DOI: /j.cnki.bingduxuebao CHINESEJOURNALOF VIROLOGY Vol.34 No.1 January 2018 RT-PCR *,,,,,, (, ) : (ZIKV virus,zikv) (

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1 DOI: /j.cnki.bingduxuebao CHINESEJOURNALOF VIROLOGY Vol.34 No.1 January 2018 RT-PCR *,,,,,, (, ) : (ZIKV virus,zikv) (Denguevirus,DENV) (Chikungunyavirus,CHIKV) ZIKV NS1 DENV NS5 CHIKV E1 RT-PCR ZIKV DENV CHIKV RNA, : RT-PCR 90%, RNA 15 /PCR, 10PFU/ ml, 2% 95% RT-PCR, : RT-PCR; (ZIKV); (DENV); (CHIKV) :R392 :A : (2018) , (Chikungunyavirus,CHIKV) [2] ,, RNA, * : (1966),,,Tel:010-, 85, (Zikavirus,ZIKV) (Den- guevirus,denv), RNA, 11kb,,, [4,5] ( ), RT-PCR WHO RT-PCR,,, [1],,, 1 : : ; : : ( 2016) 273 ), : : (1991),,, fenfeisining@sina.com , wangshiwencdc@163.com [3] ; NS1 E1 NS5 FAM HEX TEXAS- [6] RED : MR766

2 1,. RT-PCR 53 Ⅰ Ⅱ Ⅲ Ⅳ GD113 ( ; ; ; ; ) DENV ZIKA CHIKV 1 RT-PCR [7,8] 20 Figure1 SpecificityanalysisforTriplexReal-time 2 RNA RT-PCRassayswithdiferentvirus RiboMAX TM LargeScale RNA Produc- tionsystems SP6andT7 2 PCR, RNA Nano- (Ⅰ Ⅱ Ⅲ Ⅳ ) drop RNA RNA, 4, / μ L / μ L QIAamp RNA ViralKit AVE RNA,-80 4 RT-PCR AgPath-ID OneStepRT-PCR Kit(Ambi- on) PCR, : 12.5μl 2μl PFU/mL PFU/mL PFU/mL, 0.2μM PFU/mL 0.1μM dntp0.1mm MgCl 21 mm RNA 5μl, 25μl BIO-RADCFX96 PCR, 90% :50,30min,95 10min;95 15s, s, 45 RNA 3 (Ⅰ Ⅱ Ⅲ Ⅳ ) 9.05 / / μ L / μ L / μ L / (QiagenGmbH,Heiden,Germany) μ L RNA 10, / 140μl, 40μl μ L / μ L 9 3, RNA, C T PFU/mL Ⅰ Ⅱ Ⅲ Ⅳ PFU/mL , 7,, C T PCR 9.47 /PCR 8.63 /PCR 4.21 / PCR /PCR 2.91 /PCR; 1 RT-PCR, 5.51PFU/mL 1.91PFU/ ml, Ⅰ Ⅱ Ⅲ Ⅳ 1.7 PFU/mL 9.1 PFU/mL 0.4 PFU/mL 3.7PFU/mL RT-PCR,, DENV ( 1) ZIKV CHIKV

５４病毒学报３４卷图２ＤＥＮＶ体外转录ＲＮＡ对三重荧光定量ＲＴＰＣＲ灵敏性评价注Ａ登革病毒 Ⅰ 型Ｂ登革病毒 Ⅱ 型Ｃ登革病毒 Ⅲ 型Ｄ登革病毒 Ⅳ 型２ＳｎｖＴｍｒｔＰＣＲｗｈｕｄｏｎｂｄｒｎａｏＤＥＮＶＡＤＥＮＶ Ⅰ ＢＤＥＮＶ Ⅱ ＣＤＥＮＶ Ⅲ ＤＤＥＮＶ Ⅳ 图３ＺＫＶＣＨＫＶ体外转录ＲＮＡ

