KS J ISO 21569-2011 Foodstuffs-Methods of analysis for the detection of genetically modified organisms and derived products-Qualitative nucleic acid based methods《食品 基因改良有机物和衍生产品的检.pdf

1、 KSKSKSKSKSKSKSK KSKSKS KSKSK KSKS KSK KS KS J ISO 21569 KS J ISO 21569： 2011 2011 8 25 http:/www.kats.go.krKS J ISO 21569:2011 : ( ) ( ) ( ) : (http:/www.standard.go.kr) ： ：2006 12 28 ：2011 8 25 2011-0293 ： ： ( 02-509-7270) (http:/www.kats.go.kr). 10 5 , . KS J ISO 21569:2011 i .1 1 1 2 1 3 .1 4 .2

2、 4.1 .2 4.2 PCR .2 4.3 PCR 2 5 2 6 .3 7 3 7.1 , 3 7.2 .3 7.3 PCR .4 7.4 PCR 5 7.5 .5 7.6 PCR , PCR .5 8 6 8.1 .6 8.2 .6 9 .6 9.1 .6 9.2 .7 9.3 .7 9.4 7 10 .7 A( ) 8 B( ) .25 C( ) 41 D( ) 63 68 KS J ISO 21569:2011 Foodstuffs Methods of analysis for the detection of genetically modified organisms and

3、derived products Qualitative nucleic acid based methods 2005 1 ISO 21569, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Qualitative nucleic acid based methods , . 1 (GMOs) (derived products) . (PCR) . DNA . , , , , . , ( ： ). A D . 2 . . ( )

4、. KS J ISO 21571, KS J ISO 242761), 3 1) . KS J ISO 21569:2011 2 KS J ISO 24276 . 4 4.1 . . (controls) . 4.2 PCR DNA 1,2,3. DNA . DNA DNA . . PCR DNA DNA . 4.3 PCR PCR . ( , , DNA ) . PCR , . 5 20 . / . 5.1 DNA/ (target DNA/control) 5.2 (water) 5.3 (dNTP) deoxyribonucleoside triphosphate(dNTP) solut

5、ion, dATP, dCTP, dGTP, dTTP dUTP . KS J ISO 21569:2011 3 dUTP PCR . 5.4 PCR (PCR buffer solution) PCR DNA , MgCl2 . MgCl2 , . , , . 5.5 DNA (thermostable DNA polymerase) 5.6 (forward primer) 5.7 (reverse primer) 6 KS J ISO 24276 A D . 7 7.1 , . . DNA , (integrity) PCR . , , DNA . PCR PCR 4 . PCR KS

6、J ISO 24276 . 7.2 KS J ISO 24276 . A D ( 5). , MgCl2 / . , (matrix) PCR ( ) . , 10 40(cycle) PCR . , (non-specific products) . PCR PCR 100 DNA 40 PCR . KS J ISO 21569:2011 4 / , PCR . , , PCR(hot-start PCR) . PCR DNA (mispriming) (primer-oligomerization) . (validation study) . 7.3 PCR 7.3.1 PCR PCR

7、PCR . 7.3.2 PCR . DNA PCR 60 150 bp . PCR 250 bp . PCR . 7.3.3 7.3.3.1 A D . 7.3.3.2 . ： 18 30 60 ( ), , ： 65 GC： AT 50： 50 . ( G C ). 3 . PCR ( ) . KS J ISO 21569:2011 5 7.3.3.3 7.3.3.3.1 . . 7.3.3.3.2 , EMBL, GenBank2) , FastA, Blast2) . . 7.3.3.3.3 . ( ) . 7.4 PCR GMO PCR , . , , . 7.5 PCR (KS J

8、ISO 24276). , . 7.6 PCR , PCR A D PCR . PCR PCR DNA PCR . . . . 2) Blast GenBank . ISO . .KS J ISO 21569:2011 6 DNA PCR . , ( ) PCR PCR : PCR DNA PCR ( ) , DNA GMO . DNA . 35S (CaMV) , CaMV 35S GMO / CaMV DNA6 . CaMV DNA CaMV DNA CaMV GMO . 8 8.1 PCR a) PCR KS J ISO 24276( 2 ) . b) PCR , KS J ISO 24

9、276( 2 ) . (gene stacking)7 GMO . (KS J ISO 24276). 8.2 7.6 . 9 9.1 , “” . “GMO ” . , (LOD) . KS J ISO 21569:2011 7 , DNA / . , / . (LOD) , ( DNA , , 100 ng 0.01 % GTS 40-3-2 DNA). 9.2 . “ , X Y . ABC x % .” PCR DNA (LOD) . “ X DNA (LOD) ( ).” (LOD) DNA ( ) GM( ) (LOD) 7. 9.3 . “ X Y .” GMO . 9.4 .

