KS J ISO 21570-2011 Foodstuffs－Methods of analysis for the detection of genetically modified organisms and derived products－Quantitative nucleic acid based methods《食品 转基因有机体和衍生物探测分.pdf

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1、KSKSKSKSKSKSKSK KSKSKS KSKSK KSKS KSK KS KS J ISO 21570 KS J ISO 21570： 2011 2011 8 25 http:/www.kats.go.krKS J ISO 21570:2011 : ( ) ( ) ( ) : (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 21570:2011 i .1 1 1 2 1 3 .1 4 2 4

2、.1 .2 4.2 PCR , , 2 4.3 PCR .2 5 2 6 .2 7 3 7.1 .3 7.2 .3 7.3 3 7.4 3 7.5 .4 8 4 9 .5 10 .5 A( ) 6 B( ) 12 C( ) 20 D( ) Event 88 101 KS J ISO 21570:2011 Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Quantitative nucleic acid based methods 200

3、5 1 ISO 21570, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Quantitative nucleic acid based methods , . 1 (PCR) (GMOs) . GMO DNA DNA DNA . , , . . A D . 2 . . ( ) . KS A ISO Guide 32, KS J ISO 21569, KS J ISO 21571, KS J ISO 24276, 3 KS J IS

4、O 21570:2011 2 KS J ISO 24276 . 4 4.1 DNA , . PCR1, 2 PCR3, 4 . , , (dynamic range) . PCR PCR , , , . 4.2 PCR , , DNA , KS J ISO 21569 . 4.3 PCR DNA , ( ) ( ) . (matrix) . , (CRMs) (traceable) . , . 5 . (validation study) (measurement uncertainty) 6, 7, 8, 9 . 5 . , . . . 6 AD KS J ISO 24276 . KS J

5、ISO 21570:2011 3 7 7.1 GMO PCR KS J ISO 21569 . AD PCR , . DNA . (taxon) DNA GMO ( ), (quantity) . DNA . (test portion) DNA (duplicate) . (KS J ISO 24276 1 ). 7.2 (allelic) , (cultivars) . 7.3 2 4 ( 42 ) 6 ( 6 ). 9 . DNA dsDNA , DNA DNA . 7.4 PCR (variability) . / , (stacked genes) , GMO . (LOQ) , K

6、S J ISO 24276 . GM (species) , . AD . (assay) GMO , Ct( ) . KS J ISO 21570:2011 4 7.5 (measurements) . ( KS A ISO 5725 ). , (feasible) . , GMO DNA ( ) , . a) (test portion) , 33 % b) a) GMO DNA ( PCR 1 Ct .) 50 100 % ( , 1.0 % 2.0 % 0.9 % 2.1 % ). , event (RM), (CRM) . , , , (traceability) . (calibr

7、ation) (KS A ISO Guide 32 ). 8 PCR . a) . KS J ISO 21569： 2011 8.1 (positive) . . . . (7.3 ) . b) . . AD DNA . DNA GMO . . GM , . KS J ISO 21570:2011 5 GMO DNA . 9 GM . , . , (LOD) (LOQ) (LOD) (LOQ) . , . 1 . 1 . KS J ISO 21569 “ x DNA .” , GM . KS J ISO 21569 . “ x , GMO DNA .” , “ X % ( ).” GM , L

8、OQ . GMO . “( ) ( ) GMO(GMO ) DNA .” ： “ X % ( ).” GM , LOQ . GMO . “( ) ( ) GMO(GMO ) DNA X % ( ).” GMO DNA . 10 KS J ISO 24276 KS J ISO 21569 , . a) (LOQ) b) (LOQ) c) DNA d) e) 9. KS J ISO 21570:2011 6 A ( ) A.1 PCR (maize) adh1 DNA A.1.1 DNA , DNA DNA PCR / , ( , housekeeping) (Zea mays) adh1 ( 1

9、： alcohol dehydrogenase 1 ) . A.1.8 . A.1.2 A.1.2.1 (ground) , (IRMM 411, IRMM 412, IRMM413 10)11 DNA . (type) DNA (U1U6) (A.1.2.2 ) , Bt11(D.1 ) GM events . (haploid genome) 1 11. (allelic stability) 11. A.1.2.2 (collaborative trial) (international harmonized protocol) (EC-JRC), (IHCP) 12. (haploid

10、 maize genomes) DNA (183 486, 61 162, 20 387, 6 796, 2 265, 755) 13 . 6 (S1 S6) . DNA (PicoGreen, Molecular Probes, Cat. Number P-7589 dsDNA .) 1C (2.725 pg)14. DNA( 13 .) 6 (U1 U6) . . KS J ISO 21570:2011 7 A.1 . , Bt11 (sweet maize) GMO event . ( ) 15 16, D.1 . A.1 U1 U2 U3 U4 U5 U6 12 12 12 12 12

