytotoxin genes and eaeA genes of Escherichia coli O26:H11, O111:H8, and O157:H7 in a multiplex PCR assay. This assay successfully detected E. coli O26:H11 in bloody stool specimens in which other enteric pathogens were not detected by culture-based methods. Rapid assays to detect non-O157:H7 verocytotoxin-producingE. coli is important to improve methods for the etiologic diagnosis of hemorrhagic colitis.

Usefulness of a Multiplex PCR for Detection of Diarrheagenic Escherichia coli in a Diagnostic Microbiology Laboratory Setting

2016 ◽  
Vol 1 (2) ◽  
pp. 38-42 ◽  
Author(s):  
Khairun Nessa ◽  
Dilruba Ahmed ◽  
Johirul Islam ◽  
FM Lutful Kabir ◽  
M Anowar Hossain
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Clinical Laboratory ◽  
Proteinase K ◽  
Microbiology Laboratory ◽  
Pcr Assay ◽  
E Coli ◽  
Diarrheagenic Escherichia Coli ◽  
Multiplex Pcr Assay ◽  
Diagnostic Microbiology

A multiplex PCR assay was evaluated for diagnosis of diarrheagenic Escherichia coli in stool samples of patients with diarrhoea submitted to a diagnostic microbiology laboratory. Two procedures of DNA template preparationproteinase K buffer method and the boiling method were evaluated to examine isolates of E. coli from 150 selected diarrhoeal cases. By proteinase K buffer method, 119 strains (79.3%) of E. coli were characterized to various categories by their genes that included 55.5% enteroaggregative E. coli (EAEC), 18.5% enterotoxigenic E. coli (ETEC), 1.7% enteropathogenic E. coli (EPEC), and 0.8% Shiga toxin-producing E. coli (STEC). Although boiling method was less time consuming (<24 hrs) and less costly (<8.0 US $/ per test) but was less efficient in typing E. coli compared to proteinase K method (41.3% vs. 79.3% ; p<0.001). The sensitivity and specificity of boiling method compared to proteinase K method was 48.7% and 87.1% while the positive and negative predictive value was 93.5% and 30.7%, respectively. The majority of pathogenic E. coli were detected in children (78.0%) under five years age with 53.3% under one year, and 68.7% of the children were male. Children under 5 years age were frequently infected with EAEC (71.6%) compared to ETEC (24.3%), EPEC (2.7%) and STEC (1.4%). The multiplex PCR assay could be effectively used as a rapid diagnostic tool for characterization of diarrheagenic E. coli using a single reaction tube in the clinical laboratory setting.Bangladesh J Med Microbiol 2007; 01 (02): 38-42

scholarly journals Molecular Detection of Diarrheagenic Escherichia coli from Children with Acute Diarrhea in Tertiary Care Hospitals of Dhaka, Bangladesh.

2013 ◽  
Vol 5 (2) ◽  
pp. 59-66 ◽  
Author(s):  
Sushmita Roy ◽  
SM Shamsuzzaman ◽  
Kazi Z Mamun
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Acute Diarrhea ◽  
Tertiary Care ◽  
Medical Science ◽  
Pcr Assay ◽  
E Coli ◽  
Diarrheagenic Escherichia Coli ◽  
Multiplex Pcr Assay ◽  
Stool Samples

Objective: Multiplex PCR assay was used for diagnosis of diarrheagenic Escherichia coli (DEC) in stool samples of children (under 5 years) with acute diarrhea.  Methods: Samples were collected from January 2011 to December 2011, from Dhaka Medical College Hospital and Dhaka Shishu Hospital. Multiplex PCR with five specific primer pairs to detect enteropathogenic E. coli (eae, bfp), enterotoxigenic E. coli (lt, st) and enteroaggregative E. coli (aat) were used. However, enteroinvasive E. coli, enterohemorrhagicE. coli and diffusely adhererentE. coli were not sought. Result: In total, 135 (67.5%) E. coli were isolated from 200 stool samples. The prevalence of DEC was 68 (34%). Among DEC, most frequently isolated pathotype was EPEC 40 (58.82%), followed by ETEC 24 (35.29%) and EAggEC 18 (26.47%). Among the EPEC, 5 (12.5%) were typical EPEC. Among the 68 DEC positive cases, 22 samples contained more than one pathogenic gene in various combinations. Among the combination of DEC, EPEC+ETEC combination was 6 (27.27%) followed by ETEC+EAggEC 4 (18.18%), EPEC+EAggEC and ETEC+EPEC+EAggEC were both in 3 (13.6%). Conclusion:This study shows that DEC is a common cause of childhood diarrhea in Dhaka city of Bangladesh. By using multiplex PCR assay, DEC can be diagnosed in one PCR reaction that makes a conclusive diagnosis of diarrhea. DOI: http://dx.doi.org/10.3126/ajms.v5i2.8576 Asian Journal of Medical Science, Volume-5(2) 2014: 59-66

scholarly journals A combined AFLP–multiplex PCR assay for molecular typing of Escherichia coli strains using variable bacterial interspersed mosaic elements

