CHINESEJOURNALOF VIROLOGY Vol.34 No.5 September2018 TaqManqRT-PCR,, * *, (, ) : TaqManqRT-PCR, L, PCR, RT-PCR N qrt-pcr L qrt- PCR 9

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1 CHINESEJOURNALOF VIROLOGY Vol.34 No.5 September2018 TaqManqRT-PCR,, * *, (, ) : TaqManqRT-PCR, L, PCR, RT-PCR N qrt-pcr L qrt- PCR 90%; FFU/mL, N qrt-pcr RT-PCR100; 100, RNA, ;, DFA qrt-pcr : (Rabies); (RABV); PCR :R373.9 :A : (2018) DOI: /j.cnki.bingduxuebao (Rabies) (Rabies RNA (Polymerasegene,Lgene) virus,rabv), TaqManqRT-PCR,,, 100%, (Directfluorescentantibodytest,DFA) [1] 59000,,, [2],, DFA RT-PCR (Nucleoproteingene,N gene)qrt-pcr,,,, 1 [3], [4], (CVS-11), DFA 1~4 (Denguevirus1~4,DENV1~4) PCR(Real-timePCR,qPCR) (Japaneseencephalitisvirus,JEV), 9, GenBank : ; : L (1) : ( :2017YFC ), : ; ( :2017ZX ), : : (1993-),,,, syzxgx123@163.com * : (1985-),,, @163.com; sina.com 2.1 GenBank 732, ClustalX2.1, L, PrimerExpress3.0.1 BLAST,

2 5,. TaqManqRT-PCR 505 (2) N (2) qrt-pcr RT-PCR 1 Table1 Informationofreferencesequencesoflyssavirusesfrom GenBank IsolateName Virus GenBankNo. Host Bokelohbatlyssavirus(BBLV) JF Bat RV2416 Europeanbatlyssavirus1(EBLV-1) LT RV1333 Europeanbatlyssavirus2(EBLV-2) NC Human - Ikomalyssavirus(IKOV) NC Cat IRKV-THChina12 Irkutlyssavirus(IRKV) JX Bat - Khujandlyssavirus(KHUV) NC CAM Mokolavirus(MOKV) EU Shrew - Shimonibatvirus(SHBV) NC Bat - WestCaucasianbatvirus(WCBV) EF Table2 Oligonucleotidesequencesofprimersandprobesinthisstudy Name Type Length (bp) Oligonucleotide sequences(5 to3 ) Gene Location 4 Tm Value ( ) 5 Product Size(bp) Source RVLF 1 Forwardprimer 23 GACATTATATTRGCRAGGTTGAC Polymerase 7900~ ~ This study JW(12) 2 Forwardprimer 19 ATGTAACACCYCTACAATG Nucleoprotein 55~ ~51.8 RVLR 1 Reverseprimer 22 GARGARACMATGTGYAGTTATG Polymerase 7755~ ~58.8 RVLP 1 Probe 29 FAM-TGGTCRTTRGAGATRCAR- GAGACTCTRGC-BHQ1 Polymerase 7873~ ~71.0 N165~146 2 Reverseprimer 20 GCMGGRTAYTTRTAYTCATA Nucleoprotein 165~ ~56.4 RABVP 2 Probe 34 FAM-CCGAYAAGATTGTAT- TYAARGTCAAKAATCAGGT -BHQ1 Nucleoprotein 78~ ~ [5,6] N127 3 Forwardprimer 20 ATGTAACACCTCTACAATGG Nucleoprotein 55~ N8m 3 Reverseprimer 18 CAGTCTCYTCNGCCATCT Nucleoprotein 1586~ ~58.1 N577 3 Forwardprimer 20 AAGATGTGYGCYAAYTGGAG Nucleoprotein 644~ ~61.0 N829 3 Reverseprimer 19 GCCCTGGTTCGAACATTCT Nucleoprotein 899~ [7] : 1 L TaqManqRT-PCR ; 2 N TaqManqRT-PCR ; 3 RT-PCR; 4 CVS-11 ; 5 Geneious Tm Note: 1 usedforlgenetaqmanqrt-pcrassay; 2 usedforngenetaqmanqrt-pcrassay; 3 usedfornestedrt-pcrassay; 4 refertorabiesvirusstraincvs-11 (GenBankAcNo.GQ918139); 5 calculatedbygeneious11.0.4program. 2.2 RNA [8-10] (Ambition),CVS-11 PCR CVS-11 QIAamp ( ) ( ViralRNA MiniKit(Qiagen) TrizolRea- ), ct gent (Invitrogen) RNA, L TaqManqRT-PCR AgPath-ID One-Step RT-PCR Reagents RVLF RVLR CVS-11 L (7845~7900), TaqManqRT-PCR pgem-teasy (Promega)

