六种禽病原检测的基因诊断芯片的研制及临床应用

©1994-2009ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(5):108-113JournalofAgriculturalScienceandTechnology:2009206230;:2009209203:(QN2902011):,,,:,,,Tel:[phone];E2mail:[e-mail]1,2,2,1,1(1.,250100;2.,266101):RT2PCRPCR(newcastlediseasevirus)(avianinfluenza)(infectiousbronchitisvirus)(eggdropsyndrome)(infectiouslaryngotracheitis)(mycoplasmagallisepticum),,DNA,(genechip),RT2PCRbio2112dUTP,PCR,RT2PCR,RT2PCR102104,,95%:;;;:S851:A:100820864(2009)0520108206EstablishmentandClinicalApplicationofGeneChipsforDiagnosingSixKindsofPoultryDiseasePathogensZHANGWei1,TIANZhen2yu2,ZHENGTong2tong2,ZHANGXiu2mei1,HUBei2xia1(1.ShandongAcademyofAgriculturalSciences,Jipnan250100;2.ShandongOlandBioengineeringInstitute,ShandongQingdao266101,China)Abstract:Theconservedsequencesofnewcastlediseasevirus,avianinfluenza,infectiousbronchitisvirus,eggdropsyndrome,infectiouslaryngotracheitisandmycoplasmagallisepticumwereobtainedbypolymerasechainreaction(PCR)orreversetranscription2polymerasechainreaction(RT2PCR).Atwo2dimensionalDNAmicroarray,thatisgenechip,wasproducedbyusingcellulosemembraneasbio2chipsubstrateandmicro2spottingtechnology.Pathogenpolynucleotidewasextractedfromsuspectedsamples,thenlabeledwithbio2112dUTPbyRT2PCRincorporationmethod.ThelabeledPCRproductandpreparedgenechipwerehybridized.Therapidandefficientdetectionandanalysiswereachievedbydetectingthestrengthofhybridizationsignals.ThegenechipsensitivityishigherthanthatofRT2PCRandvirusisolation,by102and104fold,respectively.Ithasfaithfulspecificity.Thecoincidencerateofanimalexperimentswasover95%.Keywords:eggdropsyndromepathogen;genechip;identificationdiagnosis;clinicalapplication,,,(newcastlediseasevirus,NDV)(avianinfluenza,AIVH9)(infectiousbron2chitisvirus,IBV)(eggdropsyn2drome,EDS)(infectiouslaryngotracheitis,ILT)(mycoplasmagallisepticuminfection,MG),,,6()(nitrocellulosefiltermembrane),,©1994-2009ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.();DNASigama;Millipore;HRPSepharos2BOPD;Bio2112dUTPENZO;NDV(F48E9)H9(A/Chicken/Guangdong/SS/94)IBV(M41)EDS276(B4)ILT(Blen)MG(NB72);()[1];[2]PCR,Spectrumlab6116101.2GenBankNDVAIVIBVEDS276ILTMG,DNAStar[3],Oligo6.0,(),11DNATable1Microarrayprimersequence.PathogenPrimersequncePCR(bp)PCRproductlength(bp)NDV52CTTGACACCTCATACTGTAT2352CCCAAGCCATAATAAGGTCTT23622AIV52CTGGTGCACTTGCCAGTTG2352GATCAAGAATCCACAATATC23460IBV52CATAACTAACATAAGGGCAA2352TGGTCCTCTTCAAGGTG23602EDS7652AAACGCCAAACGGCAATCA2352GGAACTGTGCGAGCGAAGAA23430ILT52ACGATGACTCCGACTTTC2352CGTTGGAGGTAGGTGGTA23643MG52GGATCCCATCTCGACCAGGAGAAA2352CTTTCAATCAGTGAGTAACTGATG237341.3,RT2PCR,,PMD182T,JM109,,PCR,[4]1.46,PCR;100ng/L,;802h,4,,,;[5]12A1.51.5.1[6],ddH2O11.5L,10PCRbuffer4L,dNTP(10mmol/L)2L,RNA015L,M2MLV0.5L,(20pmol/L)1.5L,,,421h[7]1.5.2Bio2112dUTP,9015:©1994-2009ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(A)(B)Fig.1Distributiondotsofthegenechip(A)andapictureresult(B).1:ND;2:AI;3:IB;4:EDS276;5:ILT;6:MGa:,;b:,a:Positivecontrol,thepointfortheswinesamplesglobin;b:Negativecontrol,thepointfortheplantproteinsample:ddH2O6L,10PCRbuffer3L,Mg2+(25mmol/L)4L,dNTP(2.5mmol/L)2.5L,Bio2112dUTP8L,TaqDNA(5U/L)0.5L,(10pmol/L)3L,2LPCR:952min;94.530s,5540s,721min,30;7210min[8]1.6[1]()DNA,612nm633nm,20m,:0.1,0.2,[9],(12B)1.7,NDVEDS27610-110-10,3,,RT2PCR,91.81.8.1,,1.8.2611,1.8.3IBDREV(E.coli),,1.920120SPF2,10,H9EDS2765,0.5mL,,,,10mLPBS,,,3000rpm10min,3,RT2PCR,,22.1RT2PCR3(DNA)(EDS276),RT2PCR,RT2PCR102104RT2PCR10-8,10-6,10-10(2,3)2Table2SensitivitydetectionofNDV.DetectionmethodDilution10-110-210-310-410-510-610-710-810-910-1010-1110-12Chipdetection++++++++++--RT2PCR++++++++----Virusisolation++++++------01111©1994-2009ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(4),,,(5)6,IBDVREVE.coli3,,,,4Table4Thespecifictestresultsforsinglepathogendetection.PathogenTestresultsSNDVIBVH9EDS276MGILTNDV++-----IBV+-+----H9+--+---EDS276+---+--MG+----+-ILT+-----+5Table5Thespecifictestresultsformultiplepathogendetection.PathogenTestresultsSNDVIBVH9EDS276MGILTNDV+IBV+++----H9+MG+--+-+-EDS276+ILT+---+-+ILT+IBV+H9+-++--+NDV+MG+EDS276++--++-H9+IBV+MG+-++-+-EDS276+IBV+ILT+-+-+-+IBV+H9+EDS276+ILT+-+++-+NDV+H9+MG+ILT++-++-+NDV+IBV+H9+ILT+MG++++-++NDV+H9+EDS276+MG+ILT+++++++NDV+IBV+H9+EDS276+MG+ILT+++++++2.3,RT2PCR,RT2PCR102103(7),:2.3.1200726Table6Thespecifictestresultsforunrelatedpathogendetection.PathogenTestresultsSNDVIBVH9EDS276MGILTIBDV+------REV+------E.coli+------1115:©1994-2009ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.