33 1 2017 1 CHINESEJOURNALOF VIROLOGY Vol.33 No.1 January 2017 PCR! ( 102206) : PCR(DigitalPCRdPCR) (InfluenzaAvi- rusflua) dpcr dpcr 64.4 ; FluA dpcr FluA 37.7~8.22 " 10 4 /#L 3.77 / dpcr R 2 = 0.9988 dpcr FluA dpcr FluA : PCR; :R373.1 :2016-11-03; :2016-12-24 :A :1000-8721(2017)01-0001-05 DOI:10.13242/j.cnki.bingduxuebao.003095 5%~10% 20% ~30% 300 dpcr 500 25 50 [1] : ( :D151100002115003) : ; ( :2015BA/12B11) : : (1990-) Tel:010-58900852E-mail:fengzhaomin@cnic.org.cn * : (1970-) Tel:010-58900850E-mail: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 2016 1 ILI real- timert-pcr QX200DropletReader ;Ep- pendorfpcr Eppendorf ;RNA (RNeasy minikit) ; One-step RT-ddPCR Advanced Kitfor Probes
2 33 Dropletgenerationoilforprobe Dropletreader oil 3 1.03 "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-3 3 96 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 55-65 1min - Enzymedeactivation 98 10min 1 Hold 4 $ 1-6 dpcr RNA NanoDropLite 31.44 /#L S0-7 RNA RNA dpcr 4 dpcr dpcr QX200 dpcr : 1 1 20 #l 55.2 55.4 56 57 58.2 ( 5 #l 2 #l300nm DTT1 #l 59.6 61 62.3 63.5 64.4 65 65.1 12 900nM 0.9 #l250nm 0.25 #l2 #l RNA 7.95 #l) 187 182 185 191 195 190 219 209 70 #l 210 221 210 219 /#L 1 64.4 2 PCR 96 64.4 PX1 96 EppendorfPCR PCR 1 12 5 dpcr dpcr Figure1 One-dimensionalscaterplotofselected wels/samplesattwelvetemperaturegradientsfor quantificationofrnaininfluenzaaviruses :55-65 usingdropletdigitalpcr EppendorfPCR 55.2 55.4 56 57 58.2 59.6 61 2 dpcr 62.3 63.5 64.4 65 65.1 12 RNA RNA PCR (48 ) 16390( 12141 19640)
1 : PCR 3 S12 RNA 8.22 "10 4 10000 ~37.7 /#L 16390 dpcr 2.a dpcr ( ) ; ( ) S0 dpcr 2.b R 2 =0.9988 3 dpcr Figure3 DynamicrangeofdropletdigitalPCRfor quantificationinfluenzaaviruses 4 dpcr 3 3 S12 S1~S12 S1-S12 dpcr S12 3.77 / 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"10 5 81000 77500 88000 82166.7 84.05 5346.3 6.51 38900 S2 2.575"10 5 39700 38166.7 85.18 2003.3 5.25 35900 20680 S3 1.2875"10 5 18870 18890 85.33 1780 9.42 17120 9970 S4 6.4375"10 4 10340 10066.7 84.36 240.1 2.39 9890 4930 S5 3.2188"10 4 4560 4580 85.77 340.4 7.43
4 33 3 UV dpcr Mean Bias No. measured measured copies (copies/#l) (copies/#l) (copies/#l) SD (S) CV 4250 2320 S6 1.6094"10 4 2240 2186.7 86.41 166.53 7.62 2000 1210 S7 8.047"10 3 1270 1233.3 84.67 32.15 2.61 1220 643 S8 4023.5 686 630.3 84.33 62.96 9.99 562 356 S9 2011.75 304 313.3 84.43 38.85 12.4 280 162 S10 1005.88 158 150.6 85.03 16.29 10.82 132 77 S11 502.94 70 75.3 85.03 4.73 6.28 79 36 S12 251.47 38 37.7 85.01 1.53 4.06 39 26 S13 125.74 23 23 81.71 3 13.04 20 20 S14 62.87 13 18 71.37 4.36 24.22 21 13 S15 31.44 5.7 13.9 55.79 8.69 62.52 23 Note: NoCal denotesthatthevirusload wasbeyondthe detectionrange. [3]DeJongJCBeyer W ERimmelzwaan G Fetal. 