ヨーネ病検査マニュアル - 農業・食品産業技術総合研究機構
Contents
1. II is OD LTS FHC 2 8 1 1 2 32. ParaTB supplement 50 9P HZ 281E RR a BD BACTEC MGIT Para TB Supplement Code 245156 50 ey ancomycin Nalidixic acid sodium salt Amphotericin B 1 1 5 ml a 2 5 Vancomycin 40 ul 60 ul 120 ul 240 ul 25 mg ml 20 10 mg ml 20 2 0 1 ml BD BACTEC MGIT ParaTB Mediume
3. 1 DNA DNA 0 0025cc DNA 0 001pg 4 EDO DNA PCR DNA EE DNA 4 si N H R firas oa v nade 5 PC
4. 2 Threshold cycle Ct 3 A CHS DNA Ct DNA OF B PCR gt TTT LL LL LL LL LL 1 1 1 1 1 1 ut TTT HERE WIIS A TTT T TT ei 1 SYBR Green I PCR I 80 PCR Ct yay WI DNA gt iA iA 2 DNA 1 SYBR Green I TTT oa ee kee S TTT a es ee rit OT Ct PCR V Tm ff 2 AA DNA
5. FE 1 g 20 ml OME RTS APSR LIA 2 0 ml 1 OPFER AER 1 0 ml 20K xg 5 600 ul D 1 DNA A We LD Lie ted H a 9 4 75 ul OF E 13KXg 60 HAI 10 12 HI 6 1 30 30 1 5 5 20KXg 10 Ey 500 ul amp 400 ul amp 9 900 600 ul Bey WM 10 12 ul 13KXg
6. 2 7H10MEY Middlebrook 7H10 Agar medium with mycobactin and egg yolk 2 1 0 1 ml 2 37 5 7 37 sly le Ars OI EN ota 5 4 ee eee DNA PCR 5 6 PCR i 1 QuantiTect SYBR Green PCR Kit Cat No 204143 KS
7. DNA Reaction PCR 1S900 IS900 PCR 94 IS Z Z Mycobacterium sp 2333 1 A EML IS Al TRA IS Polymerase Chain 15 20 Green IS900 DNA Mycobacterium sp 2333 Engl
8. 37 3 DAIZ KER 365 nm 400 450nm UV LED HE UV 10 20 c m A 2 SERGE 24 HONAN Ko TIELT 24 NE A A rtp artn s
9. 5 170ul 1 50 DNA PCR IS900 4 5 2 2 3 1 DNA i 100C8 Be 13 000 rpm 5 PCR DNA DNA BioRad LD InstaGeneY DNA ii PCR 2 6 3 BETO I XA DNA PCR 6
10. 3 10 000 rpm 5 4 1mL 10 000rpm 5 5 700 1 1 700 6 1 10 000rpm 5 7 350 L 1 5mL
11. Melt curve 11 2 PCR PCR PCR III 1 BD BACTEC MGIT UE Medium Code 245154 MGIT Ash Medium
12. 6 PCR 96 well 3 1 DNA DNA DNA 1 DNA 1 2 mL 0 3mL 1 5 g 2 1 g 20ml OPA EPR CARL HL IRATRAET 6 30 30 1 m
13. 50 C Cycle of A 1 000 0 001pg 2 5 0 9 11 DNA 4 1 DNA DNA DNA DNA DNA DNA DNA 0 001pg 2 5zL
14. 1 PCR PCR 1 PCR Mx3000p ABI7300 ABI7500 ABI7500Fast ABI7900HT StepOnePlus Dice TP800 iCycler Chromo4 CFX96 LightCycler 480 LightCycler 96 LightCycler Nano 2 2 mL Code 72 693 100 3 24 4 1 5 ml 5
15. fl E All rau 2 PCR A LightCycler Nano H R iciency 80 120 SG CR as Mx3000p ABI7300 ABI7500 ABI7500Fast ABI7900HT StepOnePlus Dice TP800 iCycler Chromo4 CFX96 LightCycler 480 LightCycler 96 R Ct 88 C 1 5 C 87C 1 5C 87C 1 5C 91C 1 5 C 87C 1 5C 86 5C 1 5 C 88 C 1 5 C E 95C30 68C 60C 98C Melt curve Dissociation curve