10、, (KS J ISO 24276). . , PCR . . 10 KS J ISO 24276 , . , 9. KS J ISO 21569:2011 8 A ( ) A.1 A.1.1 (glycine max) (single copy) . DNA . A.1.2 (validation status) A.1.2.1 35 German Federal Institute for Health Protection of Consumers and Veterinary Medicine(BgVV) “ ” 89. DNA KS J ISO 21571：2006 A.3 CTAB

11、 100 mg . A.1 . A.1 199781998/19999 25 27 22 20 10 3 220 60 220 50 0 1(2 %) 1(2 %) A.1.2.2 A.1.2.2.1 7. . GenBank3) K008213) GenBank BlastN . ISO . , .KS J ISO 21569:2011 9 M30884 . A.1.2.2.2 Le110 . (EMBL) NCBI BlastN2) , ( , , ) DNA . GM03 100 % . AX033509( DE19906169 17), AX033507( DE19906169 15)

12、, AX033501( DE19906169 9), , GM04 No. AX033509( DE19906169 17) . , No. M30884 1993 GenBank K00821 . , . A.1.2.2.3 ( , , ) (beef) (pork) DNA , . PCR DNA 1011. A.1.2.3 (LOD) , , DNA 0.1 ng . A.1.3 . A.1.4 118 bp PCR , . A.1.5 (reagents) KS J ISO 24276 . A.1.5.1 A.1.5.2 PCR (MgCl2) , 104)A.1.5.3 (MgCl2

13、solution), c(MgCl2) 25 mmol/L A.1.5.4 dNTP (dNTP solution), c(dNTP) 2.5 mmol/L( ) A.1.5.5 (oligonucleotides) 4) 10 10-fold, PCR 1.5 mol/L Tris-HCl, pH 8.3 KS J ISO 21569:2011 10 A.1.5.5.1 (Soya bean lectin gene)(GenBank K00821) GM03： 5-gCC CTC TAC TCC ACC CCC ATC C-3 A.1.5.5.2 (GenBank K00821) GM04：

14、 5-gCC CAT CTg CAA gCC TTT TTg Tg-3 A.1.5.6 DNA ( PCR ), 5 IU/L A.1.5.7 (hybridization probe)(GM), 5-ggT AgC gTT gCC AgC TTC g-3 A.1.5.8 (saline sodium citrate buffer)(SSC), 5, pH 7.0 SSC 0.75 mol/L 0.075 mol/L . A.1.5.9 (prehybridization solution), 5 SSC 0.1 % N- , 0.02 % SDS, 1 % 5) 5 % 12A.1.5.10

15、 (hybridization solution), 2.5 mL 10 pmol (A.1.5.9). 50 . 12 A.1.6 A.1.6.1 (thermal cycler) A.1.6.2 A.1.7 A.1.7.1 PCR A.2 PCR 25 L , PCR . A.2 . 5) Boehringer . ISO . .KS J ISO 21569:2011 11 A.2 (L) DNA 10 50 ng 1 15.9 10PCR (MgCl2 ) 1 2.5 MgCl2 a, 25 mmol/L 1.5 mmol/L 1.5 dNTP , 10 mmol/L 0.8 mol/L

16、 2 GM03, 5 mol/L 0.2 mol/L 1 GM04, 5 mol/L 0.2 mol/L 1 Taq DNA , 5 IU/L 0.5 IU 0.1 aPCR MgCl2 , MgCl2 1.5 mmol/L . A.1.7.2 PCR (IRMM 410) CRM GTS 40-3-2 . KS J ISO 24276 . A.1.7.3 A.3 GeneAmp2400, 9600, AmpliTaq GoldDNA polymerase6) . . / . (hot-start polymerase) , . A.3 / 10 /95 30 /95 30 /60 60 /7

17、2 35 3 /72 A.1.8 DNA PCR . , digoxigenine 6) GeneAmp2400, 9600 AmpliTaq GoldDNA polymerase Applied Biosystems . , ISO . .KS J ISO 21569:2011 12 GM(A.1.5.7 A.1.5.10) (southern hybridization) PCR , A.1.5.5 GenBank . A.1.9 PCR GTS 40-3-2( , IRMM 410, ) DNA . A.1.8 . 118 bp A.1.2.2 DNA DNA . KS J ISO 21571： 2006 B.2 . A.2 (multicopy) DNA A.2.1 ( trnL ) DNA . DNA DNA