ヨーネ病検査マニュアル (2014年10月17日版)[PDFファイル]
Contents
1. 24 4 1 5 ml 5 6 PCR 96 well 3 1 DNA DNA HS B DNA ver2 DNA HH HH 1 337v v TOZ Hoa AED 0 DNA 1 2 mL 0 3mL 1 5 g N SOEUR 3Ek3 5 3EfB 1 g 20ml2. 1 2 BHT B 0 1 2 BH 1 BHI B 1H 20 SE BE TUBETE Z2 SCA Stabel J Vet Diagn Invest 9 375 380 1997 II 1 Middlebrook 7H10 7H10MEY 1 2 7H10MEY Middlebrook 7H10 Agar medium with mycobactin and egg yo
3. 1 1 10cm 2 30cm 3 BI E EB 2 55 50cm 4 He 2 5 Im 5 6 EIA Y v7 ff 7 8 iila 9 10cm 50ml nuoc ou LU NM 2 2 SFZ Tn y ZER Ut UR 2 3
4. 13 000 rpm 5 PCR DNA BioRad InstaGene DNA ii PCR 2 6 Ev ZO3 XBBSEASOu AS 2e iude d OE PCR t Jed A T 3 DNA 4 7 1 1
5. 30 1 mL 3 10 000 rm 5 THER 30 ES 4 InL 10 000rpm 5 AOTH TD H 5 700u L 1 1 700 L 6
6. 10 20 c m 4 2 24 24 5 170ul 1 50 DNA PCR IS900 DNA OR i 100 8 D A 2
7. 1 10 000rpm 5 1 Jv350u L Co OTH L lt RR 350 L 1 5mL 15 000rpm 10 10 15 000rpm 3 15 000rpm 30 1 E JC D ES GB 709 1m RET D F 70 A 11 TE p
8. 1 4 5 20 000 x g 5min 450 1 1 5mL 6 Buffer C 450 1 Buffer B 300 1 TERRE le SERTI 7 15 000 x g 1 8 Buffer D 50041 LET 15 000 x g 1 i 1 5mL 9 Buffer E 50u 1 15 000 x g 1 a a 2O HE JER amp E
9. PCR 50C Cycle of A 1 000 E ER E GS RE 0 001pg 2 5 0 9 12 FAC FE 5 SERIE ORD e 2 PCR Mx3000p 88 C 4 1 5 C ABI7300 ABI7500 ABI7500Fast 87 C 1 5 C ABI7900HT StepOnePlus Dice TP800 87C 1 5 C iCycler 91 C c 1 5 C
10. DNA Reaction PCR 1S900 Z Z Mycobacterium sp 2333 E PCR IS900 PCR 1H IS TUE PRI IS Polymerase Chain 15 20 Green IS900 DNA Mycobacterium sp 2333 1989 94 IS Englund 2002 EMG 1
11. 15 000 x g 1 l Buffer D 500 u l 15 000 x g 1 l 1 5mL l Buffer E 50 gl l 15 000 x g 1 l DNA l PCR DNA 15 1 Takara DEXPAT Y Code TKR 9091 DNA 1 2 DE 3 10 4 12 v v v DNA fill 5 10um 1 5 1 5 ml XPAT 0 5 ml 0 10 000 rpm 10 5 DNA 2 DNA 5 10um 1 5 iml Voltex 37 5 1 x 2 E 12 000rpm 5 100 1ml Voltex 1 x 2 12 000rpm 5 DNA
12. E 18900 SH DNA SYBR Green I Eo T S NEZAR DNA PCR E H DNA SYBR Green I Ed 1 PCR ofh 1H PCR JL ER Haie H3 5 M CEU PCR DNA Z a 2 ES PCR X 1 DNA a
13. 1 DNA DNA 0 0025cc DNA 0 001pg 4 WEDO DNA PCR DNA DNA TOR 5 PCR 1 DNA PCR 2 PCR DNA PCR 3 PCR 4 5
14. 50 90gL 10 EBERT S KOT 10 DNA 45 L DNA 5zL PCR 96 1 Bul 2 0 2mLPCR 1 Bul 2 DNA 5zL 45 L 25 xL DNA TE 5L 25 L 2 PCR i d PCR Jj f RR ERN 50C 95 C 95 C l 68 C 60 C 98 C 3 PCR
15. PCR 7 LAMP i a LAMP Loopampe DNA Loopampe b UV LAMP c DNA BioRad InstaGene d i DNA 6 i DNA ii LAMP LAMP 2x Reaction Mix 12 5 gal Primer Mix Map 2 0 Bst DNA Polymerase 1 0 Fluorescent Detection Reagent Map i 0 D W 2 0 DNA 5 0 Total db LAMP 66 C 45 8090 527 UI
