Rapid Multi-Locus Sequence Typing Using Microfluidic Biochips 英文参考文献.docVIP

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Rapid Multi-Locus Sequence Typing Using Microfluidic Biochips 英文参考文献.doc

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Rapid Multi-Locus Sequence Typing Using Microfluidic Biochips 英文参考文献

RapidMulti-LocusSequenceTypingUsingMicrofluidic Biochips TimothyD.Read1,2*,RosemaryS.Turingan3,ChristopherCook1,HeidiGiese3,UlrichHansThomann3, CatherineC.Hogan3,EugeneTan3,RichardF.Selden3 1BiologicalDefenseResearchDirectorate,NavalMedicalResearchCenter,Rockville,Maryland,UnitedStatesofAmerica,2DivisionofInfectiousDiseases,Departmentof Medicine, and Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia, United States of America, 3Network Biosystems Inc, Woburn, Massachusetts,UnitedStatesofAmerica Abstract Background: Multiple locus sequence typing (MLST) has become a central genotyping strategy for analysis of bacterial populations.Theschemeinvolvesdenovosequencingof6–8housekeepinglocitoassignuniquesequencetypes.Inthis workweadaptedMLSTtoarapidmicrofluidicsplatforminordertoenhancespeedandreducelaboratorylabortime. Methodology/PrincipalFindings:Usingtwointegratedmicrofluidicdevices,DNAwaspurifiedfrom100Bacilluscereussoil isolates,usedasatemplateformultiplexamplificationof7lociandsequencedonforwardandreversestrands.Thetimeon instrument from loading genomic DNA to generation of electropherograms was only 1.5hours. We obtained full-length sequence of all seven MLST alleles from 84 representing 46 different Sequence Types. At least one allele could be sequencedfromafurther15strains.ThenucleotidediversityofB.cereusisolatedinthisstudyfromonelocationinRockville, Maryland(0.04substitutionspersite)wasfoundtobeasgreatastheglobalcollectionofisolates. Conclusions/Significance:Biogeographicalinvestigationofpathogensisonlyoneofapanoplyofpossibleapplicationsof microfluidics based MLST; others include microbiologic forensics, biothreat identification, and rapid characterization of humanclinicalsamples. Citation: Read TD,Turingan RS,CookC, GieseH, ThomannUH,etal.(2010)Rapid Multi-LocusSequenceTypingUsingMicrofluidicBiochips.PLoSONE5(5): e10595.doi:10.1371/journal.pone.0010595 Editor:NiyazAhmed,UniversityofHyderabad,India ReceivedNovember20,2009;Acc