KS J ISO 21571-2006 Foodstuffs－Methods of analysis for the detection of genetically modified organisms and derived products－Nucleic acid extraction《食品 基因改良有机物及其衍生产品检测的分析方法 核酸萃取》.pdf

1、KSKSKSKS SKSKSKS KSKSKS SKSKS KSKS SKS KS 2006 12 28 http:/www.kats.go.krKS J ISO 21571 KS J ISO 21571： 2006 (2011 ) J ISO 21571： 2006 : ( ) ( ) ( ) ( ) : (http:/www.standard.go.kr) ： ：2006 12 28 ：2011 12 29 2011-0652 ： ： ( 02-509-7273) (http:/www.kats.go.kr). 10 5 , . ICS 67.050 KS J ISO 21571： 200

2、6(2011 )Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Nucleic acid extraction 2005 1 ISO 21571 Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Nucleic acid extraction . 1. DNA / . A B .

3、. KS H ISO 21569 KS H ISO 21570 . 2. . . KS H ISO 24276 ISO 21568 Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Sampling 3. 3.1 (KS H ISO 24276). DNA DNA , , DNA ( , DNA DNA , DNA , ) . . 3.2 DNA DNA ( ) DNA , PCR DNA . DNA A , . 3.3 DNA DNA

4、PCR . DNA ( ), ( ), ( ) PCR . DNA DNA , DNA . B DNA . J ISO 21571： 2006 2 , DNA . 4. DNA . DNA , a) b) (forward flow) . DNA PCR DNA . 5. 5.1 5.1.1 ( ) DNA . DNA . . ( 0.1 % 3 000 ) . / 2 g . . A DNA DNA 200 500 mg . DNA DNA . DNA 2 . , KS H ISO 24276 . 5.1.2 ( , , , ) KS H ISO 24276 . . . , , . , DN

5、A . . DNA ISO 21568 . , ISO 21568 . . , . (hexane) . J ISO 21571： 2006 3 . 40 20 . . , DNA . , . . , DNA , , , A . , ( ) DNA . 5.2 DNA 5.2.1 . DNA ( , , , ) (CTAB/chloroform) ( , , , , ) . NA (RNase), (proteinase) RNA . (n ) . / . , / DNA . DNA , , , ( ) , / , , . DNA , . DNA PEG t RNA DNA . PCR , P

6、CR PCR . (carrier) DNA DNA . DNA , . DNA DNA . DNA A , DNA . DNA DNA . J ISO 21571： 2006 4 DNA DNA , DNA DNA . DNA , . DNA (trans-acting) PCR (KS H ISO 24276, KS H ISO 21569, KS H ISO 21570 ). DNA DNA . 5.2.2 KS H ISO 24276 1 . , . 5.2.3 DNA ：PCR DNA DNA PCR DNA (KS H ISO 24276, KS H 21570) . DNA PC

7、R . . , PCR . DNA . , . 5.3 DNA 5.3.1 , , . . DNA . DNA ( ) . 5.3.2 (dynamic range) . 5.3.3 (standards) . , / . / . (KS A ISO Guide 30). (intercalating agent) , DNA DNA . DNA DNA . DNA . DNA DNA ( ), DNA . , DNA . , 3 ( , ) . DNA . J ISO 21571： 2006 5 5.4 DNA DNA . DNA . 6. DNA . ( , PCR , PCR DNA )

8、. KS H ISO 21569, KS H 21570, KS H 24276 . 7. KS H ISO 24276 . (derivation), (raw data) KS A ISO/IEC 17025 . . J ISO 21571： 2006 6 A() DNA A.1 / DNA ：PCR DNA A.1.1 / A.1.1.1 ( 5) DNA (A.1.1.8 ). . ( , ) , PCR DNA . , PCR DNA . , CTAB PVP, DNA . A.1.1.2 40 , , , GMO . A.1.1.3 (SDS) EDTA ( ) , , DNA .