3 ５４病毒学报３４卷图２ＤＥＮＶ体外转录ＲＮＡ对三重荧光定量ＲＴＰＣＲ灵敏性评价注Ａ登革病毒 Ⅰ 型Ｂ登革病毒 Ⅱ 型Ｃ登革病毒 Ⅲ 型Ｄ登革病毒 Ⅳ 型２ＳｎｖＴｍｒｔＰＣＲｗｈｕｄｏｎｂｄｒｎａｏＤＥＮＶＡＤＥＮＶ Ⅰ ＢＤＥＮＶ Ⅱ ＣＤＥＮＶ Ⅲ ＤＤＥＮＶ Ⅳ 图３ＺＫＶＣＨＫＶ体外转录ＲＮＡ对三重荧光定量ＲＴＰＣＲ灵敏性评价注Ａ寨卡病毒Ｂ基孔肯雅病毒３ＳｎｖＴｍｒｔＰＣＲｗｈｕｄｏｎｂｄｒｎａｏＺＫＶｎｄＣＨＫＶＡＺＫＡＢＣＨＫＶ表１病毒培养物和体外转录参考品对三重检测和单重检测方法灵敏度比较Ｔｂ１ＳｎｖｈｔｘｎｄｍｘｗｈｈｖｕｎｄｕｄｏｎｂｄｒｎａＰＣＲｏｎｂｄｒｎａＣｏＬＯＤＴｘＲＴＰＣＲＭｘＲＴＰＣＲＣＨＫＶＺＫＶ２９１４２４ＶｕＰＵｍｌＤＥＮＶＣＨＫＶＺＫＶ４２１１９１２５８０４１９Ｖ１４７５９０５９４７８６３２２４９２１０８１１９７２２３ＤＥＮＶＶ５５１１７９１４３７８５４２５２２１８７９４３

１期龚雪蕊等寨卡病毒登革病毒基孔肯雅病毒三重荧光定量ＲＴＰＣＲ检测方法的建立及评价５５图４ＤＥＮＶ病毒核酸对三重荧光定量ＲＴＰＣＲ灵敏性评价注Ａ登革病毒 Ⅰ 型Ｂ登革病毒 Ⅱ 型Ｃ登革病毒 Ⅲ 型Ｄ登革病毒 Ⅳ 型４ＳＴｍｒｔＰＣＲｗｈｄｎｖｕＡＤＥＮＶ Ⅰ ＢＤＥＮＶ Ⅱ ＣＤＥＮＶ Ⅲ Ｄ

浓度的核酸进行２０次独立的三重荧光定量ＲＴ自云南的２０份ＤＥＮＶＮＳ１抗原检测阳性的急性期ＰＣＲ检测的重复实验评价三重体系的稳定性结病人血清进行检测其中１９份样本为阳性Ｃ值介果显示表２ＣＨＫＶＺＫＶ和ＤＥＮＶ病毒核酸高于１４２２到３７７９之间１份

4 １期龚雪蕊等寨卡病毒登革病毒基孔肯雅病毒三重荧光定量ＲＴＰＣＲ检测方法的建立及评价５５图４ＤＥＮＶ病毒核酸对三重荧光定量ＲＴＰＣＲ灵敏性评价注Ａ登革病毒 Ⅰ 型Ｂ登革病毒 Ⅱ 型Ｃ登革病毒 Ⅲ 型Ｄ登革病毒 Ⅳ 型４ＳＴｍｒｔＰＣＲｗｈｄｎｖｕＡＤＥＮＶ Ⅰ ＢＤＥＮＶ Ⅱ ＣＤＥＮＶ Ⅲ ＤＤＥＮＶ Ⅳ 图５ＺＫＶＣＨＫＶ病毒核酸对三重荧光定量ＲＴＰＣＲ灵敏性评价注Ａ寨卡病毒Ｂ基孔肯雅病毒５ＳＴｍｒｔＰＣＲｗｈｚｋｖｕｎｄｃｈｋｕｎｎｖｕＡＺＫＡＢＣＨＫＶ３重复性４临床样本验证取寨卡病毒基孔肯雅登革病毒高中低三个采用单重和三重实时荧光定量ＲＴＰＣＲ对来浓度的核酸进行２０次独立的三重荧光定量ＲＴ自云南的２０份ＤＥＮＶＮＳ１抗原检测阳性的急性期ＰＣＲ检测的重复实验评价三重体系的稳定性结病人血清进行检测其中１９份样本为阳性Ｃ值介果显示表２ＣＨＫＶＺＫＶ和ＤＥＮＶ病毒核酸高于１４２２到３７７９之间１份样本为阴性ＺＫＡ临中低三个浓度所对应的Ｃ值的标准差均在５以床模拟标本２０份阳性Ｃ值介于２５６至３５８９内变异系数均在２以下可见三重体系对于三种之间与已报道文献检测值相似对ＣＨＫＶ模拟标病毒核酸均具有良好的稳定性本进行检测其中１９份阳性Ｃ值介于１８７１至

