Multiplex PCR-DNA probe assay for the detection of pathogenic Escherichia coli

Watterworth, Leigh; Topp, Edward; Schraft, Heidi; Leung, Ka Yin

doi:10.1016/j.mimet.2004.08.016

Cited by 31 publications

(23 citation statements)

References 34 publications

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“…However, the PCR methodology was modified to a three-sample reaction in order to increase the specificity and sensitivity of the reaction. mPCR is specific ISSN 2157-6076 2016 as multiple PCR primers were specific for each target, and no cross-reactivity was detected with heterologous DNA and validation of this PCR method confirmed that approximately 100 strains previously characterized were correctly identified by the three-sample PCR method, this is in keeping with other studies (Watterworth et al 2005, Oscar et al, 2009. mPCR identification is rapid, as in 4 hours it is possible to isolate crude DNA preparations from a bacteria suspension, amplify the DNA, separate the DNA fragments in an agarose gel, and have a visual report of the banding pattern.…”

Section: Journal Of Biology and Life Sciencesupporting

confidence: 84%

Molecular Characterization of Diarrheagenic Bacteria Isolated from Stool of Under-five Children in Dar Es Salaam. Tanzania

Ngoso

Namkinga²,

Nkwengulila

2015

JBLS

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Diarrhea is a daily public health song in developing countries like Tanzania. The causative agents are theoretically known almost to everybody. However, the eradication of this killer disease for the under-fives is an enigma. This study aimed to provide update advantages of molecular diagnostic versus conventional methods as regards to acute diarrhea, and to determine bacterial causes of diarrhea among children aging five years and below in Dar Es Salaam, Tanzania, using multiplex PCR technique.Samples were collected from the under-fives from district hospitals in Dar Es Salaam city between June 2010 and February 2014. This included children admitted due to acute and/ or Journal of Biology and Life Science ISSN 2157-6076 2016 www.macrothink.org/jbls 72 chronic diarrhea. A total of 3600 stool samples were analyzed, of which 1800 samples were from diarrhea cases and 1800 samples from normal control cases. About 1080 (60%) of the patients recruited were aged less than 3 years and 983 (54.6%) were males. Diarrheagenic bacteria were isolated and identified using conventional stool cultures then were characterized by mPCR.Pathogenic bacteria were detected in 67.7% of the cases and in 20% of the controls. The pathogenic bacteria most strongly associated with diarrhea disease were diarrheagenic Escherichia coli (21.6% of cases, 6% of controls), Shigella spp. (16.1% of cases, 5% of controls) and Salmonellae, (10.6% of cases, 3% of controls. The pathogenic bacteria were mostly from children aging from 24 months and above.Diarrheagenic bacteria play an important role in relation to childhood diarrhea aging from two years and above. Proper diagnostic methods, prevention and control through fostering good hygiene and sanitation to water and food should be emphasized especially to oral-faecal age.

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Section: Journal Of Biology and Life Sciencesupporting

confidence: 84%

Molecular Characterization of Diarrheagenic Bacteria Isolated from Stool of Under-five Children in Dar Es Salaam. Tanzania

Ngoso

Namkinga²,

Nkwengulila

2015

JBLS

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“…These assays target common genes for most but not all of the currently recognized classes of diarrheagenic E. coli or require additional steps (4,5,6,15,(25)(26)(27)(28)30). Traditional PCR methods require amplification in a thermocycler followed by product separation by gel electrophoresis (19) or fluorescent capillary electrophoresis (7), time-consuming and laborious processes.…”

Section: Discussionmentioning

confidence: 99%

Detection of Diarrheagenic Escherichia coli by Use of Melting-Curve Analysis and Real-Time Multiplex PCR

