数字PCR标准The-Digital-MIQE-Guidelines-Minimum-Informa

TheDigitalMIQEGuidelines:MinimumInformationforPublicationofQuantitativeDigitalPCRExperimentsJimF.Huggett,1*CaroleA.Foy,1VladimirBenes,2KerryEmslie,3JeremyA.Garson,4RossHaynes,5JanHellemans,6MikaelKubista,7ReinholdD.Mueller,8TaniaNolan,9MichaelW.Pfaffl,10GregoryL.Shipley,11JoVandesompele,6CarlT.Wittwer,12andStephenA.Bustin13ThereisgrowinginterestindigitalPCR(dPCR)be-causetechnologicalprogressmakesitapracticalandincreasinglyaffordabletechnology.dPCRallowstheprecisequantificationofnucleicacids,facilitatingthemeasurementofsmallpercentagedifferencesandquantificationofrarevariants.dPCRmayalsobemorereproducibleandlesssusceptibletoinhibitionthanquantitativereal-timePCR(qPCR).Consequently,dPCRhasthepotentialtohaveasubstantialimpactonresearchaswellasdiagnosticapplications.However,aswithqPCR,theabilitytoperformrobustmeaningfulexperimentsrequirescarefuldesignandadequatecon-trols.Toassistindependentevaluationofexperimentaldata,comprehensivedisclosureofallrelevantexperi-mentaldetailsisrequired.TofacilitatethisprocesswepresenttheMinimumInformationforPublicationofQuantitativeDigitalPCRExperimentsguidelines.ThisreportaddressesknownrequirementsfordPCRthathavealreadybeenidentifiedduringthisearlystageofitsdevelopmentandcommercialimplementation.Adoptionoftheseguidelinesbythescientificcommu-nitywillhelptostandardizeexperimentalprotocols,maximizeefficientutilizationofresources,anden-hancetheimpactofthispromisingnewtechnology.©2013AmericanAssociationforClinicalChemistryDigitalpolymerasechainreaction(dPCR)14(1)ispro-gressingfromamethodthatislimitedbytechnicalcomplexitytowardamainstreamtechnologythathasuniqueadvantagesandapplications.Ithasthepoten-tialtohaveamajorimpactonmolecularanalysesrang-ingfromclinicalapplications,suchasbiomarkeranal-ysis(2),viraldetection(3),prognosticmonitoring(4),andfetalscreening(5),toresearchapplicationssuchasphage–hostinteractions(6)andintracellularprofiling(7).dPCRcanalsobeappliedtoassistwiththelibrarypreparationneededformassivelyparallel(ornextgen-eration)sequencingmethods(8).dPCRinvolvesperformingPCRwithreal-timeorend-pointdatacollectioninalargenumberofseparatereactionchambers,alsotermedpartitions.Hundredstomillionsofthesereactionpartitionsarecreatedwithdilutionsoftemplatesuchthatatleastsomeofthemcontainnocopiesofthetargetse-quence(s)ofinterest.Resultsareobtainedbycount-ingthenumberofpartitionsinwhichtheamplifiedtargetsequenceisdetected(regardedaspositive)andthenumberofpartitionsinwhichthereisnoamplification(regardedasnegative).Quantificationofthemeannumberoftargetsequencesperparti-tionisachievedbyapplyingaPoissoncorrectiontothefractionofthepositivepartitions(9).Thiscom-pensatesforthefactthatmorethanonecopyoftem-platemaybepresentinsomepartitions.Becausetheuseof96-or384-wellplatesforasin-glesampleisneitherpractical,affordable,norveryac-curate,morewidespreadimplementationofdPCRhasrequiredtheintroductionofnanofluidictechniquesand/oremulsionchemistries.Threeenhancementsas-sociatedwithdedicatedinstrumentshavehelpedpro-motetheuseofdPCR:1LGC,Teddington,Middlesex,UK;2GenomicsCoreFacility,EMBLHeidelberg,Heidelberg,Germany;3NationalMeasurementInstitute,Lindfield,Australia;4ResearchDepartmentofInfection,DivisionofInfectionandImmunity,UCL,London,UK;5NationalInstituteforStandardsandTechnology,Gaithersburg,MD;6CenterforMedicalGeneticsGhent(CMGG),GhentUniversityHospital,Ghent,Belgium;7TATAABiocenter,Sweden,andInstituteofBiotechnologyoftheCzechAcademyofSciences,Gothenburg,Sweden;8SequenomCenterforMolecularMedicine,SanDiego,CA;9SigmaCustomProducts,Haverhill,Suf-folk,UK;10PhysiologyWeihenstephan,CenterofLifeandFoodSciencesWeihenstephan,Germany;11ShipleyConsulting,LLC,Houston,TX;12Depart-mentofPathology,UniversityofUtah,SaltLakeCity,UT;13PostgraduateMedicalInstitute,FacultyofHealth,SocialCare&Education,AngliaRuskinUniversity,Essex,UK.*Addresscorrespondencetothisauthorat:LGC,QueensRd.,Teddington,Middlesex,TW110LY,UK.Fax
44-(0)20-8943-2767;[e-mail]17,2013;acceptedMarch29,2013.PreviouslypublishedonlineatDOI:10.1373/clinchem.2013.20637514Nonstandardabbreviations:dPCR,digitalpolymerasechainreaction;qPCR,quantitativereal-timePCR;Cq,quantificationcycle;MIQE,MinimumInforma-tionforPublicationofqPCRExperiments;gDNA,genomicDNA;BLAST,BasicLocalAlignmentSearchTool;RT-dPCR,reversetranscriptiondigitalPCR;gDNA,genomicDNA;RT-qPCR,reverse-transcriptionqPCR;dMIQE,MinimumInformationforthePublicationofDigitalPCRExperiments.ClinicalChemistry59:6892–902(2013)SpecialReport892Partitionvolumeshavebeenloweredtoaslittleas5pL.Thepartitioningprocesshasbeenautomated.Thenumberofpartitionshasbeenincreasedto,insomecases,over100000forasingleexperiment.These3elementshavesimplifieddPCRandin-creaseditsprecisionwhilekeepingthetotalreactionvolumeofasingleexperimentsimilartothatofacon-ventionalqPCR.CurrentlydPCRinstrumentsachievepartitioningeitheronchips(10–12)orthroughwater-in-oilemul-sionsordroplets(13–16):A.Usingchip-basedmethods,dPCRisperformedinsmall-volume,solidpartitionsthatalloweitherreal-timeorend-pointanalysisofthe