1、#%S*S&NB%B*ZtB PCR&jJ!g:g Protocol of the qualitative polymerase chain reaction for detecting genetically modified plant components in food 3nf+t:)1mkaa*km1milfiljt%*h%m%&n m fl:.asrsssmxFa+le.mmi%H#&t&#R&)#t5%4BH;f.;4%t%TmtBfiRB2P;ZB&B%3iWf%ABRf!HBBAt+;9%E*e RR+%f:t g l 5 l M F f R 1 z b:H *.GB/T 6
2、682 j)$i%&SH7k%#j#IBtk&(eqv IS0 3696:1987)SNIT 1193 J#B!&%&3Z&*Bjk SNIT 1194 M B#I%&B!#!#-)%SN/T 1195 hEi+$SL!4&3)Z?4 ICR UB!YJ&SN/T 1196 39%PCR%!h?&SN/T 1197?h%RR&B3&3)2#PCR 3$ildh k&SN/T 1199#l%+R&B3&3)PCR&I%-&SN/T 1204 t$B#%fiSC&%3&r3t3)TdU%X PCR%&%t%?f?&TPl%&HT*&%*3.1 PCRI polymerase chain react
3、ion,m%PCR.3.2 DNA 1 dexribonucleic acid,%j&.3.3 dNTP:deoxyribonucleoside triphosphate,8#$t%3?.3.4 dATPldeoxyadenosine trphosphateLlk!%IlT T.3.5 dCTP:deoxycytidine triphsphate,a#SmBE-R 3.6 dGTPIdeoxyguanosine triphosphate,R#$7 3.7 dUTP deoxurdine triphosphate,Jl$!$G$e3.aft 3.8 bp base pair,SN.3.9 Tag
4、 BrDNA J*m.3.10 Irls,tris(hydroxymethyl)amlnoniethanc,!-:(%!p g)&,%V%$.,3.11 TE:TI-CI.EDTA$!$.3.12 I.ectinl&44&l%.3.13 ZEINi#&%B$#E$.3.14 3-case1hoshoenilruvate-carboxylahc,b#aj;)fat.(t:.3.15 a t a t n,#&Z E%Z?l A Palatin BH#&jttlR%q%BHli4%fl:e 3.16 tRNAeu&kQdWflW#4f!il?il.3.17 18s rRNAl&%118s RNA%a
5、.3.18 CaMV 35S135S promoter from cauliflower mosaic virus,?f a T8%lJt$i%l8J 3 5 S B a T.3.19 NOS,terminator of nopaline synthase gene,M R B -&l i l t k .3.20 FMV 35S135S promoter from a modified figwort mosaic virus(caulimovirus group),g8ZR-t R*35s a a .3.21 NPTII:neomycin-3-phoephotrmsforase gene,#
6、$#-3-RBS 88.3.22 BAR:phosphinothricin acetyltransferase gene from Bacillus amylolique facions,?#Tfim Bacillus amylolique facisns VTj#(Pt l%E#SBj.3,23 PAT I phosphinothricin acetyltransferase gene,9TM 2 E?Rl$&B.3.24 COX I glyphosate oxidoreductaae gene,WFMgtEHll#ftlE.3.25 CryIA(b)l a synthetic gene e
7、ncodes the firet 648 amino acids,insecticidal-active ftruncated product identical to that of cryIA(b)gene of Bacillus thuringiensis subsp,%2$?$84HB%&i s I3 crylA(b)SB.3.26 Cry3AlB,thuringiensis subsp,Tenebrionis(B,t,t,)strain QI 256-82 B&s fi,&#k*#bjQ&Il*a Efl&(Colorado potato beetle larvae).3.27 Ep
8、SPSI 5-enolpyruvylehikimate-3-phosphate synthaee gene,5-%9?-3-.ftj%jtS B9.3.28 PVY-cpl potato Y virus coat protein,$a Y#8%4/9!iSb.3.29 PG I plgalacturonase,%#$SL#km$I$f&qRSB&3);BBa8mmmffl!#BBiBR8 SNIT 1193 l8J%4Se 6.1 Q(fP WdiBS&i DNA g,HNrf&WMBe$ABCI4bWS$IM#B#flR#3lC,NU PCR$,DNA H W B PCR r)11,lfia
