GOST R 56058-2014 Feed and feed additives Methods of identification and determination GMO of plant origin《饲料和饲料添加剂 植物来源GMO的识别和测定方法》.pdf

1、 56058 2014 2015 560582014 II 1 - ( ) 2 454 - - 3 9 2014 . 705- 4 1.02012 ( 8). ( 1 ) , -. () - . , (gost.ru) , 2015 , - 56058 2014 1 Feed and feed additives. Methods of identification and determination GMO of plant origin 2015 07 01 1 , - - ( ) - ( ) ( ) - (Real Time PCR). 2 : 317192012 . - () 6497

2、2011 . / 170252009 - 518482001 . 521732003 . - () 532442008 ( 21570:2005) . - . , 555762013 . - - - - , 1 , , , . , , (). , , , , , - . , , , , . 3 , 6497, 51848, 55576 31719. 4 55576 ( 4, - ). 560582014 2 5 , 5.1 / 17025. 5.2 31719, : - II ; - - -; - - 1,5 3;- 0,2 3;- -; - *;- . - . 5.3 55576, , .

3、5.3.1 : - ; - -; - () 10 (10- 1,76 10-3 /3);- Taq- ; - , 0,1 % 5,0 % *. - . 5.3.2 ( ) ( FAM R6G): 5.3.2.1 40-3-2: ) : 1) 1: 5- GCC ATG TTG TTA ATT TGT GCC AT 3;2) 2: 5- GAA GTT CAT TTC ATT TGG AGA GGA C 3;3): 5- FAM-CTT GAA AGA TCT GCT AGA GTC AGC TTG TCA GCG BHQ1-3;) (lec1): 1) 1: 5-TCC ACC CCC ATC

4、 CAC ATT T -3;2) 2: 5- GGC ATA GAA GGT GAA GTT GAA GGA-3;3): 5 - R6G-AAC CGG TAG CGT TGC CAG CTT CG -BHQ1 -3;5.3.2.2 2704-12: ) : 1) 1: 5- GCA AAA AAG CGG TTA GCT CCT-3;2) 2:5- ATT CAG GCT GCG CAA CTG TT-3;3): 5- FAM-CGG TCC TCC GAT CGC CCT TCC-BHQ1-3;) (lec2): 1) 1: 5-CAC CTT TCT CGC ACC AAT TGA CA

5、-3;2) 2: 5-TCA AAC TCA ACA GCG ACG AC-3;3): 5-R6G-CCA CAA ACA CAT GCA GGT TAT CTT GG -BHQ1 -3.5.3.2.3 5547-127: ) : 1) 1: 5- GCT ATT TGG TGG CAT TTT TCC A 3;2) 2: 5- CAC TGC GGC CAA CTT ACT TCT 3;3): 5- FAM-CC GCA ATG TCA TAC CGT CAT CGT TGT-BHQ1-3;) (lec3): 1) 1: 5- CCT TCT CGC ACC AAT TGA CA -3;2)

6、 2: 5- TCA AAC TCA ACA GCG ACG AC -3;* Rotor-Gene 2000/3000/6000 (Corbett Research, ). - . * ,CCM JRC, IRMM. - . 560582014 3 3): 5-R6G-CC ACA AAC ACA TGC AGG TTA TCT TGG-BHQ1 -3;5.3.2.4 MON 810: ) : 1) 1: 5-TCG AAG GAC GAA GGA CTC TAA CGT-3;2) 2: 5-GCC ACC TTC CTT TTC CAC TAT CTT-3;3): 5-FAM-AAC ATC

7、 CTT TGC CAT TGC CCA GC-BHQ1-3;) (hmg): 1) 1: 5-TTG GAC TAG AAA TCT CGT GCT GA-3;2) 2: 5-GCT ACA TAG GGA GCC TTG TCC T-3;3): 5-R6G-CAA TCC ACA CAA ACG CAC GCG TA-BHQ1-3;5.3.2.5 NK 603: ) : 1) 1: 5-ATG AAT GAC CTC GAG TAA GCT TGT TAA-3;2) 2: 5-AAG AGA TAA CAG GAT CCA CTC AAA CAC T-3;3): 5-FAM-TGG TAC