16. 2 I 1 E 1g 20 ml 2 30 5ml 20ml 0 75 EL VY HPC HPC 1 2 Brain Heart Infusion BHT DIFCO Code 237300 3 16 18 37C 4 900 g 830 HPC 25 1ml 37 2 vancomycin 50 ug ml nalidixic acid 50 ug ml B amphotericin B 50 ug ml 1 2 BHI Vancomycin Sigma V 2002 Nalidixic acid sodium salt Sigma N 4382 Amphotericin B KK 4 E
17. B 1 2 B H I 1 2 BH I B 1 2 BH I BHI B 1 20C Stabel J Vet Diagn Invest 9 375 380 1997 II 1 1 ote ES KYRYSIM Middlebrook 7H10 7H10MEY
18. 1 AR 2 1 1 10cm 2 30cm 3 50cm 4 EED Im 5 6 7 8 ZE dl 9 10cm 50m lt
19. 5 10 hice FLA 2 Wx bal EEREN TERN gee F 1ml 0 1 0 2 ml Eee ee 20 000xg 5 l 10 000 rpm 5 Vv Voltex HYZ I 600w1 6 ul Vv 10 000 rpm5 700u 700u Vv DNA Vv Vv 5 IH I 75 wl 10 000 rpm 5 20 000xg 5 350u 500u1 1 5ml Fu Vv l WEM 400wl 15 000 rpm 10 Vv 709 1m1l 15 000 rpm 3 70 15 000 rpm 30 1 Vv LT 70
20. zl WA 50ul TE DNA Vv PCR Vv LAW 900ul 13 000xg 60 1V 600ul Vv 13 000xg 60 1 5ml Vv Vv 50m 3 13 000xg 60 Vv DNA PCR xia FLAI C DNA W H DNA PCR 13 1 Takara DEXPAT Y Code TKR 9091 DNA 1 2 DE 3 10 4 12 Vv Vv Vv DD OF MEA DNA 5 10um 1 5 1 5 ml XPAT 0 5 ml 0 10 000 rpm 10 5 DNA 2 DNA WO Le 5 10um 1 5
21. iml Voltex 37 5 1 x 2 E 12 000rpm 5 100 1ml Voltex 1 x 2 12 000rpm 5 DNA 4600rpm 3 v 10 00 Orpm 5 Vv 15 000rpm 10 x IA bai v 15 000rpm 3 70 1ml D ap bai 50ml TE buffer DW 14 1 Plante Y Remenda BW Chelack BJ Haines DM 1996 Detection of Mycobacterium paratuberculosis in formalin fixed paraffin embedded tissues by the polymerase chain reaction Can J Vet Res 1996 Apr 60 2 115 20 2 Coetsier C Vannuffel P Blondeel N Denef JF Cocito C Gala JL 2000 Duplex PCR for differential identification of Mycobacterium bovis M avium and M avium subsp paratuberculosis in formalin fixed paraffin embedded tissues from cattle J Clin Microbiol 2000 Aug 38 8 3048 54 15
22. 2 1S900 PCR 88 1 RENZ 1 X3 B S BR Fluorescence Primary Curve ss ao Ct ias d RFU dT IEE RSHA ab HB 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 BD 62 84 86 88 90 92 94 9 52 54 56 58 60 62 64 66 68 70 72 7476 78 6082 64 86 88 90 92 9496 98 ere eaae d T 2 yo N WwW WwW e Tl A I Sf 2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 Cycles 45 p RSq 0 991 Kres Y 3 482 LOG
23. 1pg 2 5 prL 0 001 1 pg 2 100gzL 200uL F 5pL EDNA10L TE EC 10 TERK IO UL DNA 10 HAV 3 DHS CHET A 13Kxg 60 L WE 90 45 x2 90L gL 50 90L 10 10 45uL cee FH 96 1 D H H DNA 5gL 25guL 2 E DNA 5uL k lt htt m ETS PTS KOT E DNA 1 Bul Po RIZ 0 2mLPCR lt HERP TS PCR x 45 L
