Alignment-Free Design of Highly Discriminatory Diagnostic Primer Sets for Escherichia coli O104H4 Outbreak Strains 英文参考文献.docVIP

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Alignment-Free Design of Highly Discriminatory Diagnostic Primer Sets for Escherichia coli O104H4 Outbreak Strains 英文参考文献.doc

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Alignment-Free Design of Highly Discriminatory Diagnostic Primer Sets for Escherichia coli O104H4 Outbreak Strains 英文参考文献

Alignment-FreeDesignofHighlyDiscriminatory DiagnosticPrimerSetsforEscherichiacoliO104:H4 OutbreakStrains LeightonPritchard1*.,NicolaJ.Holden2.,MartinaBielaszewska3,HelgeKarch3,IanK.Toth2 1Information and Computational Sciences, James Hutton Institute, Dundee, Scotland, United Kingdom, 2Cellular and Molecular Sciences, James Hutton Institute, Dundee,Scotland,UnitedKingdom,3InstituteforHygieneandtheNationalConsultingLaboratoryforH?molyticUr?micSyndrome,UniversityofMu¨nster,Mu¨nster, Germany Abstract Background: An Escherichia coli O104:H4 outbreak in Germany in summer 2011 caused 53 deaths, over 4000 individual infectionsacrossEurope,andconsiderableeconomic,socialandpoliticalimpact.Thisoutbreakwasthefirstinapositionto exploit rapid, benchtop high-throughput sequencing (HTS) technologies and crowdsourced data analysis early in its investigation,establishinganewparadigmforrapidresponsetodiseasethreats.Wedescribeanovelstrategyfordesignof diagnosticPCRprimersthatexploitedthisrapiddraftbacterialgenomesequencingtodistinguishbetweenE.coliO104:H4 outbreakisolatesandotherpathogenicE.coliisolates,includingthehistoricalh?molyticur?micsyndrome(HUSEC)E.coli HUSEC041O104:H4strain,whichpossessesthesameserotypeastheoutbreakisolates. Methodology/PrincipalFindings:Primersweredesignedusinganovelalignment-freestrategyagainstelevendraftwhole genomeassembliesofE.coliO104:H4GermanoutbreakisolatesfromtheE.coliO104:H4GenomeAnalysisCrowd-Sourcing Consortiumwebsite,andanegativesequencesetcontaining69E.colichromosomeandplasmidsequencesfrompublic databases.Validationinvitroagainst21‘positive’E.coliO104:H4outbreakand32‘negative’non-outbreakEHECisolates indicatedthatindividualprimersetsexhibited100%sensitivityforoutbreakisolates,withfalsepositiveratesofbetween9% and22%.Aminimalcombinationoftwoprimersdiscriminatedbetweenoutbreakandnon-outbreak E.coliisolateswith 100%sensitivityand100%specificity. Conclusions/Significance: Draftgenomesofisolatesofdiseaseoutbreakbacteriaenablehighthroughputpr