9、fie1$egae:itt3f.6.2 PZNlll 6.2.1 PCR%fi.6.2.2 DNANBfi.6.2.3 B?9lt#b.6.2.4 E$%!X.6.2.5 8W9i%&o 6.2.6#iRB$IR.6.207 MB!BB3)fi.oY*6.2.0&i$*l&sba,6S/jB$fb#l,Mini/.6.2.9 B%$#,W;%#B.6.2.10 4$7Jc#,Xa#k#6.2.11#11bfon.6,2.12&ASB#.2 6.2.13 s?J#,B;5 6 B M%H#&f#6.2.14$FRY.6.2.15 BB&#!qPC%fB%l-6.2.16#*%#80.5 pL.2
10、 pL.10 pL.20 pL.100 pL.200 pL.1 000 PI.6.3 24iitIfd RBGMBM Eff41%+BTM&!k21:&fpl a 6.3.1%R&flf:GB/T 6682 q-%7JCb9B#fO 6.3.2%llBMF.6.3.3 RNase H%tli20 W m g DTICIHMSJYI%&-&FF&B!%#I RNAsc-A MlB%10 g/I+%fiT-2O#%EB%H 6.3.4 CTAB%:CTA13 20 g/I.,#&+&1.4 nol/I-lris 0.1 rnol/L,Nal EDTAO.02 mol/I.,Rj&%$f,ikQlt
11、jNPi p H 35 8.0.6.3.5 CTAH W,s#i ICTAB 5 g/L,%kM 0.04 mol/I.6.3.6#,4&9!ia-#kj l.,2 mol/l.,6.3.7 BifiiYl K K R I I Q M Wlf 20 m g/m i.%T 1 f l S k l&b N%l M W T-2 0 CC,B%lZ$Z%fil4.6.3.0 B$#t&4 F 4.4 -#b 9&3#DNAe 6.3.9 TE BNj&tTris 0.010 rnol/l.,Na,EDTAO.001 mol/LlJa&S,lTB-#ki141 pH H 8.0.6.3.10%7)CtS
12、.WTJ4W%#i.Uliieffi1.6.3.11&!iEl&DNA RlrC%!f.6.3.12 PCR a3PJ&lrq Mgf PCR#.dATP.dTTP,dCTP.dGTP,Tag A.6.3.13 Q#br#N%(I,oading Huffer).6.3.14 51%e s +a m a m a *b a w f i m i:d a s 1 v x x m w f f i NbUIb9.%tpa+#ttk%+I BINSBWr3I?aFFM!&SN/T 1196-2003$U SN/T 1197-2003.1&NJ*rffr$PlB$5BitW1*4ESNH3I3$#Pl 48t
13、iU!lM26!3i CaMV35S1 FMV35Sa.NOS NPTII PATm 35S/Petu CaMV35YCTP ol WSPS COX CP4-EPSPS($#I BAR LBB#R 9bm$h%N ma Hd9S$1 9i-H W#J%m fin W H l i l f i Bk 1%BE B!Eib!lJ&Bis8 fi&W bQbB;8H%F!B 1?1H%BBkEL(Roundup Ready),%(Bt176,Btll,Mon8lO,T14/25).%IH,?#%,4+$#%JX#-Bra cZlR&faB5&4+4f*#RJIR;jl PIBP*WI z R%A x,
14、A I R.I B%I B Wi?-btrsA H%Rkl2%Et#bQf&cZlRBB%l?t&URi%B#EHMttll3iXBl 176,CBH-35l)%#+M.BtWI Yl#3U:5-tcatcccttacgtcagtggag-3 I j i 15-ccatcattgcgataaaggaea-3 E,5-aagacatccaccgaagactte-3 bi 15-aggacagctcttttccacgtt-3 iE I 5-nagcctcaacaaggtcsg-3 g I 5-ctgctcgatgttgacaag-3 E 15-gaatcctgttgccggtcttg-3 15-t
15、tatcctagtttgcgcgct&3 E 5-atcgttcaaacatttggca-3 R 15-attgcgggactctaatcata-3 E I 5-ggatctcctgtcatct-3 g 15-gatcatcctgatcgac3-iE 5-sggatctcgtcgtgscccat-3*,5-gcacgnggaagcggtca-3 iE I 5-gtcgacatgtctccggagag-3 15 J-gcaaccaaccaagggtatc-3 e 15-tgatgtsatatctccactgacg-3 15-tgtatcccttgagccstgttgF3 iE 15-ccacta
16、tccttcgcaagacccttcc-3 15-ttgtatcccttgegccatgttgt-3 5-gtcttcgtgttgctggaaccgtt-3 fi 15-gaactggcaggagcgagagct-3 IE,5-gacttgcgtgttcgttcttc-3&1 5-aacaccgttgagcttgegac-3 H 5-acasgcacggtceacttcr3 5-ectcggccgtccagtcgta-3 PCR PQk/l/bp 165 210 196 180 165 173 183 191 171 120 120 204 175 I 5-ggatccttagaagcatctagt-3 PC/NOS 350 62:5-catcgcaagaccggcnacag-3 RB1114581.#&f%ilmilr#ttl383 bp%I 180 bp ik4+;l;k/I)NA+;.ee:.c%ss8.nnmrq3a3 I,P tttl DNA 6.4&M?b%6.4.1#Y#H#J#&H%tfill&%#3b!J%R&H$SGHid 120C.so min&Em%.6.4.1