Assessing the Performance Capabilities of LRE-Based Assays for Absolute Quantitative Real-Time PCR 英文参考文献.docVIP

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Assessing the Performance Capabilities of LRE-Based Assays for Absolute Quantitative Real-Time PCR 英文参考文献.doc

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Assessing the Performance Capabilities of LRE-Based Assays for Absolute Quantitative Real-Time PCR 英文参考文献

AssessingthePerformanceCapabilitiesofLRE-Based AssaysforAbsoluteQuantitativeReal-TimePCR RobertG.Rutledge*,DonStewart CanadianForestService,NaturalResourcesCanada,Quebec,Quebec,Canada Abstract Background: Linear regression of efficiency or LRE introduced a new paradigm for conducting absolute quantification, which does not require standard curves, can generate absolute accuracies of 625% and has single molecule sensitivity. Derived from adapting the classic Boltzmann sigmoidal function to PCR, target quantity is calculated directly from the fluorescence readings within the central region of an amplification profile, generating 4–8 determinations from each amplificationreaction. Findings: Based on generating a linear representation of PCR amplification, the highly visual nature of LRE analysis is illustratedbyvaryingreactionvolumeandamplificationefficiency,whichalsodemonstrateshowLREcanbeusedtomodel PCR.ExaminingthedynamicrangeofLREfurtherdemonstratesthatquantitativeaccuracycanbemaintaineddowntoa single target molecule, and that target quantification below ten molecules conforms to that predicted by Poisson distribution.Essentialtotheuniversalityofopticalcalibration,thefluorescenceintensitygeneratedbySYBRGreenI(FU/bp) isshowntobeindependentofGCcontentandampliconsize,furtherverifyingthatabsolutescalecanbeestablishedusing asinglequantitativestandard.Twohigh-performancelambdaampliconsarealsointroducedthatinadditiontoproducing highlypreciseopticalcalibrations,canbeusedasbenchmarksforperformancetesting.Theutilityoflimitingdilutionassay for conducting platform-independent absolute quantification is also discussed, along with the utility of defining assay performanceintermsofabsoluteaccuracy. Conclusions:FoundedontheabilitytoexploitlambdagDNAasauniversalquantitativestandard,LREprovidestheability to conduct absolute quantification using few resources beyond those needed for sample preparation and amplification. Combined with the quantitative and quality control capabilitie