et al. 2008

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Diarrheagenic Escherichia coli strains are important causes of diarrhea in children from the developing world and are now being recognized as emerging enteropathogens in the developed world. Current methods of detection are too expensive and labor-intensive for routine detection of these organisms to be practical. We developed a real-time fluorescence-based multiplex PCR for the detection of all six of the currently recognized classes of diarrheagenic E. coli. The primers were designed to specifically amplify eight different virulence genes in the same reaction: aggR for enteroaggregative E. coli, stIa/stIb and lt for enterotoxigenic E. coli, eaeA for enteropathogenic E. coli and Shiga toxin-producing E. coli (STEC), stx 1 and stx 2 for STEC, ipaH for enteroinvasive E. coli, and daaD for diffusely adherent E. coli (DAEC). Eighty-nine of ninety diarrheagenic E. coli and 36/36 nonpathogenic E. coli strains were correctly identified using this approach (specificity, 1.00; sensitivity, 0.99). The single false negative was a DAEC strain. The total time between preparation of DNA from E. coli colonies on agar plates and completion of PCR and melting-curve analysis was less than 90 min. The cost of materials was low. Melting-point analysis of real-time multiplex PCR is a rapid, sensitive, specific, and inexpensive method for detection of diarrheagenic E. coli.Escherichia coli strains associated with diarrhea have been classified into six groups, based on clinical, epidemiological, and molecular criteria. These E. coli strains are commonly isolated from children with gastroenteritis in the developing world. Recent data suggest that these strains are common in the United States in children less than 5 years of age with acute diarrhea (10, 17). However, diarrheagenic E. coli strains are not routinely sought as stool pathogens in clinical laboratories. Some of these pathogens respond to antimicrobial agents, while for others (e.g., Shiga toxin-producing E. coli [STEC]), antibiotics should be avoided. Because the time frame in which treatment choices must be made is short, there is a need for a rapid, sensitive, and inexpensive detection technique. We have developed a monochromatic, fluorescence-based real-time PCR procedure to simultaneously identify eight virulence genes associated with the six classes of diarrheagenic E. coli. In this assay, the post-PCR products are identified based on melting-point curve analysis. This assay is simple, rapid, inexpensive, and reliable. It is suitable for use in clinical laboratories as well as research facilities. MATERIALS AND METHODSBacterial strains. One hundred twenty-six E. coli strains (Table 1) were analyzed, including strains representing all six of the currently recognized classes of diarrheagenic E. coli as well as commensal organisms. The prototypical strains, used in laboratories worldwide, included enterotoxigenic E. coli (ETEC) H10407; enteropathogenic E. coli (EPEC) 2348/69; STEC strains H30, HW1, and 86-24; and enteroaggregative E. coli (EAEC) strains O42 and 221. Addit...

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“…The E. coli O157:H7 H32 strain we employed was obtained from C. Gyles at the University of Guelph (Guelph, ON, Canada) and corresponds to the strain identification number EC920004 from this culture collection. H32 is a bovine isolate and possesses genes for Shiga-like toxins I and II (55). It also contains the eae gene for intimin, the virulence factor responsible for mediating the attachment of E. coli to colonic epithelial cells (22,28).…”

Section: Methodsmentioning

confidence: 99%

Role of rpoS in Escherichia coli O157:H7 Strain H32 Biofilm Development and Survival

Sheldon

Yim

Saliba

et al. 2012

Appl Environ Microbiol

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bThe protein RpoS is responsible for mediating cell survival during the stationary phase by conferring cell resistance to various stressors and has been linked to biofilm formation. In this study, the role of the rpoS gene in Escherichia coli O157:H7 biofilm formation and survival in water was investigated. Confocal scanning laser microscopy of biofilms established on coverslips revealed a nutrient-dependent role of rpoS in biofilm formation, where the biofilm biomass volume of the rpoS mutant was 2.4-to 7.5-fold the size of its rpoS ؉ wild-type counterpart in minimal growth medium. The enhanced biofilm formation of the rpoS mutant did not, however, translate to increased survival in sterile double-distilled water (ddH 2 O), filter-sterilized lake water, or unfiltered lake water. The rpoS mutant had an overall reduction of 3.10 and 5.30 log 10 in sterile ddH 2 O and filter-sterilized lake water, respectively, while only minor reductions of 0.53 and 0.61 log 10 in viable counts were observed for the wild-type form in the two media over a 13-day period, respectively. However, the survival rates of the detached biofilm-derived rpoS ؉ and rpoS mutant cells were comparable. Under the competitive stress conditions of unfiltered lake water, the advantage conferred by the presence of rpoS was lost, and both the wild-type and knockout forms displayed similar declines in viable counts. These results suggest that rpoS does have an influence on both biofilm formation and survival of E. coli O157:H7 and that the advantage conferred by rpoS is contingent on the environmental conditions.

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Multiplex PCR-DNA probe assay for the detection of pathogenic Escherichia coli

Cited by 31 publications

References 34 publications

Molecular Characterization of Diarrheagenic Bacteria Isolated from Stool of Under-five Children in Dar Es Salaam. Tanzania

Molecular Characterization of Diarrheagenic Bacteria Isolated from Stool of Under-five Children in Dar Es Salaam. Tanzania

Detection of Diarrheagenic Escherichia coli by Use of Melting-Curve Analysis and Real-Time Multiplex PCR

Role of rpoS in Escherichia coli O157:H7 Strain H32 Biofilm Development and Survival

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