Multiplex PCRs for Identification of
            <i>Escherichia coli</i>
            Virulence Genes

Pass, Michael A.; Odedra, Rajesh; Batt, R. M.

doi:10.1128/jcm.38.5.2001-2004.2000

Cited by 151 publications

(57 citation statements)

References 13 publications

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“…Each of the 11 Aeromonas isolates was screened for the presence of genes associated with pathogenic E. coli. These included genes verotoxins (VT) or Shiga toxins (Stx) (VT1/Stx1, VT2/Stx2 and VT2e/STx2e), heat-labile (LT)1 toxin, heat-stable (ST) 1 and 2 toxins, cytotoxicnecrotizing factors (CNF) 1 and 2, enteroaggregative (Eagg) and enteroinvasive (Einv) genes, using primer sequences described by Pass et al (2000). These virulence genes and/or virulence traits have been commonly reported to be associated with the bacteria causing gastrointestinal infections (Nataro and Kaper 1998).…”

Section: Detection Of Virulence Genes Using Pcrmentioning

confidence: 99%

Prevalence of environmental Aeromonas in South East Queensland, Australia: a study of their interactions with human monolayer Caco-2 cells

et al. 2006

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Aims: To investigate the prevalence of Aeromonas in a major waterway in South East Queensland, Australia, and their interactions with a gut epithelial model using Caco-2 cells. Methods and Results: A total of 81 Aeromonas isolates, collected from a major waterway in South East Queensland, Australia, were typed using a metabolic fingerprinting method, and tested for their adhesion to HEp-2 and Caco-2 cells and for cytotoxin production on Vero cells and Caco-2 cells. Aeromonas hydrophila had the highest (43%) and Aeromonas veronii biovar sobria had the lowest (25%) prevalence. Four patterns of adhesion were observed on both HEp-2 and Caco-2 cell lines. Representative isolates having different phenopathotypes (nine strains) together with two clinical isolates were tested for their translocation ability and for the presence of virulence genes associated with pathogenic Escherichia coli. The rate and degree of translocation across Caco-2 monolayers varied among strains and was more pronounced with LogA pattern. Translocation was associated with the adherence of strains to Caco-2 cells microvilli, followed by internalization into Caco-2 cells. Two Aer. veronii biovar sobria strains were positive for the presence of heat-labile toxin genes, with one strain also positive for Shiga-like toxin gene. Conclusions: Pathogenic strains of Aeromonas carrying one or more virulence characteristics are highly prevalent in the waterways studied and are capable of translocating across a human enterocyte cell model. Significance and Impact of the Study: This study indicates that Aeromonas strains carrying one or more virulence properties are prevalent in local waterways and are capable of translocating in a human enterocyte cell culture model. However, their importance in human gastrointestinal disease has yet to be verified under competitive conditions of the gut.

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Section: Detection Of Virulence Genes Using Pcrmentioning

confidence: 99%

Prevalence of environmental Aeromonas in South East Queensland, Australia: a study of their interactions with human monolayer Caco-2 cells

et al. 2006

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“…PCR analyses for identification of DEC groups have been designed that detect one or a few genes per reaction [10,[12][13][14][15][16][17][18][19][20][21][22][23][24]. However, PCRs are now being designed to detect multiple genes in the same reaction, thereby further reducing the cost and time required for the experimental procedure [22,[25][26][27][28][29][30][31][32][33].…”

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A method for fast and simple detection of major diarrhoeagenic Escherichia coli in the routine diagnostic laboratory

Persson

Olsen

Scheutz

et al. 2007

Clinical Microbiology and Infection

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A multiplex PCR was developed for the detection of the following genes characteristic of diarrhoeagenic Escherichia coli (DEC): verocytotoxins 1 (vtx1) and 2 (vtx2), characteristic of verocytotoxin-producing E. coli (VTEC); intimin (eae), found in enteropathogenic E. coli (EPEC), attaching and effacing E. coli and VTEC; heat-stable enterotoxin (estA) and heat-labile enterotoxin (eltA), characteristic of enterotoxigenic E. coli (ETEC); and invasive plasmid antigen (ipaH), characteristic of enteroinvasive E. coli (EIEC) and Shigella spp. The method allowed the simultaneous identification of all six genes in one reaction, and included a 16S rDNA internal PCR control. When applied to pure cultures from a reference strain collection, all virulence genes in 124 different DEC strains and 15 Shigella spp. were identified correctly, and there were no cross-reactions with 13 non-E. coli species. The detection limit of the method was 10(2)-10(3) DEC CFU/PCR in the presence of 10(6) non-target cells. When the multiplex PCR was tested with colonies from plate cultures of clinical stool samples, it was a faster, more sensitive, less expensive and less laborious diagnostic procedure than DNA hybridisation. When used with DNA purified from spiked stool samples (by two different commercial kits), the method had a detection limit of 10(6) CFU/mL stool sample.

