副猪嗜血杆菌上海分离株的生物学特性与基因型分析

万世平1,2,王建3,葛菲菲3,刘佩红3,齐新永3,徐锋3,沈莉萍3,费荣梅1*(1.,210095;2.,201200;3.,201103):(HPS),,16SrRNA821bpPCR,,PCRHPS973%~100%ERICPCR,,ERICPCR202,APCRHPS,:;;PCR;ERICPCR:S85261:A:05295130(2010)04006505(Haemophilusparasuis,HPS),,,V,,,,(Glassersdisease)[1],,,,,10,,PCR(enterobacterialrepetitiveintergenicconsensusPCR,ERICPCR)HPS111HPS(HS83HS79HS80HS1072HS1073HS1075HS1080HS1081HS197HS1079);:20090916;:20100113:2007(07dz19508):(1982),,*(SH0854PSH080402SH0812SH0803SH08145SH08432SH0883SH08107SH08106SH08012)12BD,,;NAD(V);;PCR();()13HPS,[2][3]14VX,12h,15PCROliveira[4],:5!GTGATGAGGAAGGGTGGTGT3!,:5!GGCT∀65∀2010424TCGTCACCCTCTGTA3!,821bpDNAPCR:10#PCRbuffer5L,25mmol/LMgCl24L,25mmol/LdTNP4L,DMSO25L,1L,rTaq05L,DNA2L,ddH2O50L95∃2min;94∃30s,54∃1min,72∃15min,30;72∃10min,PCR4∃PCR(APP)(PM)16PCRSH0854P,HPS16SrRNA17ERICPCRERICPCRRafiee[5]:ERIC1:5!ATGTAAGCTCCTGGGGATTCAC3!ERIC2:5!AAGTAAGTGACTGGGGTGAGCG3!DNA:10#PCRbuffer5L,25mmol/LMgCl25L,25mmol/LdTNP8L,1L,rTaq05L,DNA4L,ddH2O50L95∃2min;94∃30s,50∃30s,72∃6min,35;72∃6min,PCR4∃NTSYS210e,(SM)(UPGAMA),221HPS,(11),,,,,(12);,(13);(14)22221培养特性NAD(V)BHIVTSA,NAD,,X,HPSVHPS,;HPS,222生化特性检测,,,,,[6-7],(1)1;2(1000#);3(1000#);4(10000#)1SH0854P1SH0803SH0812SH0854PSH080402HS83HS79HS1075+++-+-+---------------------++++++++++++++--------+-----223药敏试验,42,(5mm),,,2∀66∀AnimalHusbandry&VeterinaryMedicine2010Vol42No42mmSH0803SH0812SH080402SH0854PHS79HS107520163814334517201964647205215494526264119354559175424115122712393081421941221934423040462830393437471752212484081010124515551552223205215282225213530234121842444643131455464025203717184225193924473723PCRPCR,821bp(2),,,PCRPCR,M:Marker2000;1~15:HPS1~15;16:APP;17:PM216SrRNAPCR24PCR,GenBank(EF3963061DQ2290761DQ2290011AY3629091AY1267101),973%~100%25ERICPCR10HPS10ERICPCR,7~16,100~6000bp,,(3)(4),20:AB,CD3,7;10A,HS83HS197A;058~094M1M2.DL15000Marker;M3.DL2000Marker;1~10.HPS;11~20.HPS320ERICPCR420ERICPCR∀67∀20104243,HPS,10,HPS,,,Turni[8],(4),,,(12),,HPS,HPS,,;PCR,PCR,16SrRNAGenBank973%~100%16SrRNAPCR,16SrRNA,974%~977%[9],PCR,,,ERICPCRHPS,,ERICPCR,HPSRafiee[10]ERICPCR15,14,3HPS122,15,,3HPS12,3HPSOliveira[11]HPS,(1~3)4,ERICPCRERICPCR20,100%,HS83HS197A,HS83HS197,HPS,ERICPCR3,,ERICPCRHPSDNA,15,20%,[12]ERICPCRMEE(multilocusenzymeelectrophoresis),RAPD(randomamplifiedpolymorphicDNA),MLST(multilocussequencetyping),PCRRELP(PCRrestrictionfragmentlengthpolymorphism)[13-15],,,,,:[1].[M].4.:,2007:144146.