5 dpcr Neuraminidaseinhibitorsoseltamivirandzanamivir:new meansofdefenceagainstinfluenza[j].ned Tijdschr 8 Geneeskd200414148(7):348. [4]HindsonBJNessK DMasquelierD Aetal.High- dpcr :62.5 55.5 4 throughput droplet digital PCR system for absolute 080 2735 1540 287 27400 22615 /#L quantitationof DNA copy number[j].anal Chem dpcr 201183:8604-8610. [5]HuY WLuS HSongZJetal.Associationbetween adverseclinicaloutcomeinhumandiseasecausedbyno- [15-17] dpcr dpcr dpcr dpcr dpcr [18] dpcr dpcr RNA DNA FluA dpcr FluA : 097808/en/. [1]World HealthOrganizationInfluenza (Seasonal)[OL]. FactsheetNo.2112009. htp://www. who. int/buletin/volumes/90/4/11- [2]MontoAS.Theroleofantiviralsinthecontrolofinflu- enza[j].vaccine20031:21(16):1796-1800. velinfluenzaa H7N9virusandsustainedviralshedding andemergenceofantiviralresistance[j].lancet2013 S0140-6736 (13):61123-61125. dpcr [6]YanYJiaXJWangH [7-8] [9-10] Hetal.DynamicQuantifica- tionofavianinfluenzah7n9(a)virusinahumanin- [11] [12] fectionduringclinicaltreatmentusingdropletdigital dpcr GBV-CRNA RNA PCR[J].JVirolMethods2016234:22-27. [13-14] HIV HBV [7]WangJRamakrishnanRTangZetal.Quantifying
1 : PCR 5 EGFR alterations in the lung cancer genome with nanofluidicdigitalpcrarrays[j].clinchem201056 (4):623-632. [8]YungT KChanK AMokTSetal.Single-molecule detectionofepidermalgrowthfactorreceptormutations inplasmabymicrofluidicsdigitalpcrinnon-smalcel lung cancer patients [J].Clinical Cancer Research 200915(6):076-2084. [9]LoY MLunF MChanK Cetal.DigitalPCRfor themoleculardetectionoffetalchromosomalaneuploidy 13121. [J].Proc Natl Acad Sci USA2007104:13116- [10]BarretA NMcDonnelTCChanK Cetal.Digital PCRanalysisofmaternalplasmafornoninvasivedetec- tionofsicklecelanemia[j].clin Chem201258: 1026-1032. vides sensitive and absolute calibration for high throughputsequencing[j].bmc Genomics200910 (1):116. [12]Srain M CLadaS MLuongTetal.Highlyprecise measurementofhiv DNA bydropletdigitalpcr[j/ OL].PloSOne20138(4):e55943. [13]WhiteR AQuakeSRCurrK.DigitalPCRprovides absolutequantitationofviralloadforanoccultrna virus[j].jvirolmethods2012179:45-50. step RT-dropletdigitalPCR:abreakthroughinthe quantificationof waterborne RNA viruses[j].anal BioanalChem2014406:661-667. [11]WhiteRBlaineyPFan H Cetal.DigitalPCRpro- [14]RackiNMorissetDGutierrez-AguirreIetal.One- [15]HenrichTJGalienSLiJZetal.Low-leveldetec- LTR circles using dropletdigitalpcr [J].J Virol Methods2012186:68-72. ficationofnucleicacidswithlargedynamicrangeusing multivolumedigitalrt-pcr onarotationalslipchip testedwith HIVandhepatitisCviralload[J].JAm ChemSoc2011133:17705-17712. tionandquantitationofcelular HIV-1 DNA and2- [16]ShenFSunBKreutzJEetal.Multiplexedquanti- [17]HuangJTLiuYJWangJetal.NextGeneration DigitalPCR Measurementof HepatitisB VirusCopy NumberinFormalin-FixedParafin-EmbeddedHepato- celularcarcinomatissue[j].clinchem201561 1:290-296. [18]BosmanKJNijhuisMVan Ham P Metal.Com- parisonofdigitalpcrplatformsandsemi-nestedqpcr asatooltodeterminethesizeofthehivreservoir[j]. ScientificReports20155:13811. 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 was37.7~8.22 10 4 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 YuelongE-mail:yshu@cnic.org.cn