8、 CAC GCG ACA CAC TTC CAC TC-BHQ1-3;) (adhl 1): 1) 1: 5-CCA GCC TCA TGG CCA AAG-3;2) 2: 5-CCT TCT TGG CGG CTT ATC TG-3;3): 5-R6G-CTT AGG GGC AGA CTC CCG TGT TCC CT-BHQ1-3;5.3.2.6 Bt 11: ) : 1) 1: 5-GCG GAA CCC CTA TTT GTT TA-3;2) 2: 5-TCC AAG AAT CCC TCC ATG AG-3;3): 5-FAM-AAA TAC ATT CAA ATA TGT ATC

9、 CGC TCA-BHQ1-3;) (adhl 2): 1) 1: 5-CGT CGT TTC CCA TCT CTT CCT CC-3;2) 2: 5-CCA CTC CGA GAC CCT CAG TC-3;3): 5-R6G-AAT CAG GGC TCA TTT TCT CGC TCC TCA-BHQ1-3;5.3.2.7 T 25: ) : 1) 1: 5- ACA AGC GTG TCG TGC TCC AC-3;2) 2: 5- GAC ATG ATA CTC CTT CCA CCG-3;3): 5-FAM-TCA TTG AGT CGT TCC GCC ATT GTC G-BH

10、Q1-3;) (adhl 3): 1) 1: 5-CGT CGT TTC CCA TCT CTT CCT CCT-3;2) 2: 5-CCA CTC CGA GAC CCT CAG TC-3;3): 5-R6G-AAT CAG GGC TCA TTT TCT CGC TCC TCA-BHQ1-3;5.3.2.8 GA 21: ) : 1) 1: 5-CTT ATC GTT ATG CTA TTT GCA ACT TTA GA-3;2) 2: 5-TGG CTC GCG ATC CTC CT-3;3): 5-FAM-CAT ATA CTA ACT CAT ATC TCT TTC TCA ACA

11、GCA CCT GGG-BHQ1-3;) (adhl 4): 1) 1: 5-CCA GCC TCA TGG CCA AAG-3;2) 2: 5-CCT TCC TTG GCG GCT TAT CTG-3;3): 5-R6G-CTT AGG GGC AGA CTC CCG TGT TCC CT-BHQ1-3;5.3.2.9 MIR 604: ) : 1) 1: 5-GCG CAC GCA ATT CAA CAG-3;2) 2: 5-GGT CAT AAC GTC ACT CCC TTA ATT CT-3;3): 5-FAM-AGG CGG GAA ACG ACA ATC TGA TCA TG-

12、BHQ1-3;) (adhl 5): 1) 1: 5-CGT CGT TTC CCA TCT CTT CCT CC-3;2) 2: 5-CCA CTC CGA GAC CCT CAG TC-3;3): 5-R6G-AAT CAG GGC TCA TTT TCT CGC TCC TCA-BHQ1-3;5.3.2.10 MON 863: ) : 1) 1: 5-GTA GGA TCG GAA AGC TTG GTA C -3;2) 2: 5-TGT TAC GGC CTA AAT GCT GAA CT-3;3): 5-FAM-TGA ACA CCC ATC CGA ACA AGT AGG GTC

13、A-BHQ1-3; 560582014 4 ) (adhl 6): 1) 1: 5-CCA GCC TCA TGG CCA AAG-3;2) 2: 5-CCT TCT TGG CGG CTT ATC TG-3;3): 5-R6G-CTT AGG GGC AGA CTC CCG TGT TCC CT-BHQ1-3.6 6.1 - (Real Time PCR) - , , - , , - , . 6.2 53244. 7 , , - 55576. 8 52173 55576. 9 - (Real Time PCR) 9.1 - - 1 3 , 1 3 1 5.3.2 5 10-6 /3, 1 3

14、 2 5.3.2 5 10-6 /3, 1 3 5.3.2 3 10-6 /3, 3 3 () - 1,5 3. - 18 12 . 9.2 9.2.1 , ( ), - , . 9.2.2 10 3 - 9.1, 5 3 - 0,5 3 (TaqF). - 1530 . 9.2.3 0,2 3 15 3 , 9.2.2, -, , 10 3 , - (-) 8. 25 3.9.2.4 : - (-) - 9.2.2 10 3 -;- (+) - 9.2.2 10 3 1 %- .9.2.5 - . 9.2.2 10 3 , 0,1 % 5,0 % . . -. 9.3 . 1. 560582014 5 1 95 /5 45 95 /15 60 /30 72 /30 60 FAM/Green, JOE/Yellow. 2. 2 95 /15 10 95 /15 60 /20 72 /15 35 95 /15 55 /20 72 /15 55