GB-T 19439-2004 H5亚型禽流感病毒NASBA检测方法

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1、ICS11.220B41GB/T19439-2004H5NASBAProtocolofdetectingavianinfluenzavirussubtypeH5usingNASBA2004-2-152004-2-15GB/T19439—2004AOIEAÒßÍÞßOIEîðððßÞC2GB/T19439—2004H5NASBA1NASBAH5Øë2GB/T1893620033NASBANucleicacidsequence-basedamplification,NASBAHT75’T7DNARNA5’5’RNAECLECLECLRNA44.14.25L200LRNA1.

2、5mL1.5mL5mL2C5mLVWRECLNucliSensELISA3GB/T19439—20044.3AATTCTAATACGACTCACTATAGGGAGAAGGCCAIAAAGA(C/T)AGACCAGCTAGATGCAAGGTCGCATATGAGGAGAGAAGAAGAAAAAAGAGAGGAC4.4:a)(lysisbuffer)5M10%TritonX-10010mMTris/HClb)(washbuffer)5M10mMTris/HClc)500mg/mL(Silica)500mg/mld)(elutionbuffer)10mMTris/HCle)Af

3、)H5(H5captureprobe)10mMBiotin-GC(A/G)AGTTC(C/T)CTAGCACTGGCAATg)10.9mL(10mMgenericECLdetectionprobe)DNAECLGATGCAAGGTCGCATATGAGGTGA(C/T)AATGAATG(C/T)ATGGAAh)21.7mL(instrumentreferencesolution)i)115mLdetectiondiluent15mMTris-/HClj)assaybuffer(50mMTris-HCl);k)cleanersolution100mMKOH,10%SDS,1

4、0%TritonX-100l)(alcohol)4.5GB/T1893620032.155.1a)0.1mL0.9mLb)50Lc)d)10min2min4GB/T19439—2004e)12,000r/min30secf)1mLg)h)12,000r/min30seci)f)h)70100j)k)5610minl)50Lm)n)5610min5mino)12,000r/min2minp)5L1hQiagen5.2a)5L5.1p10Lb)655minc)415mind)5Le)415minf)4190ming)h)-2030d5.3ELISA*a)N25mL1b)12

5、0LBc)25Ld)5LRNAe)f)4130min10ming)10.3mLh)0.25mLIRS15GB/T19439—2004i)j)NucliSensC*N6NASBA/ECL0.15ÈH5H56GB/T19439—2004A()A.1a)enzymesspheres6.5mg1.3U/LAMVRT0.51.3U/LRNaseH151.3U/LT7RNA,100mMTris/HCl,10%BSAb)enzymespherediluent0.42g/LBSAc)(reagentsphere)10mg4mMNTP,15mDTT,40mMMgCl2d)(reagent

6、spherediluent)100mMTris/HClpH8.3,2mMdNTPe)(100mMKClsolution)f)(10mMPrimerMixture)g)RNApositivecontrolRNAh)DEPCA.2A.2.1a)80Lb)16LKCl14LDEPCc)10LH5d)e)30minA.2.2a)55L20b)c)d)e)60min7GB/T19439—2004B()B1a)H5ECLb)NH5N210LH5N210LECLc)d)60min8GB/T19439—2004C()NucliSensC.1C.1.1NucliSensC.1.2Prim

7、eC.1.3Start3minC.1.4log-inusernameserviceengineerloginC.1.5RoutineNewRunC.1.6yesC.1.78okC.1.8selectedassayFreetubeC.1.9AddtolistC.1.10IDC.1.11closeC.1.12OKC.2instrumentcarouselIDC.3RoutineRunWorklistC.4ProceedC.51.5minC.6RoutineSampleresultsAssayr