平成26年11月20日 厚生労働省医薬食品局食品安全部監視安 …

1 1120 1 26 11 20 ( ) O26 O103 O111 O121 O145 O157 24 12 17 1217 O26 O111 O157 O103 O121 O145 O 1 O26 0103 O111 O121 0145 O157

2 O26 0103 O111 O121 0145 O157 VT O 200 cm 1 25 VT O 225 ml 42 1 C 22 VT O VT O 10 ml C (-75 C ) DMSO 10 mEC ( )

3 G g g 1 2004 2 K2 HPO4 KH2PO4 NaCl pH g g g 1,000 ml 121 C 15 mEC (USDA )( ) g (Bile salts ) g K2 HPO4 KH2PO4 NaCl pH g g g g 1,000 ml 121 C 15 mEC 36 1 C mEC 42 1 C DNA DNA.

4 VT DNA DNA DNA DNA VT . ml 10,000Xg 10 50 mM NaOH 85l 100 C 10 M Tris-HCl(pH )15 l 2,000 10,000Xg 10 VT DNA 3 C PrepMan Ultra Sample Preparation Reagent DNeasy Blood & Tissue Kit High Pure PCR Template Preparation Kit NucleoSpin Tissue (.)

5 VT DNA VT VT VT O O Real-time PCR VT CycleavePCR O-157 VT gene Screening Kit Ver. CY217A CY217B Thermal Cycler Dice Real Time System II applied biosystems 7500 applied biosystems 7500 Fast applied biosystems 7900HT applied biosystems ViiA7 LightCycler 480II applied biosystems 7500 applied biosystems 7500 Fast applied biosystems 7900HT applied biosystems ViiA7 manual baseline threshold LightCycler 480II Abs Quant/2nd Derivative Max auto Abs

6 Quant/Fit Points Background 4-10 cycle foodproof STEC 3216D60211 5'- applied biosystems 7500 applied biosystems 7500 Fast applied biosystems ViiA7 LightCycler 480II ROX eae ROX applied biosystems 7500 applied biosystems 7500 Fast applied biosystems ViiA7 auto 4 threshold LightCycler 480II Abs Quant/2nd Derivative Max auto Abs Quant/Fit Points background 3-15 cycle VT1 VT2 Nielsen Clin.

7 Microbiol. 41:2884-2893 2003 16 SrRNA United States Department of Agriculture USDA MLG 5B Appendix PCR 5'- . applied biosystems 7500 applied biosystems 7500 Fast applied biosystems 7900HT applied biosystems ViiA7 LightCycler 480II Thermal Cycler Dice Real Time System II ABI PRISM 7500 Fast standard chemistry . TaqMan Environmental Master Mix Product No. 4396838.

8 L . DNA l l . 50 C 95 C 10 95 C 15 60 C 1 45 .. applied biosystems 7500 applied biosystems 7500 Fast applied biosystems 7900HT applied biosystems ViiA7 auto threshold baseline 3-10 cycle Ct LightCycler 480II Abs Quant/2nd Derivative Max auto Abs Quant/Fit Points background 4-10 cycle Thermal Cycler Dice Real Time System II CP auto l 5 TaqMan Environmental Master Mix l

9 VT1-F (50 pmol/l) l VT1-R (50 pmol/l) l VT2-F (50 pmol/l) l VT2-R (50 pmol/l) l 16 SrRNA-F (20 pmol/l) l 16 SrRNA-R (20 pmol/l) l ( pmol/l) VT1-P l VT2-P l 16 SrRNA-P l l VT1-F: 5 -GGA TAA TTT GTT TGC AGT TGA TGT C-3 VT1-R: 5 -CAA ATC CTG TCA CAT ATA AAT TAT TTC GT-3 VT1-P: 5 -FAM-CCG TAG ATT ATT AAA CCG CCC TTC CTC TGG A-BHQ1-3 VT2-F: 5 -GGG CAG TTA TTT TGC TGT GGA-3 VT2-R: 5 -GAA AGT ATT TGT TGC CGT ATT AAC GA-3 VT2-P: 5 -FAM-ATG TCT ATC AGG CGC GTT TTG ACC ATC TT-BHQ1-3 16 SrRNA-F: 5 -CCT CTT GCC ATC GGA TGT G-3 16 SrRNA-R: 5 -GGC TGG TCA TCC TCT CAG ACC-3 16 SrRNA-P.

10 5 -HEX-GTG GGG TAA CGG CTC ACC TAG GCG AC-BHQ1-3 applied biosystems 7500 7500 Fast 7900HT ViiA7 FAM-BHQ1 FAM-None HEX-BHQ1 VIC-None LC480 FAM-BHQ1 Excitation 465 nm Emission 510 nm HEX-BHQ1 Excitation 533 nm Emission 580 nm DiceII FAM-BHQ1 FAM HEX-BHQ1 HEX LightCycler Nano applied biosystems 7000 7300 7700 VT1 VT2 16 SrRNA simplex FAM l TaqMan Environmental Master Mix l VT1-F (50 pmol/l) l VT1-R (50 pmol/l) l 6 VT2-F (50 pmol/l) l VT2-R (50 pmol/l) l ( pmol/l) VT1-P l VT2-P l l l TaqMan Environmental Master Mix l 16 SrRNA-F (20 pmol/l) l 16 SrRNA-R (20 pmol/l) l ( pmol/l)