3 506 34, Trans5α (Transgen) vitrogen), PCR, QIAprepSpin MiniprepKit(Qiagen)256bp,Pvu Ⅱ (Takara), QIAquick GelExtraction Kit (Qiagen) T7 (Tiandz) RNA, MicroEluteRNA CleanUp Kit(Omega), RNA RNA 10, RNA,,, log10( ), ct 2 TaqManqRT-PCR [7] PCR GelRedDNA (In- / μ L~1 / μ L PCR, 30, 2, log10( ), ct (1) NCBIprim- DMEM (LifeTechnology) er-blast CVS-11 10,, TaqManqRT-PCR TaqManqRT-PCR CVS-11 DENV JEV, :, 2 PCRBufer12.5μl,25 EnzymeMix1μl,RV LF (10μM)1μl,RVLR (10μM)1μl,Taq- DMEM (Life Technology) CVS-11 10, free Water7.5μl CVS-11 RNA, qrt-pcr, RT-PCR terrna, TL988 PCR ( 2.4 N TaqManqRT-PCR N TaqMan qrt-pcr :2 PCRBufer12.5μl,25 EnzymeMix1μl,JW(12), ct, S RNase-free Water7.5μ ; : 50 10min, 95 10min, 95 15s,50 30s, RT-PCR (10μM)1μl,N (10μM)1μl,Taq- Man RABVP (5μM )1μl,RNA 1μl, RT-PCR RNA qrt-pcr, PrimeScriptOneStepRT-PCR Kit FFU/mL FFU/mL (Takara) PCR :Pri-, log10( ), mescript1stepenzymemix1μl,2 1StepBufer ct (3) (DyePlus)12.5μl,N127 (10μM)1μl,N8m (10μM)1μl, RNA1μl,DEPC 8.5μl Man RVLP(5μM)1μl,RNA 1μl,RNase- RNA, RNase-free Wa- ), :50 10min,95 10min, 95 15s,52 30s, 40 ct 39,3 RNA qrt-pcr ( 2), :y = x , R , 95.55%, CVS FFU/mL,DMEM (Life Technolo- gy) 10, :y = 3.193x , R , 105.7%,

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5 L qrt-pcr RNA Figure2 StandardcurveofLgeneTaqManassay basedonthenucleicacidcopynumberofstandardrna 4 L TaqManqRT-PCR :1.CVS-11:2.JEV;3.DENV-1;4.DENV-2;5.DENV-3; 6.DENV-4;7.BBLV;8.EBLV-1;9.EBLV-2;10.IKOV; 11.IRKV;12.KHUV;13.MOKV; 14.SHBV;15.WCBV;16. Figure4 Amplificationcurveofspecificityanalysis usingthetaqmanqrt-pcrassaybasedonlgene Notes:1.CVS-11:2.JEV;3.DENV-1;4.DENV-2;5.DENV-3; 6.DENV-4;7.BBLV;8.EBLV-1;9.EBLV-2;10.IKOV; 11.IRKV;12.KHUV;13.MOKV; 14.SHBV;15.WCBV;16.Negativecontrol 3 CVS-11 3 Figure3 Standardcurvebasedonthetitrated Table3 Comparisonandanalysisofdetectionsensitivity CVS-11celsupernatant Titer N TaqManAssay LTaqManAssay Nested (FFU/mL) Meanct SD Meanct SD RT-PCR 3 L TaqManqRT-PCR Positive Positive Positive TaqMan qrt-pcr Positive DENV JEV HPV Positive,, Positive CVS-11 (4) Positive Positive L TaqManqRT-PCR Positive Noct Negative RNA 10, qrt-pcr, 100 DMEM Noct Noct Noct Negative Negative 10, 5 L TaqManqRT-PCR RNA qrt-pcr RT-PCR, ( CVS L 3) L qrt-pcr 2.7 qrt-pcr, 10-3 FFU/mL, N qrt- 10 ct 24.31, PCR RT-PCR , 1.8%,