5 ,1 ; 50, 2 PCR Table2 StabilityevaluationofTriplexReal-timeRT-PCRassayswiththreeviruses. DENV ZIKV CHIKV high medium low high medium low high medium low Ctmean SD CV 1.9% 0.4% 1.5% 0.7% 0.3% 0.6% 0.95% 1.1% 0.9% detectionrate (n/20) [10], [11-13] RT- PCR,,,,,, [9], RT-PCR, PCR,, PCR,, :, [1]SantiagoG A,VergneE,QuilesY,CosmeJ,Vazquez J,MedinaJF,MedinaF,ColonC,MargolisH,Mu-, noz-jordanjl.analyticalandclinicalperformanceof, thecdcrealtimert-pcrassayfordetectionandtyp- ingofdenguevirus[j/ol].plos Negl Trop Dis., 2013,11;7(7):e2311. PCR [2]SahadeoN,Mohammed H,AlicockO M,AugusteA RNA 2~15 J,WidenSG,BadalK,PulchanK,FosterJE,Weaver /PCR, 10PFU/ S C, Carrington C V.Molecular characterisation of ml,, Ct chikungunyavirusinfectionsintrinidadandcomparison 2% ofclinicalandlaboratoryfeatureswithdengueandother acutefebrilecases[j/ol].plosnegltropdis,2015,9,95%, (12):e RT-PCR, [3],,. [J].,,2008,24(2): [4]Teng T S,Kam Y W,Lee B,Hapuarachchi H C, WimalA,NgLC,NgLF.Asystematicmeta-analysis

6 1,. RT-PCR 57 ofimmunesignaturesinpatientswithacutechikungunya virusinfection[j].jinfectdis,2015,211(12): [5]FayeO,FayeO,DialoD,Weidmann M,SalA A. Quantitativereal-timePCRdetectionofZikavirusande- valuation with field-caught mosquitoes[j]. VirolJ, 2013,22;10: ,10:311. [6]PangZ,LiA,LiJ,QuJ,HeC,ZhangS,LiC,Zhang Q,Liang M,LiD.Comprehensive multiplexone-step real-time TaqMan qrt-pcr assaysfordetection and quantificationofhemorrhagicfeverviruses[j/ol].plos One,2014,9(4):e [7]FayeO,FayeO,Dialo D,Dialo M,Weidmann M, SalA A.Quantitativereal-timePCR detectionofzika virusandevaluation withfield-caught Mosquitoes[J], VirolJ,2013,10:311 [8]WaggonerJJ,GreshL,Mohamed-HadleyA,Bal- esterosg,davila MJ,TelezY,Sahoo M K,Balmase- daa,harrise,pinskyb A.Single-reaction multiplex reversetranscriptionpcrfordetectionofzika,chikun- gunya,anddengueviruses[j].emerginfectdis,2016, 22(7): [9]SchogginsJ W,WilsonSJ,Panis M,Murphy M Y, JonesC T,BieniaszP,RiceC M.A diverserangeof geneproductsareefectorsofthetypeiinterferonanti- viralresponse.[j].nature,2011,472(7344): [10],. [J]., 2011,26(3): getingns1gene[j].jclinvirol,2015,65: [12]KimJ,ChongC,Sinniah M,SinnaduraiJ,SongH O, Park H.Clinicaldiagnosisofearlydengueinfectionby novelone-step multiplexreal-time RT-PCR targeting NS1gene[J].JClinVirol,2015,65: [11]KimJH,ChongC K,Sinniah M,SinnaduraiJ,Song H O,Park H.Clinicaldiagnosisofearlydengueinfec- tionbynovelone-stepmultiplexreal-timert-pcrtar- [13]SimmonsM,MyersT,GuevaraC,JungkindD,Wil- liamsm,houngh S.Developmentandvalidationofa quantitative,one-step, multiplex,real-time reverse transcriptasepcr assayfordetection ofdengueand chikungunyaviruses[j].jclin Microbiol,2016,54(7):