2004 ◽  
Vol 132 (1) ◽  
pp. 61-65 ◽  
Author(s):  
R. HORVÁTH ◽  
M. DENDIS ◽  
J. SCHLEGELOVÁ ◽  
F. RŮŽIČKA ◽  
J. BENEDÍK
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Molecular Typing ◽  
Original Method ◽  
Pcr Assay ◽  
E Coli ◽  
Promising Tool ◽  
Multiplex Pcr Assay ◽  
Chromosomal Sequences

The original method for molecular typing of E. coli strains was developed using the polymorphism in chromosomal sequences of bacterial interspersed mosaic elements (BIMEs) detected by multiplex PCR and analysed by AFLP assay. The applicability of the method in the epidemiology of E. coli was tested on a group of 524 strains of human and veterinary origin. In the studied group 18 different genotypes were detected. Significant differences were found in the frequencies of the genotypes among various groups of strains, suggesting the method could be a promising tool in the epidemiology of E. coli.

scholarly journals Detection of Escherichia coli O104 in the Feces of Feedlot Cattle by a Multiplex PCR Assay Designed To Target Major Genetic Traits of the Virulent Hybrid Strain Responsible for the 2011 German Outbreak

2013 ◽  
Vol 79 (11) ◽  
pp. 3522-3525 ◽  
Author(s):  
Z. D. Paddock ◽  
J. Bai ◽  
X. Shi ◽  
D. G. Renter ◽  
T. G. Nagaraja
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Feedlot Cattle ◽  
Pcr Assay ◽  
Hybrid Strain ◽  
Genetic Traits ◽  
Fecal Samples ◽  
Content Type ◽  
E Coli ◽  
Multiplex Pcr Assay

ABSTRACTA multiplex PCR was designed to detectEscherichia coliO104:H4, a hybrid pathotype of Shiga toxigenic and enteroaggregativeE. coli, in cattle feces. A total of 248 fecal samples were tested, and 20.6% were positive for serogroup O104. The O104 isolates did not carry genes characteristic of the virulent hybrid strain.

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.

scholarly journals Rapid Identification of Different Escherichia coli Sequence Type 131 Clades

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Yasufumi Matsumura ◽  
Johann D. D. Pitout ◽  
Gisele Peirano ◽  
Rebekah DeVinney ◽  
Taro Noguchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epidemiological Studies ◽  
Rapid Identification ◽  
Sequence Type ◽  
Fluoroquinolone Resistance ◽  
Pcr Assay ◽  
Content Type ◽  
E Coli ◽  
Multiplex Pcr Assay ◽  
Clade C

ABSTRACT Escherichia coli sequence type 131 (ST131) is a pandemic clonal lineage that is responsible for the global increase in fluoroquinolone resistance and extended-spectrum-β-lactamase (ESBL) producers. The members of ST131 clade C, especially subclades C2 and C1-M27, are associated with ESBLs. We developed a multiplex conventional PCR assay with the ability to detect all ST131 clades (A, B, and C), as well as C subclades (C1-M27, C1-nM27 [C1-non-M27], and C2). To validate the assay, we used 80 ST131 global isolates that had been fully sequenced. We then used the assay to define the prevalence of each clade in two Japanese collections consisting of 460 ESBL-producing E. coli ST131 (2001-12) and 329 E. coli isolates from extraintestinal sites (ExPEC) (2014). The assay correctly identified the different clades in all 80 global isolates: clades A (n = 12), B (n = 12), and C, including subclades C1-M27 (n = 16), C1-nM27 (n = 20), C2 (n = 17), and other C (n = 3). The assay also detected all 565 ST131 isolates in both collections without any false positives. Isolates from clades A (n = 54), B (n = 23), and C (n = 483) corresponded to the O serotypes and the fimH types of O16-H41, O25b-H22, and O25b-H30, respectively. Of the 483 clade C isolates, C1-M27 was the most common subclade (36%), followed by C1-nM27 (32%) and C2 (15%). The C1-M27 subclade with bla CTX-M-27 became especially prominent after 2009. Our novel multiplex PCR assay revealed the predominance of the C1-M27 subclade in recent Japanese ESBL-producing E. coli isolates and is a promising tool for epidemiological studies of ST131.

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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