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ISSN 0255-6162 GPN 2006900031 287 93 11 20 161-2 02 26531318 http://www.nlfd.gov.tw 1

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44 93 13 1 monocrotophos 0.40 ppm 105 1300 99% 6

3 44 106 93 13 1 0.40 ppm 403 40 7

DNA (HPLC) DNA DNA DNA DNA hybridization PCR product sequencing PCR-restriction 8

fragment length polymorphism (PCR-RFLP) species-specific primers PCR PCR-SSCP random amplified polymorphic DNA (RAPDs) real-time PCR TaqMan real-time PCR real-time PCR PCR product sequencing PCR-RFLP GMO 92 8 PCR real-time PCR 酶 (Polymerase chain reaction, PCR) ( ) DNA PCR PCR ( 1) ( ) 1. 260 nm 280 nm 2. (Vortex mixer) 3. DNA 4. 55 5. 20,000 g 4 6. 7. 酶 ABI PRISM 9700 Sequence Detector 8. 酶 ( 2) ABI PRISM 7700 Sequence Detector Roche LightCycler 9. DNA 10. 11. 12. 312 nm 365 nm 13. 14. 15. ph 16. + 1.0 9

17. 2,000 g 0.1 g 100 1 mg 18. 1 2 ( ) 1. DNA RNase (96-100%) DNeasy Tissue 2. 酶 (PCR) ( 3) (1) i myostatin ) F MYF 5 -TTGTGCAAATCCTGAGACTCAT-3 R MYR 5 -ATACCAGTGCCTGGGTTCAT-3 97 bp ii growth hormone) F SWF 5 -TCAGTTTACACTCACCTGATAGCATCT-3 R SWR 5 - GGGTGGTGGAGAGGGGTGAATT-3 108 bp ( 4) (2) i myostatin ) F MYF 5 -TTGTGCAAATCCTGAGACTCAT-3 R MYR 5 -ATACCAGTGCCTGGGTTCAT-3 P MYP 5 -(FAM)-CCCATGAAAGACGGTACAAGGTATACTG -(TAMRA)-3 97 bp ii growth hormone) F SWF 5 -TCAGTTTACACTCACCTGATAGCATCT-3 R SWR 5 - GGGTGGTGGAGAGGGGTGAATT-3 P SWP 5 -(FAM)-CCTCAATACTCCAGAACCCCTCATT TTCCTC-(TAMRA)-3 108 bp 3 20 10

4 20 5 6-carboxy-fluorescein (FAM) 3 6-carboxytetramethyl-rhodamine (TAMRA) (3) 苷 (deoxyribonucleotside triphosphate, dntp) 苷 (deoxyadenosine triphosphate, datp) 苷 (deoxycytidine triphosphate, dctp) 苷 (deoxyguanosine triphosphate, dgtp) 苷 (deoxythymidine triphosphate, dttp) 2.5 mm (4) 酶 Taq DNA polymerase (2 U/µL) (5) TaqMan Universal PCR Master Mix ( ABI PRISM 7700) PCR 苷 酶 DNA (6) LightCycler-FastStart DNA Master Hybridization Probes Roche LightCycler) PCR 苷 酶 DNA 3. (ethidium bromide) (agarose) (bromophenol blue) (xylene cyanol FF) (ethylenediaminetetraacetic acid disodium salt, Na 2 -EDTA) (tris (hydroxymethyl) aminomethane (Tris)) DNA (DNA molecular weight marker) 100-bp DNA Ladder Marker 4. pidm1 ( 5) ( ) 1. (Pipette) 10 µl 20 µl 100 µl 200 µl 1000 µl 2. 3. (Pipette tips) 10 µl 20 µl 200 µl 1000 µl 4. 200 µl 600 µl 1.5 ml 2 ml 5. PCR 200 µl 500 µl 11

