GBT 19439-2004 H5亚型禽流感病毒NASBA检测方法.pdf

ICS 11.220 B41GB/T19439-2004H5NASBAProtocol of detecting avian influenza virus subtype H5 using NASBA 2004-2-152004-2-15GB/T 194392004 2AOIEAOIECGB/T 194392004 3H5NASBA1NASBAH52GB/T 189362003 3NASBANucleic acid sequence-based amplification,NASBAHT75T7DNARNA55RNAECLECLECLRNA44.1 4.2 5L200 LRNA1.5mL1.5mL5mL2 C5mLVWRECLNucliSensELISAGB/T 194392004 44.3 AAT TCT AAT ACG ACT CAC TAT AGG GAG AAG G CCA IAA AGA(C/T)AG ACC AGC TA GAT GCA AGG TCG CAT ATG AGGAGA GAA GAA GAA AAA AGA GAG GAC4.4:a)(lysis buffer)5M10%Triton X-10010mMTris/HClb)(wash buffer)5M10mMTris/HClc)500mg/mL(Silica)500mg/mld)(elution buffer)10mMTris/HCle)Af)H5(H5 capture probe)10mM Biotin-GC(A/G)AGT TC(C/T)CTA GCA CTG GCA AT g)10.9mL(10mMgeneric ECL detection probe)DNAECL GAT GCA AGG TCG CAT ATG AG GT GA(C/T)AAT GAA TG(C/T)ATG GAA h)21.7mL(instrument reference solution)i)115mLdetection diluent15mMTris-/HClj)assay buffer(50mMTris-HCl);k)cleaner solution100mM KOH,10%SDS,10%Triton X-100l)(alcohol)4.5 GB/T 1893620032.155.1 a)0.1mL0.9mLb)50Lc)d)10min2minGB/T 194392004 5e)12,000r/min30secf)1mLg)h)12,000r/min30seci)f)h)70100j)k)5610minl)50Lm)n)5610min5mino)12,000r/min2minp)5L1hQiagen 5.2 a)5L5.1 p10Lb)655minc)415mind)5Le)415minf)4190ming)h)-2030d5.3 ELISAa)N*25mL1b)120LBc)25Ld)5 LRNAe)f)4130min10ming)10.3 mLh)0.25 mL IRS1GB/T 194392004 6i)j)NucliSensC*N6NASBA/ECL0.15 H5H5GB/T 194392004 7 A()A.1 a)enzymes spheres6.5mg1.3U/L AMVRT 0.51.3U/L RNase H151.3U/L T7 RNA,100mMTris/HCl,10%BSAb)enzyme sphere diluent0.42g/L BSAc)(reagentsphere)10mg4mMNTP,15mDTT,40mMMgCl2d)(reagent sphere diluent)100mMTris/HCl pH8.3,2mMdNTPe)(100mMKCl solution)f)(10mM Primer Mixture)g)RNApositive control RNAh)DEPCA.2 A.2.1 a)80Lb)16L KCl14L DEPCc)10L H5d)e)30 min A.2.2 a)55L20b)c)d)e)60 min GB/T 194392004 8 B()B1a)H5ECLb)NH5N210L H5N210L ECLc)d)60 min GB/T 194392004 9 C()NucliSensC.1C.1.1NucliSensC.1.2 PrimeC.1.3Start3minC.1.4 log-inuser nameservice engineerloginC.1.5 RoutineNew RunC.1.6 yesC.1.7 8okC.1.8 selected assayFree tubeC.1.9 Add to listC.1.10 IDC.1.11 closeC.1.12 OKC.2instrument carouselIDC.3RoutineRun WorklistC.4ProceedC.51.5minC.6RoutineSample resultsAssayresult