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Alignment-free design of highly discriminatory diagnostic primer sets for Escherichia coli O104

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Alignment-free design of highly discriminatory diagnostic primer sets for Escherichia coli O104 : H4 outbreak strains. / Pritchard, Leighton; Holden, Nicola J.; Bielaszewska, Martina; Karch, Helge; Toth, Ian K.

In: PLoS ONE, Vol. 7, No. 4, e34498, 05.04.2012.

Research output: Contribution to journalArticle

Harvard

Pritchard, L, Holden, NJ, Bielaszewska, M, Karch, H & Toth, IK 2012, 'Alignment-free design of highly discriminatory diagnostic primer sets for Escherichia coli O104: H4 outbreak strains' PLoS ONE, vol 7, no. 4, e34498., 10.1371/journal.pone.0034498

APA

Pritchard, L., Holden, N. J., Bielaszewska, M., Karch, H., & Toth, I. K. (2012). Alignment-free design of highly discriminatory diagnostic primer sets for Escherichia coli O104: H4 outbreak strains. PLoS ONE, 7(4), [e34498]. 10.1371/journal.pone.0034498

Vancouver

Pritchard L, Holden NJ, Bielaszewska M, Karch H, Toth IK. Alignment-free design of highly discriminatory diagnostic primer sets for Escherichia coli O104: H4 outbreak strains. PLoS ONE. 2012 Apr 5;7(4). e34498. Available from: 10.1371/journal.pone.0034498

Author

Pritchard, Leighton; Holden, Nicola J.; Bielaszewska, Martina; Karch, Helge; Toth, Ian K. / Alignment-free design of highly discriminatory diagnostic primer sets for Escherichia coli O104 : H4 outbreak strains.

In: PLoS ONE, Vol. 7, No. 4, e34498, 05.04.2012.

Research output: Contribution to journalArticle

Bibtex - Download

@article{1f90c922257340cf9d019eb5ef4eecc3,
title = "Alignment-free design of highly discriminatory diagnostic primer sets for Escherichia coli O104: H4 outbreak strains",
author = "Leighton Pritchard and Holden, {Nicola J.} and Martina Bielaszewska and Helge Karch and Toth, {Ian K.}",
year = "2012",
doi = "10.1371/journal.pone.0034498",
volume = "7",
number = "4",
journal = "PLoS ONE",
issn = "1932-6203",

}

RIS (suitable for import to EndNote) - Download

TY - JOUR

T1 - Alignment-free design of highly discriminatory diagnostic primer sets for Escherichia coli O104

T2 - H4 outbreak strains

A1 - Pritchard,Leighton

A1 - Holden,Nicola J.

A1 - Bielaszewska,Martina

A1 - Karch,Helge

A1 - Toth,Ian K.

AU - Pritchard,Leighton

AU - Holden,Nicola J.

AU - Bielaszewska,Martina

AU - Karch,Helge

AU - Toth,Ian K.

PY - 2012/4/5

Y1 - 2012/4/5

N2 - <p>Background:An Escherichia coli O104:H4 outbreak in Germany in summer 2011 caused 53 deaths, over 4000 individual infections across Europe, and considerable economic, social and political impact. This outbreak was the first in a position to exploit rapid, benchtop high-throughput sequencing (HTS) technologies and crowdsourced data analysis early in its investigation, establishing a new paradigm for rapid response to disease threats. We describe a novel strategy for design of diagnostic PCR primers that exploited this rapid draft bacterial genome sequencing to distinguish between E. coli O104:H4 outbreak isolates and other pathogenic E. coli isolates, including the historical haemolytic uraemic syndrome (HUSEC) E. coli HUSEC041 O104:H4 strain, which possesses the same serotype as the outbreak isolates.</p><p>Methodology/Principal Findings: Primers were designed using a novel alignment-free strategy against eleven draft whole genome assemblies of E. coli O104:H4 German outbreak isolates from the E. coli O104:H4 Genome Analysis Crowd-Sourcing Consortium website, and a negative sequence set containing 69 E. coli chromosome and plasmid sequences from public databases. Validation in vitro against 21 'positive' E. coli O104:H4 outbreak and 32 'negative' non-outbreak EHEC isolates indicated that individual primer sets exhibited 100% sensitivity for outbreak isolates, with false positive rates of between 9% and 22%. A minimal combination of two primers discriminated between outbreak and non-outbreak E. coli isolates with 100% sensitivity and 100% specificity.</p><p>Conclusions/Significance: Draft genomes of isolates of disease outbreak bacteria enable high throughput primer design and enhanced diagnostic performance in comparison to traditional molecular assays. Future outbreak investigations will be able to harness HTS rapidly to generate draft genome sequences and diagnostic primer sets, greatly facilitating epidemiology and clinical diagnostics. We expect that high throughput primer design strategies will enable faster, more precise responses to future disease outbreaks of bacterial origin, and help to mitigate their societal impact.</p>

AB - <p>Background:An Escherichia coli O104:H4 outbreak in Germany in summer 2011 caused 53 deaths, over 4000 individual infections across Europe, and considerable economic, social and political impact. This outbreak was the first in a position to exploit rapid, benchtop high-throughput sequencing (HTS) technologies and crowdsourced data analysis early in its investigation, establishing a new paradigm for rapid response to disease threats. We describe a novel strategy for design of diagnostic PCR primers that exploited this rapid draft bacterial genome sequencing to distinguish between E. coli O104:H4 outbreak isolates and other pathogenic E. coli isolates, including the historical haemolytic uraemic syndrome (HUSEC) E. coli HUSEC041 O104:H4 strain, which possesses the same serotype as the outbreak isolates.</p><p>Methodology/Principal Findings: Primers were designed using a novel alignment-free strategy against eleven draft whole genome assemblies of E. coli O104:H4 German outbreak isolates from the E. coli O104:H4 Genome Analysis Crowd-Sourcing Consortium website, and a negative sequence set containing 69 E. coli chromosome and plasmid sequences from public databases. Validation in vitro against 21 'positive' E. coli O104:H4 outbreak and 32 'negative' non-outbreak EHEC isolates indicated that individual primer sets exhibited 100% sensitivity for outbreak isolates, with false positive rates of between 9% and 22%. A minimal combination of two primers discriminated between outbreak and non-outbreak E. coli isolates with 100% sensitivity and 100% specificity.</p><p>Conclusions/Significance: Draft genomes of isolates of disease outbreak bacteria enable high throughput primer design and enhanced diagnostic performance in comparison to traditional molecular assays. Future outbreak investigations will be able to harness HTS rapidly to generate draft genome sequences and diagnostic primer sets, greatly facilitating epidemiology and clinical diagnostics. We expect that high throughput primer design strategies will enable faster, more precise responses to future disease outbreaks of bacterial origin, and help to mitigate their societal impact.</p>

U2 - 10.1371/journal.pone.0034498

DO - 10.1371/journal.pone.0034498

M1 - Article

JO - PLoS ONE

JF - PLoS ONE

SN - 1932-6203

IS - 4

VL - 7

ER -

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