7 58 34 PCRAssaysforZika,ChikungunyaandDengueViruses GONG Xuerui,LIAqian,LIU Yang,LIChuan,ZHANG Quanfu,LIDexin, LIANG Mifang,WANGShiwen * (ChineseCenterfor DiseaseControland Prevention,NationalInstituteof Viral DiseaseControland Prevention,Key Laboratoryof Medicaland Viral Diseasesofthe Ministryof Health,Beijing102206,China) DevelopmentandEvaluationoftheDetection MethodofTriplexReal-timeRT- Abstract:ToachieverapidnucleicaciddetectionanddiagnosisofZikavirus(ZIKA),Denguevirus(DENV) andchikungunyavirus(chikv)infection,theprimersandprobestargetingthezikv NS1gene,DENV NS5geneandCHIKV E1geneweredesigned,andthenatriplexReal-timeRT-PCRassaywasdeveloped. Thespecificity,sensitivityandstabilitywereevaluatedusinginvitrotranscribedRNAandvirusisolateof ZIKV,DENVandCHIKV,folowedbyclinicalspecimenverification.Asresultsshowed,forthethreevi- rusesdetection,theamplificationeficiencyofthetriplexassaywerealabove90%.thelimitofdetection (LOD)ofinvitrotranscribedRNAswerelessthan15Copies/PCR,andtheLODofthreevirusisolates werelowerthan10pfu/ml.comparedwithrelatedmonoplexassays,therewasnosignificantdiference. Therewasnocrossreactionwithothervirus,andvariablecoeficientofCTvaluewereallessthan2%. In20serum samplesofdengueacute-phasepatients,19 weredetectedpositive.alof50healthyserum samplesweredetectednegative.inconclusion,atriplexreal-timert-pcrassayforzikv,denvand CHIKVdetectionwasestablished,andprovedtobespecificandsensitive.Sothistestmethodcanbeused forlaboratorydetectionofsuspectedzikavirusdiseaseandotherrelatedcases. Keywords:TriplexReal-timeRT-PCRassay;ZIKVvirus(ZIKV);Denguevirus(DENV);Chikungunya virus(chikv) Funding:Thepresent work wassupportedby Reform and developmentofthe ministryofscienceandtechnology *Corresponding author:wang Shiwen, wangshiwencdc@163.com

124 河南农业科学第 42 卷, (HPS) ;PRRSV PCV2, [4] PRRSV CSFV ;20, PRRSV,CSFV [5-9],, 1.2 主要试剂和仪器 Real-timePCR PRRSV CSFV PCV2, 3 RotorGene, RG-3000; ND-1000 Na

124 河南农业科学第 42 卷, (HPS) ;PRRSV PCV2, [4] PRRSV CSFV ;20, PRRSV,CSFV [5-9],, 1.2 主要试剂和仪器 Real-timePCR PRRSV CSFV PCV2, 3 RotorGene, RG-3000; ND-1000 Na 河南农业科学,2013,42(2):123-127 JournalofHenanAgriculturalSciences PCR PRRSV CSFV PCV2,, (, 450011) : 根据 TaqMan 复合荧光探针设计原则, 应用 Primer5.0 和 Oligo6.0 软件设计检测猪繁殖与呼吸综合征病毒 (PRRSV) 猪瘟病毒 (CSFV) 和猪圆环病毒 2 型 (PCV2) 的特异性引物和探针,