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通知法収載
2012
24 3 2
8
0302 2 4
19 3
30
LAMP
0330006
≪
≫
P.3
P.4
P.5
P.6,7
P.8
LAMP
P.9
LAMP
P.10,11
P.12,13
P.14,15
P.14
P.15
2
≪
≫
◆
Cryptosporidium
Giardia
Cryptosporidium
18S rRNA
LAMP
Giardia
DNA
DNA
Cryptosporidium
1 1.5
Giardia
◆
•
• LAMP
•
1
◆
DNA RNA
RT
RT
RNA
cDNA
Loopamp
cDNA
LAMP
DNA
3
≪
≫
RT
①
○
Dynabeads GC-Combo
②
○
③
○
1.5mL
④
○
⑤
○
⑥
○
⑦
⑧
⑨
1.5
1.5mL
1.5
-80℃
○
○
○
○
○
○
○
⑩
○
⑪
○
⑫
○
○
○
○
⑬
⑭
⑮
2mL
○
0.2mL
○
0.2mL8
⑯
○
①
②
③
④
⑤
⑥
⑦
⑧
⑨
⑩
⑪
⑫⑬
⑫⑭
4
⑮
⑯
≪
≫
◆
Dynabeads GCGC-Combo
DB73002
Dynabeads GC-Combo
1 kit 10tests
2
8℃
DB73012
Dynabeads GC-Combo
1 kit 50tests
2
8℃
◆
1%PET
PET
100
PET
1L
10
EDTA3Na
Tween80
20
1N HCl
30
800mL
1N NaOH
10
◆ 1×Proteinase K
20℃
20℃
1 test
600mAU/mL Proteinase K
1.5mAU/mL
0.125µL
1M NaCl
20mM
1.00µL
10% Triton X-100
0.1%
0.50µL
1M DTT
2mM
0.10µL
QIAGEN
48.275µL
TE Tris EDTA pH8.0
50µL
RT
◆ RT
pH7.4
1L
PrimeScript ® RT Master Mix
LAMP
◆ LAMP
Loopamp
Loopamp
5
≪
≫
Dynabeads GC Combo
10×
10×
A
B
1mL
1mL
1
Dynabeads anti-Cryptosporidium 100µL
Dynabeads anti-Giardia
100µ
1 /
2
オーシスト、シストの濃縮と精製
PET
①②10mL×2
③④ 2mL×2
⑤ 10mL×1
1×
A
5
10
1mL
3
1.5mL
1.5mL
180
1 /
1
1.5mL
1.5mL
-80℃⇔37℃
5
1.5mL
P15
1×Proteinase K
50µL
核酸の抽出
60℃ 30
2
75℃ 10
95℃
5
1.5mL
LAMP
RT
RT
6
≪
≫
● Loopamp
1.5mL
RT
PrimeScript ® RT Master Mix
Master Mix 2µL
RNase free dH2O 5µL
R
T
3µL
37℃ 15
反応
85℃ 5
1.5mL
LAMP
Inomata, A., et al. : Development and evaluation of a reverse transcription-loop-mediated isothermal
amplification assay for rapid and high-sensitive detection of Cryptosporidium in water samples,
Water Sci. Technol., Vol.60, 2167-2172 (2009)
7
≪
≫
Dynabeads GC-Combo
LAMP
LAMP
1.
1
→
1 /
2
2.
3.
1%PET
2mL
10mL PET 2
10mL PET 1
2mL PET 2
P.6
1.
2.
3.
80℃
4.
1×Proteinase K
⇒37℃
1
1
5
50µL
5. 60℃ 30
6.
2
7.
75℃ 10
8. LAMP
95℃ 5
RT
1.
RT
Master Mix 2µL RNase free dH2O 5µL
2.
PrimeScript ® RT Master Mix
3µL
3. 37℃ 15
4.
85℃ 5
LAMP
◆
•
• 1×Proteinase K
•
•
2
8
≪LAMP
≫
◆
Loopamp
Loopamp
1 2×Reaction Mix. RM
1 2×Reaction Mix. RM
2 Primer Mix. Cry PM Cry
2 Primer Mix. Gia PM Gia
3 Distilled Water DW
3 Distilled Water DW
4 Bst DNA Polymerase
4 Bst DNA Polymerase
5 Positive Control Cry PC Cry
5 Positive Control Gia PC Gia
LAMP
DNA
※
◆
マスターミックスの調製
≪ 1test ≫ ≪10test ≫
12.5µL
125µL
Primer Mix. Cry PM Cry
Primer Mix. Gia PM Gia
2.5µL
25µL
Bst DNA Polymerase
1.0µL
10µL
Distilled Water DW
4.0µL
40µL
20.0µL
200µL
2×Reaction Mix. RM
Total
20µL
サンプル添加
1.5mL
RT
5µL
RT
DW
63℃
63℃ 60
9
PC 5µL
≪LAMP
≫
LAMP
DNA
1. -20℃
2. 1.5mL
1
1.
Loopamp
2.
3.
20μL
RT
5μL
Distilled Water (DW)
Positive Control Gia(PC Gia) 5µL
Positive Control Cry (PC Cry)5μL
4.
3 LAMP
1.
2.
3.
4.
5.
◆ LAMP
•
×3
Loopamp
•
•
•
•
10
≪LAMP
≫
■
○
1
2
0.7
0.7
0.6
0.6
0.5
0.5
0.4
0.4
0.3
0.3
0.2
0.2
0.1
0.1
0
0
0
15
30
45
60
0
◆
•
•
/
11
15
30
45
60
≪
≫
⇒
⇒
DNase RNase
◆ DNase RNase
⇒ DNase RNase
⇒
DNase RNase
⇒
⇒
⇒
⇒
⇒
A
A
B
B
C
RT
B
C
D
B
LAMP
D
A⇒B⇒C⇒D
12
≪
≫
LAMP
•
•
•
•
D
13
≪
≫
LAMP
Loopamp
≪ 1test
≫ ≪10test
≫
12.5µL
125µL
Primer Mix. Cry PM Cry
2.5µL
25µL
Bst DNA Polymerase
1.0µL
10µL
Distilled Water DW
7.0µL
70µL
23.0µL
230µL
2×Reaction Mix. RM
Total
23µ
µL
RT
2µ
µL
Loopamp
≪ 1test
≫ ≪10test
12.5µL
125µL
Primer Mix. Gia PM Gia
2.5µL
25µL
Bst DNA Polymerase
1.0µL
10µL
Distilled Water DW
8.0µL
80µL
24.0µL
240µL
2×Reaction Mix. RM
Total
24µ
µL
1µ
µL
◆
RT
14
≫
≪
≫
1.5mL
1.5mL
50µ 0.1M HCl(+0.01% Tween80)
塩酸解離
10
10
10
50µ 0.1M HCl(+0.01% Tween80)
10
10
10µ 1M NaOH
◆ 2×Proteinase K
200µ TE
1 test
600mAU/mL Proteinase K
3mAU/mL
0.25µL
1M NaCl
40mM
2.00µL
10% Triton X-100
0.2%
1.00µL
1M DTT
4mM
0.20µL
TE Tris EDTA pH8.0
Total 310µL
10,000×g, 3
46.55µL
50µL
LAMP
260µL
P6
LAMP
210µL
50µL
2×
×Proteinase K
50µL
50µL
-80℃⇔37℃
5
15
50µL LAMP
100µL
50µL
2012 8