DOI: /j.cnki.bingduxuebao

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1 CHINESEJOURNALOF VIROLOGY Vol.33 No.1 January 2017 PCR! ( ) : PCR(DigitalPCRdPCR) (InfluenzaAvi- rusflua) dpcr dpcr 64.4 ; FluA dpcr FluA 37.7~8.22 " 10 4 /#L 3.77 / dpcr R 2 = dpcr FluA dpcr FluA : PCR; :R373.1 : ; : :A : (2017) DOI: /j.cnki.bingduxuebao %~10% 20% ~30% 300 dpcr [1] : ( :D ) : ; ( :2015BA/12B11) : : (1990-) Tel: fengzhaomin@cnic.org.cn * : (1970-) Tel: yshu@ cnic.org.cn H7N9 2 NanoDropLite ;QX200 PCR QX200 Droplet Generator PX1 Plate Sealer [2-3] [6] dpcr dpcr (Influenza [4-5] AvirusFluA) PCR (Quanti- dpcr FluA tativepcrqpcr) qpcr dpcr FluA FluA PCR(DigitalPCRdPCR) PCR(qPCR) dpcr PCR ( ) 1 RNA RNA 1 0 PCR ILI real- timert-pcr QX200DropletReader ;Ep- pendorfpcr Eppendorf ;RNA (RNeasy minikit) ; One-step RT-ddPCR Advanced Kitfor Probes

2 2 33 Dropletgenerationoilforprobe Dropletreader oil "10 6 /#L RNA ( 1) 10pM 1 S15 20 #l dpcr Table1 Primerandprobesequencesforquantification ofinfluenzaavirusesbydropletdigitalpcr RNA 2#l dpcr Primerand probe Sequence ForwardPrimer 5 -GACCRATCCTGTCACCTCTGAC-3 ReversePrimer Probe 5 -GGGCATTYTGGACAAAKCGTCTACG-3 5 -TGCAGTCCTCGCTCACTGGGCACG QX200 QuantSoft dpcr 2 2 dpcr Table2 CyclingconditionsfordropletdigitalPCR CyclingStep Temperature Time Numberofcycle Reversetranscription 45 60min 1 Enzymeactivation 95 10min 1 Denaturation 95 30sec 40 Annealing min - Enzymedeactivation 98 10min 1 Hold 4 $ 1-6 dpcr RNA NanoDropLite /#L S0-7 RNA RNA dpcr 4 dpcr dpcr QX200 dpcr : #l ( 5 #l 2 #l300nm DTT1 #l nM 0.9 #l250nm 0.25 #l2 #l RNA 7.95 #l) #l /#L PCR PX1 96 EppendorfPCR PCR dpcr dpcr Figure1 One-dimensionalscaterplotofselected wels/samplesattwelvetemperaturegradientsfor quantificationofrnaininfluenzaaviruses :55-65 usingdropletdigitalpcr EppendorfPCR dpcr RNA RNA PCR (48 ) 16390( )

3 1 : PCR 3 S12 RNA 8.22 " ~37.7 /#L dpcr 2.a dpcr ( ) ; ( ) S0 dpcr 2.b R 2 = dpcr Figure3 DynamicrangeofdropletdigitalPCRfor quantificationinfluenzaaviruses 4 dpcr 3 3 S12 S1~S12 S1-S12 dpcr S / 3.77 / 3 dpcr Table3 DilutionseriesoutputofdropletdigitalPCR forquantificationofinfluenzaaviruses No. UV measured dpcr measured Mean copies (copies/#l) (copies/#l) (copies/#l) Bias SD (S) CV S0 1.03"10 6 NoCal NoCal NoCal 2 FluARNA dpcr (a) (b) Figure2 One-dimensionalscaterplot(a)and histograms(b)ofselectedwels/samplesfor quantificationdiferentconcentrationsofinfluenza AvirusesRNAbydropletdigitalPCR 3 dpcr 3 dpcr S1 S1 5.15" S " S " S " S "

4 UV dpcr Mean Bias No. measured measured copies (copies/#l) (copies/#l) (copies/#l) SD (S) CV S " S " S S S S S S S S Note: NoCal denotesthatthevirusload wasbeyondthe detectionrange. [3]DeJongJCBeyer W ERimmelzwaan G Fetal. 5 dpcr Neuraminidaseinhibitorsoseltamivirandzanamivir:new meansofdefenceagainstinfluenza[j].ned Tijdschr 8 Geneeskd (7):348. [4]HindsonBJNessK DMasquelierD Aetal.High- dpcr : throughput droplet digital PCR system for absolute /#L quantitationof DNA copy number[j].anal Chem dpcr : [5]HuY WLuS HSongZJetal.Associationbetween adverseclinicaloutcomeinhumandiseasecausedbyno- [15-17] dpcr dpcr dpcr dpcr dpcr [18] dpcr dpcr RNA DNA FluA dpcr FluA : /en/. [1]World HealthOrganizationInfluenza (Seasonal)[OL]. FactsheetNo htp://www. who. int/buletin/volumes/90/4/11- [2]MontoAS.Theroleofantiviralsinthecontrolofinflu- enza[j].vaccine20031:21(16): velinfluenzaa H7N9virusandsustainedviralshedding andemergenceofantiviralresistance[j].lancet2013 S (13): dpcr [6]YanYJiaXJWangH [7-8] [9-10] Hetal.DynamicQuantifica- tionofavianinfluenzah7n9(a)virusinahumanin- [11] [12] fectionduringclinicaltreatmentusingdropletdigital dpcr GBV-CRNA RNA PCR[J].JVirolMethods : [13-14] HIV HBV [7]WangJRamakrishnanRTangZetal.Quantifying