6 5,. TaqManqRT-PCR L TaqManqRT-PCR 6, 9,, L qrt-pcr [11,12], 6 13,,CHINAICHINA VI (4),,9,,5 N G, 4 Table4 Informationanddetectionresultsofrabiesvirusisolatesusedinthisstudy LgeneTaqManassay Strain GenBank Province Host Year Lineage Standard Name No. Meanct Deviation(SD) Source CHN0635H Hunan Human 2006 JN (Ngene) ChinaI [13] CJX0903D Jiangxi Dog 2009 JQ970484(genome) ChinaI 25, [12] CJS0621D Jiangsu Dog 2006 JQ970481(genome) ChinaI [12] CSC1016D Sichuan Dog 2010 JN974863(Ngene) ChinaI [13] CSX0904D Shanxi Dog 2009 JQ970487(genome) ChinaI 21, [12] CHN0802D Hunan Dog 2008 JQ970480(genome) ChinaⅡ [12] CGD0801D Guangdong Dog 2008 JN974823(Ngene) ChinaⅡ [13] CGX0614D Guangxi Dog 2006 JN936693(Ggene) ChinaⅡ [11] CNM1103C InnerMonogolia Cow 2011 KC252633(genome) ChinaⅢ [12] CQH1202D Qinghai Dog 2012 KM272192(genome) ChinaIV [12] CNX8511 Ningxia Human 1985 AY009098(Ggene) ChinaV [14] CNX8601 Ningxia Human 1986 GU345747(genome) ChinaV [15] CYN1009D Yunnan Dog 2010 JQ730682(genome) ChinaVI [16] DFA qrt-pcr 10qRT-PCR (5),DFA, DFA, 5 Table5 Informationofsamplesandresultsofdetection LgeneTaqManAssay Sample Year Host Province Sample DFA Standard Name MeanCt Diagnosis Deviation(SD) ZJ Dog Zhejiang Braintissue Negative Noct - Negative ZJ Dog Zhejiang Braintissue Negative Noct - Negative ZJ Dog Zhejiang Braintissue Negative Noct - Negative ZJ Dog Zhejiang Braintissue Negative Noct - Negative ZJ Dog Zhejiang Braintissue Negative Noct - Negative ZJ Dog Zhejiang Braintissue Negative Noct - Negative ZJ Dog Zhejiang Braintissue Negative Noct - Negative GS Dog Gansu Braintissue Positive Positive GS Dog Gansu Braintissue Negative Noct - Negative GS Dog Gansu Braintissue Positive Positive GS Dog Gansu Braintissue Positive Positive BLAST, GenBank L 732 TaqManqRT-PCR,, L,,

7 510 34, ; fera,atlan M,BarratJ,BlantonJD,BriggsDJ, RNA CleavelandS,CostaP,FreulingC M,HibyE,Knopf, FFU/mL, L,LeanesF,MeslinF X,Metlin A,Miranda M E, RT-PCR Wakeley [5] MülerT,NelL H,RecuencoS,RupprechtCE,Schu- macherc,taylorl,vigilato M A,ZinsstagJ,Dushof N qrt-pcr 100 ; J.Estimatingtheglobalburdenofendemiccaninerabies RNA, [J/OL].PLoSNeglTropDis,2015,9(4):e , 90%, [2]WangL,TangQ,LiangG.Rabiesandrabiesvirusin [17] SYBRgreen Ⅰreal- wildlifeinmainlandchina, [j].iintjinfect [18] timepcr Dis,2014,25: PCR ; [3],,, [19], [J].,2018,39 TaqManPCR (1):40-43., ;[4]JacksonA C,WarrelMJ,RupprechtCE,ErtlH C, DFA, DietzscholdB,O Reily M,LeachRP,FuZF,Wun- nerw H,BleckT P,WildeH.Managementofrabies, ; 13 inhumans.[j].clininfectdis,2003,36(1):60-63., 6, [5]WakeleyP R,Johnson N,McElhinneyL M,Marston 9, qrt-pcr D,SawyerJ,FooksA R.Developmentofareal-time,, diferentialrt-pcr TaqManassayforlyssavirusgeno-,, types1,5 and 6[J].Dev Biol,2006,126(126): , [6]NagarajT,VasanthJP,DesaiA,KamatA,Madhusu- WHO, danasn,raviv.antemortemdiagnosisofhumanra-, biesusingsalivasamples:comparisonofrealtimeand DFA conventionalrt-pcr techniques.[j].j Clin Virol, [20], 2006,36(1): RT- [7],,,,,, PCR,,,,., [J].,2008,22(3): ,, [8],,,,,, Ashutosh [21] LNA. [J]. PCR, 14,2015,28(10): L [9],,,,,. RT-PCR,,, DFA, [J].,2014,27(11): [10],,,,. [J].,,2006,33(6): PCR [11]TaoX Y,TangQ,RaynerS,GuoZY,LiH,LangS, L,YinCP,HanN,Fang W,AdamsJ,Song M,Li-, ang G D. Molecularphylodynamicanalysisindicates,, lineagedisplacementoccurredinchineserabiesepidem- icsbetween1949to2010[j/ol].plos NeglTrop Dis,2013,7(7):e2294. : [12]LiH,GuoZY,ZhangJ,TaoX Y,Zhu W Y,Tang [1]HampsonK,CoudevileL,LemboT,Sambo M,Kief- Q,Liu H T.Wholegenomesequencingandcompari-