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“…Several PCR methods for detecting the various virulence factors have been reported elsewhere (1,6,22).However, conducting the separate PCR reactions that are required for the detection of the virulence factors in order to assign an isolated E. coli strain to one of the five categories is very laborious and time comsuming. Therefore, various multiplex PCR methods have been developed for the simultaneous detection of several pathogenic genes in one PCR reaction (15,18,20,23). Using the multiplex PCR method, we will be able to save the time and effort involved in analyzing various virulence factors.…”

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Rapid Categorization of Pathogenic Escherichia coli by Multiplex PCR

Kimata

Shima

Shimizu

et al. 2005

Microbiology and Immunology

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Escherichia coli, which causes diarrhea in humans, can be classified into the following heterogenous groups: enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAggEC), enteroinvasive E. coli (EIEC), and enterotoxigenic E. coli (ETEC). These diarrheagenic E. coli categories are differentiated on the basis of their infection and pathogenic mechanisms. Serotyping, phenotypic assays, and molecular detection assays are very useful in identifying these diarrheagenic E. coli categories (2, 12).Since several virulence factors and DNA sequences of diarrheagenic E. coli have been identified, their molecular analyses have been performed using genetic detection assays, including PCR and DNA hybridization (5, 21, 26). The PCR method, which is a rapid gene detection assay, is particularly effective in identifying these diarrheagenic E. coli categories. Several PCR methods for detecting the various virulence factors have been reported elsewhere (1,6,22).However, conducting the separate PCR reactions that are required for the detection of the virulence factors in order to assign an isolated E. coli strain to one of the five categories is very laborious and time comsuming. Therefore, various multiplex PCR methods have been developed for the simultaneous detection of several pathogenic genes in one PCR reaction (15,18,20,23). Using the multiplex PCR method, we will be able to save the time and effort involved in analyzing various virulence factors. Some multiplex PCR systems have been reported for the rapid detection of specific virulence factors that distinguish EHEC O157 from other serotypes of EHEC (10,11,14,16,17). Cebula et al. (3) reported the multiplex PCR systems for the simultaneous detection of stx1, stx2, and uidA genes, which are specific to EHEC O157:H7. Wang et al. (27) have also reported the combination of certain multiplex PCR systems for the detection of stx1, stx2, stx2 variants, eaeA, EHEC hlyA, and rfbE O157 , which are specific to the O157 serotype, and fliC H7 , which is specific to the flagellum H7 serotype. Pass et al. (15) and Toma et al. (25) reported the use of multiplex PCR systems for the detection of 11 and 6 virulence genes, respectively. However, the systems described by them for simultaneous categorization of E. coli have not been completely effective for the simultaneous categorization of all diarrheagenic E. coli. The multiplex PCR system reported Abstract: A one-shot multiplex polymerase chain reaction (PCR) was developed for detecting 12 virulence genes of diarrheagenic Escherichia coli. In order to differentiate between the five categories of diarrheagenic E. coli, we selected the target genes: stx1, stx2, and eaeA for enterohemorrhagic E. coli (EHEC); eaeA, bfpA, and EAF for enteropathogenic E. coli (EPEC); invE for enteroinvasive E. coli (EIEC); elt, estp, and esth for enterotoxigenic E. coli (ETEC); CVD432 and aggR for enteroaggregative E. coli (EAggEC); and astA distributed over the categories of diarrheagenic E. coli. In our multiplex PCR ...

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Multiplex PCRs for Identification of Escherichia coli Virulence Genes

Cited by 151 publications

References 13 publications

Prevalence of environmental Aeromonas in South East Queensland, Australia: a study of their interactions with human monolayer Caco-2 cells

Prevalence of environmental Aeromonas in South East Queensland, Australia: a study of their interactions with human monolayer Caco-2 cells

A method for fast and simple detection of major diarrhoeagenic Escherichia coli in the routine diagnostic laboratory

Rapid Categorization of Pathogenic Escherichia coli by Multiplex PCR

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