5 1 : PCR 5 EGFR alterations in the lung cancer genome with nanofluidicdigitalpcrarrays[j].clinchem (4): [8]YungT KChanK AMokTSetal.Single-molecule detectionofepidermalgrowthfactorreceptormutations inplasmabymicrofluidicsdigitalpcrinnon-smalcel lung cancer patients [J].Clinical Cancer Research (6): [9]LoY MLunF MChanK Cetal.DigitalPCRfor themoleculardetectionoffetalchromosomalaneuploidy [J].Proc Natl Acad Sci USA : [10]BarretA NMcDonnelTCChanK Cetal.Digital PCRanalysisofmaternalplasmafornoninvasivedetec- tionofsicklecelanemia[j].clin Chem201258: vides sensitive and absolute calibration for high throughputsequencing[j].bmc Genomics (1):116. [12]Srain M CLadaS MLuongTetal.Highlyprecise measurementofhiv DNA bydropletdigitalpcr[j/ OL].PloSOne20138(4):e [13]WhiteR AQuakeSRCurrK.DigitalPCRprovides absolutequantitationofviralloadforanoccultrna virus[j].jvirolmethods : step RT-dropletdigitalPCR:abreakthroughinthe quantificationof waterborne RNA viruses[j].anal BioanalChem : [11]WhiteRBlaineyPFan H Cetal.DigitalPCRpro- [14]RackiNMorissetDGutierrez-AguirreIetal.One- [15]HenrichTJGalienSLiJZetal.Low-leveldetec- LTR circles using dropletdigitalpcr [J].J Virol Methods : ficationofnucleicacidswithlargedynamicrangeusing multivolumedigitalrt-pcr onarotationalslipchip testedwith HIVandhepatitisCviralload[J].JAm ChemSoc : tionandquantitationofcelular HIV-1 DNA and2- [16]ShenFSunBKreutzJEetal.Multiplexedquanti- [17]HuangJTLiuYJWangJetal.NextGeneration DigitalPCR Measurementof HepatitisB VirusCopy NumberinFormalin-FixedParafin-EmbeddedHepato- celularcarcinomatissue[j].clinchem : [18]BosmanKJNijhuisMVan Ham P Metal.Com- parisonofdigitalpcrplatformsandsemi-nestedqpcr asatooltodeterminethesizeofthehivreservoir[j]. ScientificReports20155: DropletDigitalPolymeraseChainReaction MethodforAbsoluteQuantification ofinfluenzaaviruses FENGZhaominZHAO XiangZOU XiaohuiZHU WenfeiCHEN Yongkun WANG DayanSHU Yuelong! (World Health Organization GlobalInfluenza Colaboration Centerfor Referenceand ResearchNationalInstitutefor Viral Disease Controland PreventionColaborationInnovation Centerfor Diagnosisand Treatmentof Infectious DiseasesChineseCenterfor DiseaseControland PreventionKey Laboratory for MedicalVirologyNational Healthand Family Planning CommissionBeijing102206China ) Abstract:Digitalpolymerasechainreaction(dPCR)isanew methodforabsolutequantification.however anabsolutequantificationmethodforinfluenzaavirusesbasedondropletdigitalpolymerasechainreaction (dpcr)hasnotbeenestablished.inthisstudywefoundthat:(i)theannealingtemperatureofdpcr wasoptimizedat64.4 C;(i)thedetectionrangeofdPCR was37.7~ copies/ μ lforinfluenzaa viruses;(i)thelimitofdetectionofdpcr was3.77copies/reaction.thelinercorrelationcoeficient (R 2 )wasfoundtobe0.9988suggestingthehighreliabilityofourdpcr method.thedpcr method couldbeusedtodetectinfluenza A virusesclinicaly.insummarywedevelopedadpcr methodfor absolutequantificationofinfluenzaaviruses.thismethodcouldbeusedefectivelytoquantifyinfluenza Avirusesinclinicalsamples.Thereforeourmethodcouldbeanewtoolfortheabsolutequantificationof viralload. Keywords:Digitalpolymerasechainreaction;InfluenzaAvirus;Absolutequantification *Corresponding author:shu Yuelong yshu@cnic.org.cn