Accurate Strand-Specific Quantification of Viral RNA 英文参考文献.docVIP

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Accurate Strand-Specific Quantification of Viral RNA 英文参考文献.doc

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Accurate Strand-Specific Quantification of Viral RNA 英文参考文献

AccurateStrand-SpecificQuantificationofViralRNA NicoleE.Plaskon,ZachN.Adelman,KevinM.Myles* FralinLifeScienceInstitute,DepartmentofEntomology,VirginiaTech,Blacksburg,Virginia,UnitedStatesofAmerica Abstract Thepresenceoffull-lengthcomplementsofviralgenomicRNAisahallmarkofRNAvirusreplicationwithinaninfectedcell. Assuch,methodsfordetectingandmeasuringspecificstrandsofviralRNAininfectedcellsandtissuesareimportantinthe studyofRNAviruses.Strand-specificquantitativereal-timePCR(ssqPCR)assaysareincreasinglybeingusedforthispurpose, buttheaccuracyoftheseassaysdependsontheassumptionthattheamountofcDNAmeasuredduringthequantitative PCR(qPCR)stepaccuratelyreflectsamountsofaspecificviralRNAstrandpresentintheRTreaction.Tospecificallytestthis assumption,wedevelopedmultiplessqPCRassaysforthepositive-strandRNAviruso’nyong-nyong(ONNV)thatwerebased uponthemostprevalentssqPCRassaydesigntypesintheliterature.WethencomparedvariousparametersoftheONNV- specific assays. We found that an assay employing standard unmodified virus-specific primers failed to discern the differencebetweencDNAsgeneratedfromvirusspecificprimersandthosegeneratedthroughfalsepriming.Further,we wereunabletoaccuratelymeasurelevelsofONNV(2)strandRNAwiththisassaywhenhigherlevelsofcDNAgenerated fromthe(+)strandwerepresent.Takentogether,theseresultssuggestthatassaysofthistypedonotaccuratelyquantify levelsoftheanti-genomicstrandpresentduringRNAvirusinfectiouscycles.However,anassaypermittingtheuseofatag- specific primer was able to distinguish cDNAs transcribed from ONNV (2) strand RNA from other cDNAs present, thus allowing accurate quantification of the anti-genomic strand. We also report the sensitivities of two different detection strategiesandchemistries,SYBRHGreenandDNAhydrolysisprobes,usedwithourtaggedONNV-specificssqPCRassays. Finally,wedescribedevelopment,designandvalidationofssqPCRassaysforchikungunyavirus(CHIKV),therecentcauseof largeoutbreaksofdiseaseintheIndianOceanregion. Citation: Plaskon NE, Adelman ZN, Myles