[2].[M].:,2002:1518.[3].[M].:,1990.[4]OliveiraS,GalinaL,PijoanC.DevelopmentofaPCRtesttodiagnoseHaemophilusparasuisinfections[J].JVetDiagnInvest,2001,13(6):495501.[5]RafieeM,BaraM,StephensCP,eta.lAmplicationofERICPCRforthecomparasionofisolatesofHaemophilusparasuis[J].AustVet,2000,78:846849.[6],,,.[J].,2007,16(4):210212.[7]KielsteinP,WutheH,MuttersR,eta.lPhenotypicandgeneticcharacterizationofNADdependentPasteurellaceaefromtheresperitorytractofpigsandtheirpossiblepathogeneticimportance[J].VetMicrobio,l2001,81:243255.[8]TurniC,BlackallPJ.ComparisonofsamplingsitesanddetectionmethodsforHaemophilusparasuis[J].AustVetJ,2007,85(5):177184.[9]MollerK,FussingV,Grimont,eta.lActinobacillusminorsp.nov.,Actinobacillusporcinussp.nov.,andActinobacillusindolicussp.nov.,threenewVfactordependentspeciesfromtherespiratorytractofpigs[J].IntJSystBacterio,l1996,46,951956.[10]RafieeM,BaraM,StephensCP,eta.lApplicationofERICPCRforthecomparisonofisolatesofHaemophilusparasuis[J].AustVetJ,2000,78:846849.[11]OliveiraS,BlackallPJ,PijoanC.CharacterizationofthediversityofHaemophilusparasuisfieldisolatesbyserotypingandgenotyping∀68∀AnimalHusbandry&VeterinaryMedicine2010Vol42No44株IBDV分离株VP2基因的序列分析及原核表达肖跃强1,管宇1,李书光1,刘吉山1,2,王金良1,郭广君1,沈志强1,2*(1.,256600;2.,256600):RTPCR4(IBDV)(GTHNZBBZ)VP2,:1356bp,452;932%~999%936%~100%;HNCu1955%,3956%;GTHNZBBZVariantE959%;41998~2008VP2,HNZBBZ(vvIBDV),GT254S(),GTVP2pBV220,BL21(DE3),42∃,SDSPAGEWesternblot,,,VP2:vvIBDV;VP2;;;Westernblot:S85265:A:05295130(2010)04006905,(infectiousbursaldiseasevirus,IBDV)(Cu1)(variantIBDV)(veryvirulentIBDV,vvIBDV)3[1]2090,vvIBDV,,IBDVRNARNA,AB,AVP5NVP2VP4VP3C,BVP1VP241ku,51%,4VP2,1VP2,:20090919;:20100112:(200703zb):(1978),,,*111IBDVGT()HN()ZB()BZ(),2007;DH5BL21(DE3);12KDEPC;AMVRNasinT4DNAExTaqpMD18TVectorDNATaKaRa;HRPIgGSigma;DABpBV22013vvIBDVVP2cDNA(GenBank:AY134874),Primer50,:[J].AmJVetRes,2003,64(4):435442.[12]Mart|!ndelaFuenteAJ,TuckerbAW,NavasJ,eta.lAntimicrobialsusceptibilitypatternsofHaemophilusparasuisfrompigsintheUnitedKingdomandSpain[J].VetMicrobio,l2001,20:184191.[13]BlackallPJ,TrottDJ,RappGabrielsonV,eta.lAnalysisofHaemophilusparasuisbymuLtilocusenzymeelectrophoresis[J].VeterinaryMicrobiology,1997,56:125134.[14],,,.6DNAPCR[J].,2007,39(3):177180.[15]OlveraA,MartaC,VirginiaA.StudyofthepopuLationstructureofHaemophilusparasuisbymuLtilocussequencetyping[J].Microbiology,2006,152:36833690.∀69∀2010424