24. 10 FUV 1 5 ml H CUR 1 5m 50 ul D RH 1 24 PCR 10 1 11 1 N 4 600 4 000 1 500 1 500 4 600 6 m sec 5 000 rpm 3 5 30 15 3 90 ZH RTS 30 WUE Bets 13KXg 60 Zg x 2 25 00 uL 0 25L 0 25L 1 unit uL 0 50uL 19 00 u L 45 00 u L GL F DNA TE
25. 4 PCR BE ii DNA InstaGene 1 10 000 rpm5 DNA 200u1 Vortex 56C 15 real N 100 C 8 i 10 000 rpm 5 Y PCR iii PCR PCR PCR Master Mix Primer 10 1 100 pmol u 1 Primer 11 1 100 pmol u 1 D W ES DNA Total CC PAE EK amp FAR KS 25 ul 0 25 0 25 19 5 50 QuantiTect SYBR Green PCR Kit Cat No 204143 Ley PCR 95 C 15 PCR J 95 C 30 JT 45 cycles 68 C 1 J Melt curve PCR PCR
26. lt H 25u L A TE 5zL 25L Al 10 2 PCR 3 PCR DNA pg 2 5L threshold Ct 24 3 D wl 2R DNA Ct D f PCR 50 2 45 98 C 1 gt co JOT PCR UNG ALH ne 95 15 DNA 1 45 PCR Eff PCR Efficiency PCR PCR 10 Estel 1 x 100 Sl1ope DNA 1og10 Y h A
27. ot EMA es Te CEN Lees mes Ab ODUM TD es 8 9 709 1m 10 1 TREE o MPZ FF 15 000rpm 3 15 000rpm 30 1 70 DNA TE MK SUIT D W 50 E 20C DNA 3 2 1 ICO eNO 11 12 13 14 2 1 1
28. 2 MP10 1 5 ATGCGCCACGACTTGCAGCCT 3 mer 21 MP11 1 5 GGCACGGCTCTTGTTGTAGTCG 3 mer 22 1 Middlebrook 7H10 Agar Difco 262710 2 3 121C 10 4 Mycobactin 1ng 5 2 Malachite green in DW Hi 6 50 7 Middlebrook OADC Enrichment BD BBL Code No 8 Vancomycin 9 Naladixic acid 10 Amphotericin B 211886 25 mg ml Ha T 1 25 mg ml 10 mg ml 55 th N 1IL 1 5 E 3L 19 g 28 5 g 57 g 5 ml 7 5 ml 15 ml 750 ml 1 125 ml 2 250 ml 2 ml 3 ml 6 ml 2 5 ml 3 7 ml 7 5 ml 150 ml 225 ml 450 ml 100 ml 150m1 300 ml 2 ml 3 ml 6 ml 2 ml 3 ml 6 ml 5 ml 7 5 ml 15 ml 10ml tube 3 DNA BioRad InstaGene 3
29. X 23 75 8 Qt lt F DNA 1e 05 0 0001 0 001 0 01 0 1 1 10 Initial Quantity IS900 DNA log M3 AbyvaveR 47 iE CUED BEKO BRIT EA BEY IS900DNA A DNA 10 well 1 0 001 pg DNA B DNA Ct Y 3 482 Log 23 75 DNA O ER 2 Pak HAO UE 1 KS 2 DNA 3 4 5 6 7 8 TE Tris HC1 pH8 0 10 mM EDTA 1 mM
30. 2013 03 29 1 5 2 HH 3 1 BE SE OD ELE hupa ls EAT D 1 2 4 5 3 4 5 6 1 7
31. R 1 DNA PCR 2 PCR DNA PCR 3 PCR PCR 4 5 6 DNA 6 TEL amp FAX 029 838 7857 Mail yamori affrc go jp 12 WE le 20m1 CAN l HL lt HE 30 l E Sista ELA D gt SD DNA
32. und 2002 E PCR A 1989 PCR 1 18900 I DNA SYBR Green I DNA SYBR Green I DNA 1 PCR PCR PCR DNA bik 1 DNA a 2
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