9、 DNA , . 5. A.1.1.4 . A.1.1.5 A.1.1.5.1 (ethanol) 96 %, 20 A.1.1.5.2 (glacial acetic acid) A.1.1.5.3 (potassium acetate) A.1.1.5.4 (hydrochloric acid) A.1.1.5.5 (isoamyl alcohol) A.1.1.5.6 (phenol) A.1.1.5.7 (chloroform) A.1.1.5.8 () () (Tris(hydroxymethyl) aminomethane(Tris) A.1.1.5.9 EDTA(K2EDTA)

10、A.1.1.5.10 (potassium hydroxide) A.1.1.5.11 (potassium chloride) A.1.1.5.12 (sodium dodecyl sulfate) A.1.1.5.13 (proteinase) K 20 Units/mg A.1.1.5.14 RNaseA DNase-free, , 50 kunits/mg A.1.1.5.15 (equilibrated phenol) pH 7.8, SDS . A.1.1.5.16 24 . A.1.1.5.17 . A.1.1.5.18 / ( ) 0.050 mol/L, K2EDTA 0.0

11、50 mol/L, (SDS) J ISO 21571： 2006 7 30 g/L, pH 8.0 A.1.1.5.19 TE 0.010 mol/L, K2EDTA 0.001 mol/L, pH 8.0 A.1.1.5.20 (proteinase)K 20 mg/L, 20 , / . A.1.1.5.21 RNaseA 10 mg/mL , 20 , / . A.1.1.5.22 volume fraction 70 %, 20 A.1.1.5.23 3 mol/L, pH 5.2 , 0.22 m . A.1.1.6 A.1.1.6.1 10 000g , . A.1.1.6.2

12、60 70 . A.1.1.6.3 () A.1.1.6.4 () A.1.1.6.5 (1) (1) . , ISO . . A.1.1.6.6 . A.1.1.7 A.1.1.7.1 , DNA . , ( ) . A.1.1.7.2 0.25 g . 1.6 mL 50 L K . 30 2 60 70 , RNase A 0.1 g/mL 5 000g 30 , . . 5 000g 15 . (phase) . / . 5 000g 10 . . 0.1 2.5 96 % . 5 ( 80 1 20 ) , 10 000g, 4 15 . DNA 2 70 % 10 000g 13

13、000g 4 15 , . PCR . DNA TE 100 L DNA RNase A 0.1 g/mL . A.1.1.8 (2) . J ISO 21571： 2006 8 (2), , (2), (2), , , / (pate)(2), (2), , (2), , , (2), , , , , (2), (2), , , (2), , (2), , (2), (maize feeding oil cake), , (feed), , , (meat), (2) ( , , , ), (fruit pulp), , , (2), , , , , , (glupacolza), , (2

14、), (2)(A.1.2 ), , sooie hoi sin(2), soup ball, (2), (2), , , , , , , (2), , ( ), ( ), , , , , (2), , , ( )(2), , , , , , (2)(A.1.3 ) (2) (batch) . , DNA DNA PCR . . A.1.2 / ： A.1.2.1 DNA DNA . PCR DNA DNA , summer sausage7 6 . , 8 S. aureus DNA . A.1.2.2 0.4 g . A.1.2.3 . . , . SDS K ( ) . / PCR ( )

15、 . DNA . A.1.2.4 . A.1.2.5 A.1.2.5.1 (isopropanol) A.1.2.5.2 (ethanol) 96 %, 20 A.1.2.5.3 (glacial acetic acid ) A.1.2.5.4 (hydrochloric acid) A.1.2.5.5 (sodium hydroxide) A.1.2.5.6 (isoamyl alcohol) A.1.2.5.7 (phenol) A.1.2.5.8 (chloroform) A.1.2.5.9 () ()Tris(hydroxymethyl) aminometh ane(Tris) J I

16、SO 21571： 2006 9 A.1.2.5.10 EDTA(Na2EDTA) A.1.2.5.11 (sodium dodecyl sulfate) A.1.2.5.12 50 000 Unit/mg 1 U Micrococcus lysodeikticus pH 6.24, 25 1 0.001 (450 nm) . A.1.2.5.13 A.1.2.5.14 (proteinase) K 20 Unit/mg A.1.2.5.15 (sodium acetate) A.1.2.5.16 (equilibrated phenol) pH 7.8, SDS . A.1.2.5.17 (chlorofo