6. PCR ( 6) Roche LightCycler 7. 50 ml 100 ml 250 ml 500 ml 1000 ml 2000 ml 8. 50 ml 9. 0.45 µm nitro-cellulose 5 DNase 6 Roche LightCycler ( ) 1. 5 TBE (Tris-borate-EDTA) 54.0 g 27.5 g 0.5 M ph 8.0 EDTA 20.0 ml 1000 5 TBE 0.5 2. 2% 2.0 g 0.5 TBE 100.0 ml 3. 6 (6 gel loading buffer) 25.0 g 0.25 g 30 ml 100 ml 4 4. 0.1 g 10.0 ml 10 mg/ml) 1 µg/ml ( 7) 5. PCR 12

(1) 10 PCR ( 15 mm MgCl 2 )...2.5 Taq DNA polymerase (2 U/µL)...1.0 2.5 mm dntp...4.0 10 µm F...1.0 10 µm R...1.0 DNA ( 100 ng)...5.0...10.5......25.0 µl (2) ABI PRISM 7700 Sequence Detector 5 µm F...1.25 5 µm R...1.25 3.3 µm P...1.7 TaqMan Universal PCR Master Mix...12.5 DNA ( 100 ng)...5.0....3.3... 25.0 µl (3) Roche LightCycler 5 µm F...1.5 5 µm R...1.5 3.3 µm P...1.5 LightCycler-FastStart DNA Master Hybridization Probes......2.0 25 mm MgCl 2...2.4 DNA ( 100 ng)...5.0....6.1...... 20.0 µl 7 PCR ( ) DNA 13

1. ( ) 2. DNA DNeasy Tissue (ATL proteinase K AL (DNeasy spin column) AW1 AW2 AE ) (1) 25 mg ( 8) 2 ml (2) ATL 180 µl proteinase K 20 µl (3) 55 (4) AL 200 µl (5) 70 10 (6) (96-100%) 200 µl (7) 6,000 g (8,000 rpm) 1 (8) AW1 500 µl 6,000 g (8,000 rpm) 1 (9) AW2 500 µl 20,000 g (14,000 rpm) 3 (10) 1.5 ml (11) AE 100 µl 1 6,000 g (8,000 rpm) 1 (12) AE 100 µl 1 6,000 g (8,000 rpm) 1 (13) 200 L 1.5 ml DNA (14) ( )3. DNA -20 8 DNA 10 mg RNA DNA ( ).2.(4). RNase (100 mg/ml) 4 L 2 3. DNA (1) DNA 14

(2) DNA 260 nm 280 nm (O.D.) DNA O.D. 260 50 ng/µl DNA DNA O.D. 260 / O.D. 280 1.7 2.0 ( 9) ( ) 1. PCR 2.5.5.1. PCR DNA PCR ( )5.1. PCR 10 PCR dntp DNA polymerase DNA PCR ( )2. PCR 2. PCR 1. 95 5 min 2. 95 30 sec 3. 60 30 sec 54 30 sec 4. 72 30 sec 2 4 40 5. 72 7 min 3. 6 ( ) PCR 2% 50 100 DNA PCR 15 365 nm DNA 15

4. DNA PCR DNA DNA DNA PCR DNA PCR 108 bp 9 PCR PCR DNA PCR DNA PCR DNA PCR ABI PRISM 9700 ( ) 1. PCR (1) 酶 (Real-time PCR) ABI PRISM 7700 Sequence Detector DNA ( )5.2. Master Mix 20 µl PCR DNA 5 µl PCR 200 g (1,500 rpm) 酶 1. 50 C 2 min 2. 95 C 10 min 3. 95 C 15 sec 4. 60 C 1 min 54 C 1 min 3 4 45 5. 35 C 45 sec 16

(2) 酶 (Real-time PCR) Roche LightCycler 2.5.5.2. PCR DNA ( )5.3. PCR LightCycler-FastStart DNA Master Hybridization Probes 15 µl DNA 5 µl 800 g (3,000 rpm) 酶 1. 95 C 10 min 2. 95 C 5 sec 3. 60 25 sec 54 25 sec 4. 72 C 8 sec 2 4 45 5. 35 C 45 sec 2. 酶 DNA 酶 酶 3. DNA PCR DNA PCR PCR 1. 0.1% 2. DNA DNA 17