16. 2 H 5 3 1 HIA 2
17. 4600rpm 3 v 10 00 Orpm 5 v 15 000rpm 10 nm bf v 15 000rpm 3 70 1ml D b bof 50ml TE buffer DW 16 AHIRE D gt D 00 a KARHE TIBAR 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 60 2 115 120 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 38 8 3048 3054 17
18. I EoLA 1 E 1g 20 ml OJATSAEZESUIRZK CARN UL WL IR GOHREP 5 2 30 5ml 20ml 0 75 EL HPC HPC 1 2 Brain Heart Infusion BHT DIFCO Code 237300 3 16 18 37C 4 900g 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 l 2 B 1 2 BH I
19. 1 4 600 rpm 3 4 000 rpm 5 1 500 rpm 30 1 500 rpm 15 4 600 rpm 3 Pug BU peu 6 m sec 90 f 45 fb x2 24 5 000 rpm 90 x 2 2 PCR 1 QD 1 11 25 00uL 10 1 0 25L 11 1 0 25 uL N 1 unit uL 0 50uL 19 00uL 45 00 uL DNA TE DNA10L TE fX fri 90 u L 1pg 2 5u L7 0 001pg 2 5u L 10 TE RE Du LO 100gL 200ugL DNA 10 pL
20. DNA 20001 Vortex 56C 15 N 3 EH 100C 8 10 000 rpm 5 PCR ii PCR PCR KS PCR Master Mix 25 ul Primer 10 1 100 pmol 1 0 25 Primer 11 1 100 pmol 1 0 25 D W 19 5 DNA 5 Total 50 QuantiTect SYBR Green PCR Kit Cat No 204143 PCR 95 C 15 PCR 959C 30 1 35 cycles 689C 1 l Melt curve ERO PCR PCR Melt curve 13 2 PCR PCR
21. DNA lpg 2 5L l threshold Ct 24 3 45 ul 2R DNA PCR Effi 1 Ct DNA 1 45 1 ED PR zn HX J R ciency 80 1209 R UNG Effe 95 30 68 C 60 98C Melt curve Dissociation curve PCR Efficiency PCR 10 Slope DNA PCR sel 1 x 100 X DNA 1og10 Y dh d Ct
22. 6 DNA PCR 6 Xp TEL amp FAX 029 838 7857 Mail yamoriQaffrc go jp 14 DNA 1g 20mL 5 10 l ALN 202 888 NN d l 1mL 0 10 2mL ie 10 000 rpm5 piss T Voltex EE E bd D AAI 700u Un mor 10 000rpm 5 m Il TR 102 m 70 1ml 3 mm
23. Cf ERMO TZ A Zid T 3UCHPBBSR IC 5 7 HERD REZAD E 7L rio TT 37 a E NN MC P dE 5 4 Tm nec i cou pP C DNA PCR LAMP 5 6 PCR i a QuantiTect SYBR Green PCR Kit Cat No 204143 KS b MP10 1 5 gt ATGCGCCACGACTTGCAGCCT 3 mer 21 MP11 1 5 gt GGCACGGCTCTTGTTGTAGTCG 3 mer 22 c DNA BioRad InstaGene d e PCR ii DNA InstaGenem 1 10 000 rpm5
24. Chromo4 87C 1 5 C CFX96 86 5 C 1 5 C LightCycler 480 LightCycler 96 88 C 1 5 LightCycler Nano DNA 4 1 DNA DNA DNA DNA DNA DNA DNA 0 001pg 2 5zL
25. 2 Threshold cycle Ct Bd 3 A DNA Ct DNA 3 B DNA gt PCR I BE an EX XE Lu 2 DNA 80 PCR Ct J NMMWMOKOME2KMN dk2K RM e PCR LL 4 TTT L L L L L L L L L L LL 1 11 pL p p s 9 SYBR Green E TTTT HERE PETINE TTT LL TTTTXYXTIS TT TTT B 1 SYBR Green I PCR 3 V T
26. 8 500 ul 1 5 ml 9 1 400 ul 10 12 E 0 9 900 600 ul ii 12 1 5 ml 11 ul 13KXg 30 WEIV 10 EV 13KXg 60 13 50 1 V 3 14 13KXg 60 3 e DNA 1 WIREK 1g 20 w v 30 1mL 2 20 000 x g 2 3 2 Buffer A 30041