8 5,. TaqManqRT-PCR 511 China[J].VirolSin,2012,27(3): [17]WangL,LiuY,ZhangS,WangY,ZhaoJ,MiaoF, entvirulence.[j].virolsin,2014,29(2): ManPCR [J]., 2006,27(10): [19],,,,. 1 RT-PCR [J].,2011,27(4): [20],,,,,,,,. (2016 ) [J].,2016,37(2): [21]WadhwaA,WilkinsK,GaoJ,CondoriCondoriRE, GiganteCM,Zhao H,MaX,ElisonJA,Greenberg L,Velasco-VilaA,OrciariL,LiY.Apan-lyssavirus Taqmanreal-time RT-PCR assayforthedetectionof highlyvariablerabiesvirusandotherlyssaviruses[j/ OL].PLoSNeglTropDis,2017,11(1):e EstablishmentofTaqManqRT-PCRAssayforDetectionofRabiesVirus GAO Xin,LIUJiajia,SONG Yun,LU Xuexin *,ZHU Wuyang * (NationalInstitutefor Viral DiseaseControland Prevention,ChineseCenterfor DiseaseControland Prevention,Beijing102206,China) sonsofdiferentchineserabiesviruslineagesincluding thefirstcompletegenomeofan Arctic-likestrainin China[J].BiomedEnvironSci,2016,29(5): [13]GuoZ,TaoX,YinC,HanN,YuJ,LiH,Liu H, Fang W, Adams J, Wang J,Liang G,Tang Q, RaynerS.Nationalbordersefectivelyhaltthespread ofrabies:thecurrentrabiesepidemicinchinaisdislo- catedfrom casesin neighboring countries[j/ol]. PLoSNeglTropDis,2013,7(1):e2039. [14]TangQ,OrciariL A,RupprechtiCE,ZhaoX Q,Li Z G,Yang W S.Sequencingandpositionanalysisof theglycoprotein geneoffour Chineserabiesviruses [J].VirolSin,2000,15(1): [15]HeC Q,MengSL,Yan H Y,DingN Z,HeH B, YanJX,XuGL.Isolationandidentificationofanovel rabiesviruslineageinchinawithnaturalrecombinant nucleoprotein gene [J/OL]. PLoS One, 2012, 7 (12):e [16]ZhangJ,ZhangH L,TaoX Y,LiH,TangQ,Jiang X Y,LiangG D.Theful-lengthgenomeanalysisofa streetrabiesvirusstrainisolatedinyunnanprovinceof HuR.A SYBR-greenIquantitativereal-timereverse transcription-pcrassayforrabiesviruseswithdifer- [18],,,,. Taq- Abstract:Arapidandsensitivereal-timeRT-PCRassayfordetectionofrabiesvirusbasedonpolymerase genewasestablishedandevaluated.specificprimersandprobesweredesignedbasedontheconservedre- gionoflgeneofrabiesvirus.theamplificationeficiency,sensitivity,specificityandaccuracyofthede- tectionsystem wereevaluated.theresultsofsensitivitywerecomparedwiththeconventionalnestedrt- PCRandTaqManqRT-PCRassaybasedonnucleoproteingenewhich werereportedbefore.theresults showedthatthelgenereal-timepcrassayestablishedinthisstudywasnegativeforjev,denvand other9lyssavirusesexceptrabv.thelimitofdetectionofviraltiterwas FFU/mL whichwas 10 2 timeslowerthantheconventionalnestedrt-pcrandthengenereal-timert-pcrassay.thelimit ofdetectionofrnastandardcopynumberwas100copies.astandardcurvebasedonthernastandard ofthetargetgene wasestablishedandcanbeusedforquantitativedetection.theestablished TaqMan qrt-pcrassayhadawiderangeofdetectionresultforepidemicrabiesvirusstrainsinchina.severalani- malsampleswerecolectedanddetected,andthedetectionresultswereconsistentwiththegoldstandard DFA.TheestablishedTaqManqRT-PCRassayfordetectionofrabiesviruscanbeappliedtolaboratory detectionofrabiesvirus. Keywords:Rabiesvirus(RABV);qRT-PCR *Corresponding author:lu Wuyang, zhuwuyang1971@sina.com

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

DOI: /j.cnki.bingduxuebao CHINESEJOURNALOF VIROLOGY Vol.34 No.1 January 2018 RT-PCR *,,,,,, (, ) : (ZIKV virus,zikv) ( 34 1 2018 1 DOI:10.13242/j.cnki.bingduxuebao.003304 CHINESEJOURNALOF VIROLOGY Vol.34 No.1 January 2018 RT-PCR *,,,,,, (, 102206) : (ZIKV virus,zikv) (Denguevirus,DENV) (Chikungunyavirus,CHIKV) ZIKV NS1