Read this instruction carefully before use
For research use only

Rev. January 2008（Ver.3）®

DW

PC WNV

0.6

Primer Set for West Nile Virus
Turbidity

0.5【Characteristics】
LAMP（Loop－ mediated Isothermal Amplification）method is a novel
gene amplification method capturing the following characteristics : ① Only
one enzyme is required and the amplification reaction proceeds underisothermal condition 1）, 2）., ② It has extremely high specificity because of
the use of 4 primers recognizing 6 distinct regions on the target., ③ It has
high amplification efficiency and enables amplification within a shortertime., ④ It produces tremendous amount of amplified products whichmakes simple detection possible 3）, 4）, 5）, 6）.
This reagent consists of the primer set for the detection of thegenomic RNA encoding envelope of West Nile Virus（WNV）7 ）, which is
specifically designed for Loopamp RNA Amplification Kit（RT－LAMP）.

0.4
0.3
0.2
0.1
0.0
05

101520253035Time（ min ）The amplification curve of positive control WNV RNA＊2（ monitored by Loopamp Realtime Turbidimeter）＊2：There is no correlation between the initial template number and the【Contents of the kit】

turbidity increment time.

48 tests（1） Primer Mix. WNV（PM WNV）＊1

0.12 mL × 1 tube

（2） Positive Control WNV（PC WNV）＊1【Caution for Handling】

60μL × 1 tube

＊1：The notation on each reagent tube is shown in（

１．This reagent is designed for research use only.

）.

２．If the operator does not have the experience or knowledge in the field of
nucleic acid testing, there’s a possibility of false judgment. Therefore,【How to use】

make sure that the reagent is used under the supervision of the１．Materials required but not provided

experienced and knowledgeable technicians.

Refer to the package insert for Loopamp RNA Amplification Kit（RT－LAMP）.

３ ． Eiken Chemical Co., Ltd. does not bear any responsibility for false
judgment or any consequential damage derived from the false

２．Preparation of the specimen

judgment caused by non－capability problems such as operation error...

Carry out the extraction of RNA from the specimen using acommercially available virus genome isolation reagent（for example,QIAamp Viral RNA Mini Kit, Qiagen）. When handling the sample, always
abide by the biohazard counter measures 8）.

４．Use the reagent before the expiration date, which is labeled on the outerbox（Exp.Date）.
５．The reagent tube is made of polypropylene and the main material for kit
case is paper. The institution disposing the reagent tube and case

３．Reagents preparation

should bear the responsibility and abide by the clinical waste disposal

１） Take out the reagents stored at － 20 ℃ , and thaw them at room
temperature. Once the reagents are thawed, keep them on ice.
２） Preparation of master mix.（Operate on ice）.
Referring to the table below, dispense the necessary amount of Primer
Mix. WNV（PM WNV）of this kit, and 2 × Reaction Mix.（RM）, EnzymeMix.（EM）and Distilled Water（DW）of Loopamp RNA Amplification Kit（RT－LAMP）, into sterilized tube for master mix.

regulations, water pollution prevention law, and any other regulation＜Reagents＞

12.5 μL

125 μL

Primer Mix. WNV（PM WNV）

2.5 μL

25 μL

Enzyme Mix.（EM）1.0 μL

10 μL

Total

【Unit，Storage，Expiration，Code No.】

＜Amount：1 test＞ ＜Amount：10 tests＞

2 × Reaction Mix.（RM）

Distilled Water（DW）

related.

4.0 μL40 μL

20.0 μL

200 μL

Product Name

UnitLoopamp ® Primer Set forWest Nile Virus

48 tests

Storage Expiration Code No.
－20℃

1 yearPM0001【References】
1）Notomi T. et al. : Nucleic Acids Research 28, No.12, e 63（2000）
2）Nagamine K. et al. : Clin. Chem. 47, No.9, 1742－1743（2001）
3）Mori Y. et al. : Biochem. Biophys. Res. Commun. 289, No.1, 150－ 154（2001）
4）Tomita N. et al. : Abstract for The 73rd Annual Meeting of the Japanese
Biochemical Society（2000）

４ ． Operation procedures （ Follow the instruction of Loopamp RNA
Amplification Kit（RT－LAMP））.
１）Mixing master mix. solution and the sample solution（Operate on ice）.
（1）Dispense 20μL of the master mix. i n to each Loopamp ReactionTube.
（2）Add 5μL of sample RNA, Positive Control WNV（PC WNV）or NegativeControl（DW）into the master mix., and the volume of the solution
should be 25μL in total. Mix the solution well by pipetting or tapping
the tube and then spin down. Be careful not to cause air－bubbles
when mixing.

5）Mori Y. et al. : Abstract for the 23rd Annual Meeting of the Molecular
Biology Society of Japan（2000）6）Tomita N. et al. : Abstract for the 26th Annual Meeting of the Molecular
Biology Society of Japan（2003）
7）Parida M. et al. : Journal of Clinical Microbiology 42, No.1, 257－263（2004）
8）The guideline for the bio－safety and bio－hazard（by the JapaneseSociety for Bacteriology）: Japanese Journal of Bacteriology 54, No.3,667－715（1999）

２）Amplification and detection（1）Incubate at 63℃ for 35 minutes.
（2）After the amplification reaction, use a heat block to inactivate thepolymerase and terminate the reaction（ 5 minutes at 80 ℃ or 2
minutes at 95℃）.
☆ If other final judgment procedures such as fluorescence visualdetection or electrophoresis is to be taken, be sure that the enzyme isinactivated.
For information on fluorescence visual detection, refer to the package
insert for Loopamp Fluorescent Detection Reagent.
☆ For examples of detection cases using this reagent, refer to theproducts introduction page in Eiken GENOME SITE （ URL; http://loopamp.eiken.co.jp/e/ ）Manufacturer
143 Nogi, Nogi－machi, Shimotsuga－gun, Tochigi, Japan

1／1
3PM0019－C