11、 12 10 10 10 10 10 10 1 1 1 1 1 1 9 9 9 9 9 9 4 4 4 4 4 4 Cochran 1 1 1 1 Grubbs 1 1 1 1 1 35 34 34 34 35 35 7 339 18 349 36 697 55 046 91 743 146 788 9 985 23 885 46 918 75 161 100 541 122 080 (bias)(%) 36.1 30.2 27.9 36.5 9.6 16.8 sra1 318.59 1 463.60 5 796.58 4 539.57 11 306.89 14 843.41 (%)b13.2

12、1 6.13 12.35 6.04 11.25 12.16 sRa2 013.12 2 083.57 6 145.39 6 806.85 14 592.04 17 777.70 (%)b20.16 8.72 13.10 9.06 14.51 14.56 a . b % . A.1.2.3 A.1.2.3.1 EMBL/GenBank/DDBJ1) 04050 . Zea mays(maize/corn) Zea mays subsp. diploperennis(teosinte) 11. A.1.2.3.2 (primers) (probes) http:/www.ncbi.nlm.nih.

13、 gov/blast/2003. 10. 9 BLASTN query sequence( ) GenBank/EMBL/DDBJ 1) . 1) . ISO . . KS J ISO 21570:2011 8 A.1.2.3.3 (non-target taxa) 20 (lines) 11. (cross-reactivity) (wild ancestor) teosinte Zea mays subsp. diploperennis .11, 17 . 11. A.1.2.4 ABI PRISM 7700 (SDS)2) TaqManchemistry2) . Primer Expre

14、ss (Applied Biosystems)2) . A.1.2.5 (LOD) , 10 copies 11. 7 399 copies . A.1.2.6 (LOQ) , 100 copies11. 7 399 . A.1.3 . A.1.4 adh1 134 bp adh1 ( A.2 ). PCR (fluorescent dyes)： (reporter dye) FAM (quencher) TAMRA( A.2 ) adh1 PCR . , TaqMan2)chemistry . (signal) (threshold) . Ct . adh1 DNA Ct Ct (： reg

15、ression analysis). A.1.5 (reagent) A.1.5.1 (quality) KS J ISO 24276： 2006 6.6 2) . ISO . . KS J ISO 21570:2011 9 A.1.5.2 (water) A.1.5.3 PCR (MgCl2 ), 10-fold A.1.5.4 MgCl2 , c(MgCl2) 25 mmol/L A.1.5.5 dNTP , c(dNTP) 2.5 mmol/L( ) A.1.5.6 A.2 . A.2 DNA PCR ADH FF3 5 CgT CgT TTC CCA TCT CTT CCT CC 3

16、300 nmol/L ADH RR4 5 CCA CTC CgA gAC CCT CAg TC 3 300 nmol/L ADH1 MDO 5 FAM AAT CAg ggC TCA TTT TCT CgC TCC TCATAMRA 3a200 nmol/L aFAM： 6-carboxyfluorescein:TAMRA:6-carboxytetramethylrhodamine adh1 (amplicon) 134 bp . A.1.5.7 DNA polymerase AmpliTaq GoldDNA polymerase3)A.1.5.8 Uracil N-glycosylase(

17、) A.1.5.9 A.3 . A.3 (reaction vial) / 25 L DNA( 250 ng) 5 L Taq DNA (uracil N-glycosylase dUTP) ( ROX )adNTP TaqManUniversal Master Mix 212.5 L(1) A.2 A.1.5.6 A.2 A.1.5.6 aROX carboxy X rhodamine 3) . ISO . . KS J ISO 21570:2011 10 A.1.6 A.1.6.1 . A.1.6.2 (thermal cycler) profile ABI PRISM7700 SDS (

18、Applied Biosystems)4). PCR . A.1.6.3 , ABI PRISM96 / (96-well optical/reaction plate) MicroAmp (optical caps)8 / , (8caps/strip, flat) PCR (Applied Biosystems)4). A.1.7 ： PCR A.1.7.1 GM GMO PCR . A.3 25 L PCR . A.1.7.2 PCR , . / , (high-quality), DNA JRC, IRMM CRM 4) 13. KS J ISO 24276 . A.1.7.3 A.4

19、 ABI PRISM7700 (Applied Biosystems)4). , AmpliTaq GoldDNA 4). . . A.4 . 4) . ISO . . KS J ISO 21570:2011 11 A.4 ： (s) ( ) Pre PCR： 120 50 Pre PCR ： DNA DNA 600 95 PCR(50 ) 1 (denaturation) 15 95 2 (annealing and elongation) 60 60 A.1.8 PCR (inhibitors) adh1 (accuracy) . PCR (absence) (KS J ISO 21571： 2006 A ), (template) DNA Ct( ) Ct (doubling) . (dilutions) (differences) . GMO DNA