Rapid detection of diarrheagenic E. coli by real-time PCR

Bischoff, Claus; Lüthy, Jürg; Altwegg, Martin; Baggi, Franca

doi:10.1016/j.mimet.2004.12.007

Cited by 61 publications

(31 citation statements)

References 16 publications

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“…The sta primers designed for the assays described here overlap with, but are not identical to, primers described elsewhere. 6,7,9,10,[12][13][14][15][16]24,25 Primer sequences used in this work are listed in Table 1. Oligonucleotides were synthesized by Invitrogen (Carlsbad, CA).…”

Section: Methodsmentioning

confidence: 99%

“…Detection assays rely on the amplification of eltA alone, eltA plus either sta1 or sta2, or all three toxin genes. [6][7][8][9][10][11][12][13][14][15][16] The DNA hybridization assay is a culture-dependent method that requires transfer of cultured lactose-positive colonies from MacConkey agar to Whatman filter paper (Fisher Scientific, Waltham, MA) followed by cell lysis and probe hybridization. Probes targeting ST P (sta1), ST H (sta2), and LT (eltA) are then used for ETEC detection 17,18 ; remarkably, many clinical manuscripts reporting the prevalence of ETEC use this less-sensitive DNA hybridization method or use PCR assays that target eltA plus only a single allele of ST. As a result, these studies are likely to have underestimated the prevalence of ETEC infections.…”

Section: Introductionmentioning

confidence: 99%

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Development and Accuracy of Quantitative Real-Time Polymerase Chain Reaction Assays for Detection and Quantification of Enterotoxigenic Escherichia coli (ETEC) Heat Labile and Heat Stable Toxin Genes in Travelers' Diarrhea Samples

Youmans¹,

Ajami²,

Jiang³

et al. 2014

The American Journal of Tropical Medicine and Hygiene

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Abstract. Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Development and Accuracy of Quantitative Real-Time Polymerase Chain Reaction Assays for Detection and Quantification of Enterotoxigenic Escherichia coli (ETEC) Heat Labile and Heat Stable Toxin Genes in Travelers' Diarrhea Samples

Youmans¹,

Ajami²,

Jiang³

et al. 2014

The American Journal of Tropical Medicine and Hygiene

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“…Notably, DEC strains cannot be easily distinguished from the normal fecal flora using conventional phenotypic methods; while in-vitro assay can detect toxins, adherence or invasion phenotypes of DEC are cumbersome [6]. This task of identification and differentiation of DEC associated with diarrhea has been greatly simplified by the advent of polymerase chain reaction (PCR) technology [7]. Molecular identification and classification of DEC is based on the presence of different chromosomal and/or plasmid-encoded virulence genes that are absent in commensal E. coli [8].…”

Section: Introductionmentioning

confidence: 99%

Diarrheagenic Escherichia coli pathotypes isolated from children with diarrhea in the Federal Capital Territory Abuja, Nigeria

Ifeanyi

Ikeneche

Bassey

et al. 2015

J Infect Dev Ctries

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Introduction: Escherichia coli are frequently isolated from diarrheic children in the Federal Capital Territory Abuja, Nigeria, but their virulent properties are not routinely evaluated. Therefore, the etiology of childhood diarrheal disease attributable to diarrheagenic Escherichia coli (DEC) in Abuja, Nigeria remains unknown. Methodology: Stool specimens from 400 acute diarrheic children between 0 and 60 months of age were studied.E. coli strains isolated were evaluated by polymerase chain reaction (PCR) for nine virulence genes and HEp-2 cell adherence to detect and identify five distinct diarrheagenic E. coli categories. Results: Diarrheagenic E.coli was detected in 51 (12.8%) of the diarrheic children. The observed DEC pathotypes were enteropathogenic E. coli (EPEC) in 18 (4.5%) children, enterotoxigenic E. coli (ETEC) in 16 (4.0%), enteroaggrative E. coli (EAEC) in 8 (2.0%), enterohaemorrhagic E. coli (EHEC) in 6 (1.5%), and enteroinvasive E. coli (EIEC) in 3 (0.8%). Four (1.0 %) EPEC strains with only the eae+ gene that adhered diffusely to HEp-2 cell were identified as atypical EPEC. All the DEC categories except atypical EPEC were identified in children between 6 and 12 months of age. Conclusions: This study underscores the need for routine evaluation of diarrheic children for virulence properties of infectious DEC. Atypical EPEC are emerging among the DEC pathotypes isolated from childhood acute gastroenteritis in Abuja, Nigeria.