酶 (Polymerase chain reaction, PCR) ( ) 12S ribosomal RNA) F BF 5 -ACATTCTCTACCCAAGAGAATCAAGC-3 R BR 5 -TCCTCTCATGTAGCTAGTGCGTTTA-3 193 bp 12S ribosomal RNA) F BF 5 -ACATTCTCTACCCAAGAGAATCAAGC-3 R BR 5 -TCCTCTCATGTAGCTAGTGCGTTTA-3 P BP 5 -(FAM)-CCCTCCTCAAATAGATTCAGTGCATCTAACCCT-(TAMRA)-3 193 bp 酶 (Polymerase chain reaction, PCR) ( ) satellite) F SGF 5 -CCTCTCCAGTGCTGACTTGGA-3 R SGR 5 -AAGCATGACATTGCTGCTAAGTTC-3 123 bp satellite) F SGF 5 -CCTCTCCAGTGCTGACTTGGA-3 R SGR 5 -AAGCATGACATTGCTGCTAAGTTC-3 P SGP 5 -(FAM)-CACGTGCATGCCCCCTCTCGA-(TAMRA)-3 123 bp 酶 (Polymerase chain reaction, PCR) 18

( ) 12S ribosomal RNA) F HosF1 5 -GTTATCCAAGTACACCTTCCGGTAT-3 R HosR1 5 -CGTCACCCTCCTTAAATATCACAAA-3 114 bp 12S ribosomal RNA) F HosF2 5 -GATGGAGAGAAATGGGCTACATTTT-3 R HosR2 5 -ACTGCTAAATCCTCCTTTAGTCTCCAG-3 P HosP 5 -(FAM)-ACCCTAAGAACAAGAACTTTAACCC GGACGA-(TAMRA)-3 99 bp 酶 (Polymerase chain reaction, PCR) ( ) mitochondrial cytochrome b) F DeerF 5 -CATTTATTATCGCAGCACTCGCT-3 R DeerR 5 -AGGTCTGGTACGAATAATACTAGTGAT-3 190 bp mitochondrial cytochrome b) F DeerF 5 -CATTTATTATCGCAGCACTCGCT-3 R DeerR 5 -AGGTCTGGTACGAATAATACTAGTGAT-3 P DeerP 5 -(FAM)-CCACTTACTCTTCCTCCACGAAACAGGA-(TAMRA)-3 190 bp 酶 (Polymerase chain reaction, PCR) 19

( ) 12S ribosomal RNA) F KanF 5 -GAGCTTAATTGAAACAGGCA-3 R KanR 5 -ACTTTTCTCCTCTTTTGTATTCC-3 106 bp 12S ribosomal RNA) F KanF 5 -GAGCTTAATTGAAACAGGCA-3 R KanR 5 -ACTTTTCTCCTCTTTTGTATTCC-3 P KanP 5 -(FAM)-TCCTCGACAAAACCTTAC-(TAMRA)-3 106 bp 1. 2003 (TaqMan ) : DOH92-FD-2076 2. Meyer, R. Candrian, U. and Luthy, J. 1993. Detection of pork in heated meat products by the polymerase chain reaction. J. AOAC Int. 77:617-622. 3. Laube, I., Butschke, A., Zagon, J., Spiegelberg, A., Schauzu, M., Bögl, K. W., Kroh, L. W. and Broll, H. 2002. Detection method to identify beef in foods by the TaqMan TM technology. Bundesgesundheitsblatt Gesundheitsforsch. Gesundheitsschutz. pp.1-25. 4. Chikuni, K., Tabata,T., Kosugiyama, M., Monma M. and Saito, M. 1994. Polymerase chain reaction assay for detection of sheep and goat meats. Meat science. 37:337-345. 20

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