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数字PCR仪校准方法研究
Introduction
DigitalPCR(dPCR)isasensitiveandaccuratetechniquethathasrevolutionizedthequantificationofnucleicacids.ItprovidesincreasedprecisionandsensitivitycomparedtotraditionalquantitativePCR(qPCR).However,toensureaccuratemeasurements,itisessentialtocalibratethedPCRinstrumentbeforeuse.Thecalibrationprocedureincludesthedeterminationofthelimitingdilution,theestimateoftheefficiencyofPCRamplification,andthecalculationoftheconcentrationofDNAtemplateinthereaction.ThispaperwilldiscussthemethodsusedtocalibratedigitalPCRinstruments.
LimitingDilution
Thelimitingdilutionisthepointatwhichasampleisdilutedtoadegreethatthereislessthanonetargetmoleculepresentinthereactionvolume.ThelimitingdilutionisdeterminedthroughaseriesofdilutionsoftheDNAsamplewhereeachdilutionisassessedusingdigitalPCR.Adilutionseriesisconstructedwhereeachdilutionisanalyzedinduplicateatleastonce.Bydeterminingthedilutionatwhichapositiveresultisnolongerdetected,thelimitingdilutioncanbeestablished.ThisvaluerepresentsthetheoreticalmaximumlevelofsensitivityofthedPCRinstrumentandisrequiredtocalculatethetemplateconcentrationthroughPoissondistribution.
AssessmentofAmplificationEfficiency
TheamplificationefficiencyofdigitalPCRiscalculatedusingthefluorescencesignalfromeachdroplet.Thefluorescencesignalisprocessedtomodelthenumberoftargetsineachdroplet,detectandremovedropletswithnofluorescence,andadjustforbackgroundsignal.Theapproachtocalculatetheefficiency,similartotraditionalqPCR,involvesfittingasigmoidalfunctiontothelogarithmoftheratioofpositivetototaldropletsforeachdilutionpoint.Theslopeofthelineistheamplificationefficiency(E),whichiscalculatedasE=10(-1/slope).Typically,anefficiencyof0.9-1.1isconsideredacceptable.
CalculatingtheConcentrationofDNATemplate
ConcentrationistypicallycalculatedbyapplyingaPoissondistributiontothenumberofpositivedropletsdetectedineachdigitalPCRreaction.ThePoissondistributionfunctioncalculatestheprobabilityofthedistributionoftargetmoleculesinthereaction.Basedonthelimitingdilution,th