27. 30 1 m Il TE ms e eA ver2W w DNA PCR IHE 2E OD 20 000xg 5 1 600l 6l 5 75 20 000xg 5 500 1 5mL 400 900 x 13 000xg 60 V 600 13 000xg 60 1 5mL 50l 3 13 000xg 60 DNA Y PCR l 20 000 x g 2 l Buffer A 300 l Buffer B 300 u IRH UIRE l 20 000 x g 5 l 450 1 5mL l Buffer C 450l l
28. 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 Cycles 45 B RSq 0 991 xm Y 3 482 LOG X 23 75 v Qt 7007 d ps cem 2 DNA 1e 05 0 0001 0 001 0 01 0 1 1 10 Initial Quantity IS900 DNA og G3 Ct IS900DNA A DNA 10 well 1 0 001 pg DNA B DNA Ct Y 3 482 Log 23 75 DNA O 2 SANE mA DW Ej DRR REA 1 KS r ud 2 3 DNA ver2 4 DNA
29. ARH Z HOD a ARRERA 1 BD BACTEC MGIT ParaTB Medium Code 245154 MGIT ParaTB Medium ParaTB supplement 509 BD BACTEC MGIT Para TB Supplement Code 245156 50 Vancomycin Nali
30. e QE REB 5 6 T 8 9 TE Tris HC1 pH8 0 10 mM EDTA 1 mM 1 PCR PCR 1 PCR 2 2 mL 3 Mx3000p ABI7300 ABI7500 ABI7500Fast ABI7900HT StepOnePlus Dice TP800 iCycler Chromo4 CFX96 LightCycler 480 LightCycler 96 LightCycler Nano Code 72 693 100 ZH
31. 2014 10 17 H JE EYILAIYJV 1 5 2 HH 3 1 WAED EC EUR Au A Ls Enn d 2 1 2 4 5 3 4 5 6 1 7 2
32. a DNA NX D W 50u L 20C 10 2 ver2 Hv 2 ED 00 DNA fi 1 FRE g 20 ml 30 FRERE 2 2 0 ml Xg 5 3 600 ul MWERI 6 ul 4 3 30 o 1 1 0 ml 20K 1 5 5 5 13KXg 60 6 75 ul 10 12E 7 20KXg 10
33. dixic acid sodium salt Amphotericin B 1 1 5 ml 2 5 Vancomycin 40 ul 60 ul 120 ul 240 ul 2 5 Nalidixic acid 40 ul 60 ul 120 ul 240 ul 1 Amphotericin B 38 ul 57 ul 114 ul 228 ul 50 MRA Bm 25 mg ml 20 C 10 mg ml 20 2 0 1 ml BD BACTEC MGIT ParaTB Mediume 37 3 365 nn UV 400 450nm UV LED E UV
34. lk lL 1 5 3L 1 Middlebrook 7H10 Agar Difco 262710 19 g 28 5 g 57 g 2 5 ml 7 5ml 15 ml 3 750 ml 1 125 ml 2 250 ml 121C 10 55 4 Mycobactin 1ng m1 2 ml 3 ml 6 ml 5 2 Malachite green in DW 2 5 ml 3 7 ml 7 5 ml 6 50 150 m 225 ml 450 ml 7 Middlebrook OADC Enrichment 100 ml 150ml 300 ml BD BBL Code No 211886 8 Vancomycin 25 mg ml 2 ml 3 ml 6 ml 9 Nalidixic acid 25 mg ml 2 ml 3 ml 6 ml 10 Amphotericin B 10 mg ml 5 ml 7 5 ml 15 ml 10m1 tube 1 0 1 ml 2
35. m fi 2 DNA 2 1 1S900 PCR 88 1 i Ct FAR e EGRE SCORE Fluorescence Primary Curve ES e Ct ias d RFU dT lel c RSHA ab ede I 50 52 54 56 56 60 62 64 66 68 70 72 74 76 78 80 82 84 86 88 90 92 94 9 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 82 04 86 88 90 92 94 96 98 TiPeH3HGT CE 5 s T 51 2 NN N t Co re o des quy E Ho dex gb cm 0 2 4 6 8 10 12
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