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“…Previously, real-time PCr based methods have been reported for detection of many pathogens of veterinary importance (aGUerO et al, 2007). SYBr green based real time PCr assay was used earlier to differentiate ePeC, Verotoxic E. coli and other enteroaggregative E. coli, and it was concluded in that study that it may be used as an effective diagnostic tool compared to conventional PCr, for detection of E. coli strains (BISChOFF et al, 2005;GUION et al, 2008) Rotavirus is responsible for causing economically significant maladies in neonates of many domestic animals (kaPIkIaN and ChaNOCk, 1996;eSteS and kaPIkIaN, 2007). In ovines, rotaviruses are known to cause enteritis and diarrhea (WaNI et al, 2004;GaZaL et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

Molecular detection of Clostridium perfringens toxinotypes, Enteropathogenic Escherichia coli, rotavirus and coronavirus in diarrheic fecal samples of neonatal goat kids

et al. 2018

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In the present study, out of 1156 neonatal goat kids, 238 showing clinical diarrhea were used for detection of toxinotypes of Clostridium perfringens, enteropathogenic E. coli (ePeC), Group a rotavirus (GarV) and Bovine coronavirus (BCV). Isolation and toxinotyping of isolates were done by multiplex Polymerase chain reaction (PCr) using primers for cpa, cpb, cpb2, etx and iap genes. For EPEC, isolation and identification were done using bfpa gene and SYBr green based real time PCr (qPCr). GarV and BCV were detected, by onestep rt-PCr (osrt-PCr). the incidence of C. perfringens was 15.13% with 75% isolates toxinotype a, 25% type D and 61.11% of isolates carrying the β2-toxin gene. The incidence of EPEC was 68.07% based on qPCr, whereas 21.85% were positive for GarV and 15.97% for BCV by osrt-PCr. there was mixed infection of C. perfringens and ePeC in 11.76% and 3.78% for C. perfringens and GarV and 2.1% of C. perfringens and BCV. ePeC and GarV was 19.74% and ePeC plus BCV positivity was 11.34%. GarV and BCV was 5.88%, and 4.20% had mixed infection of ePeC, GarV and BCV. Of the total diarrheic kids sampled, 0.84% had mixed infection of C. perfringens, GARV, BCV and EPEC. On the basis of the above findings, it may be concluded that isolation, multiplex PCr and real time PCr facilitated the characterization of circulating C. perfringens toxinotypes and ePeC in goats reared under semi-arid conditions. the importance of enteritis caused by GarV and BCV and their role in mixed infection in goats requires extensive screening and pathogenicity studies to associate the symptoms with disease.

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Rapid detection of diarrheagenic E. coli by real-time PCR

Cited by 61 publications

References 16 publications

Development and Accuracy of Quantitative Real-Time Polymerase Chain Reaction Assays for Detection and Quantification of Enterotoxigenic Escherichia coli (ETEC) Heat Labile and Heat Stable Toxin Genes in Travelers' Diarrhea Samples

Development and Accuracy of Quantitative Real-Time Polymerase Chain Reaction Assays for Detection and Quantification of Enterotoxigenic Escherichia coli (ETEC) Heat Labile and Heat Stable Toxin Genes in Travelers' Diarrhea Samples

Diarrheagenic Escherichia coli pathotypes isolated from children with diarrhea in the Federal Capital Territory Abuja, Nigeria

Molecular detection of Clostridium perfringens toxinotypes, Enteropathogenic Escherichia coli, rotavirus and coronavirus in diarrheic fecal samples of neonatal goat kids

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