５．Amplification curve pattern

For research use onlyTMControl Set RNA/DNA
This product is a reagent for research purpose. Do not
use this product for making or supporting a diagnosis.
Read this explanatory leaflet carefully before use.

【Introduction】
The LAMP（Loop－mediated Isothermal Amplification）method is a gene
amplification technique characterized by（1）isothermal gene amplification
reaction1）, 2）,（2）high specificity due to the use of 4 primers recognizing 6 regions,（3）high amplification efficiency resulting in amplification in a short time,（4）alarge amount of amplification product facilitating simple detection3）, 4）.
This product is a control reaction kit to be combined with Loopamp RNA/DNA
Amplification Reagent D（separately provided by Eiken Chemical）.

【Contents】＜Precautions for measurement＞

１．Because the LAMP reaction is very sensitive, any contamination withextremely minimum of the target gene or amplification product may result in
an incorrect result. To avoid such contamination, the use of this product andspecimen collection/nucleic acid extraction procedure should be performed inseparate rooms or in different areas by partitioning the laboratory area. Take
appropriate measures to prevent contamination including the use of clean
benches, gloves, and isolation gowns, as required.
２．Avoid the contamination by microorganisms or nucleic acid degrading enzymes（such as DNase and RNase）in handling this product.
３．Completely dissolve the dry reagent. Incomplete dissolution may result in poor
performance including low sensitivity. Do not leave more than 2 minutes instate of inverting reaction tubes.
４．Air bubbles may appear on the liquid level of the reaction solution after mixing
the sample solution. Remove them to prevent measurement errors byspinning down the reaction mixture.
５．Never open the tube cap after reaction. Particularly, carefully remove the tube
from the equipment so as not to open the cap after reaction. The contamination
with amplification products not only results in an erroneous decision, but also
causes the contamination of the measurement environment. Suchcontamination may persistently inhibit correct measurement unless it iscompletely eliminated.
６．Avoid handling amplification products using electrophoresis.

For 12 tests

１．Primer mix H1P（PM H1P）
２．Positive control H1P（PC H1P）
３．Negative control（NC）

0.72 mL × 1
0.16 mL × 1
0.16 mL × 1【Instructions for use】

１．Essential apparatus, equipment, and reagents, etc.

Refer to the instruction manual of Loopamp RNA/DNA Amplification Reagent D.

２．Reagent preparation method（follow the procedure of Loopamp
RNA/DNA Amplification Reagent D for details）.

１） Take a necessary number（total number of samples and controls）of the Dried
RNA/DNA Amplification Reagent. Immediately return the remaining reagent
to the original aluminum pack and seal it.
○ For control reaction（this product）＜Reagent＞
Primer mix H1P（PM H1P）

＜Dose＞
15.0μL/test２） Mix the reagent（PM H1P）by inverting, tapping or using a vortex mixer for 1
second 3 times and spin down before using as the primer mix（Operation onice）.

３．Operating procedure（Operation on ice）

【Precautions for handling（hazard prevention）】

１．This product is not designed as an in vitro diagnostic（IVD）.
２．Take care not to directly gaze at the ultraviolet ray（sterilizing ray）from thelamp of the ultraviolet irradiation device for fluorescent/visual evaluation
because it is dangerous. When it is necessary to gaze at the lamp that is on,
make sure to do that through a glass plate or using wide eyeglasses or shield.
３．A minute amount of sodium azide is contained as a preservative in the primer
mix H1P（PM H1P）, positive control H1P（PC H1P）, and negative control（NC）. Take care to prevent sodium azide from entering the eyes or mouth or
attaching to skin because it is toxic.
４．If the reagent accidentally enters the eyes or mouth or attaches to skin, immediately
rinse it off with a large amount of water and seek medical treatment, if needed.

Dispense 15.0μL of PM H1P to each reaction tube.
Add 10.0μL of the control to each tube.
（25.0μL in total as the LAMP reaction solution）
Use negative control（NC）for the negative control and positive control
H1P（PC H1P）for positive control.
After closing the cap, invert the reaction tube to transfer the solution onto the cap,
and allow it to stand on ice for 2 minutes.
Repeat the inversion 5 times and spin down the reaction mixture using an 8－
microtube simple centrifuge.

【Precautions】
１．This product should be stored as specified while avoiding freezing or suddenchange in temperature.
２．Keep the positive control H1P（PC H1P）away from other reagents.
３．Perform gene test with this product only under the supervision of experts withthe knowledge and experience of gene test because the lack of knowledge orexperience may result in incorrect judgment of the test result.
４．Eiken Chemical Co., Ltd. does not bear any responsibility for false judgment
or any consequential damage derived from the false judgment caused by
non-capability problems such as operation error.
５．Use this product within the expiration date.
６．Do not recycle the containers or accessories of this product or use them forother purposes.
７．Do not use it in combination other regents with the Loopamp RNA/DNA
Amplification Reagent D.

〈LAMP reaction〉
Set it in the real－time turbidimeter or incubator reaction block to start reaction.
The control reaction of this product occurs at 62.5℃ for 35 minutes.
Enzyme deactivation（80℃ for 5 minutes or 95℃ for 2 minutes）（will be automatically processed in the real－time turbidimeter）
Turbidity measurement/evaluation

４．Detection
Ａ．Real－time turbidity detection

The target gene can be detected in real time using the real－time turbidimeter（designed for the LAMP method）. Refer to the package insert or operation manual,etc. for the detailed operating procedure.

Ｂ．Fluorescent/visual detection

Judgment is made by emitting ultraviolet ray to the reaction tube from the bottom
using the ultraviolet irradiation device and observing the reaction tube from the sidewith the eyes protected by eyeglasses, etc. Evaluate the reaction tube according tothe following criteria after confirming that the positive control（PC H1P）produces
green fluorescence and the negative control（NC）produces no fluorescence. Take
the picture of the reaction tube using a digital camera, etc. for documentation
purposes, if required.
Positive: Produces green fluorescence.
Negative: Produces no fluorescence.

【Precautions for disposal】

１．Appropriately dispose of tubes after reaction with the cap closed by putting
them in double plastic bags that can be incinerated or sealed. To prevent
dispersion of amplification products, do not autoclave tubes before disposal.
２．The constituent reagent tube is mainly made of polypropylene（PP）. The kit
case is mainly made of paper.
３．Dispose of this product, containers, and materials before or after use on theresponsibility of the laboratories in compliance with applicable laws on waste
disposal and cleaning and water pollution prevention law.

【Storage method，shelf life，packaging unit，and product code】
Product nameStorage method

Shelf life

Package unit

Product code

LoopampTM
Control Set
RNA/DNA

2－8℃

1 yearFor 12 tests

LMP248【References】
1）Notomi T. et al.,: Nucleic Acids Research, 28（12）: e63, 2000.
2）Nagamine K. et al.,: Clin. Chem, 47（9）: 1742－1743, 2001.
3）Mori Y. et al.,: Biochem. Biophys. Res. Commun, 289（1）: 150－154, 2001.
4）Tomita N. et al.,: Nat Protoc, 3（5）: 877－882, 2008.
5）Nagamine K. et al.,: Molecular and Cellular Probes, 16（3）: 223－229, 2002.
Manufacturer
1／1

143 Nogi, Nogi－machi, Shimotsuga－gun, Tochigi 329－0114, Japan
380234－A

Date of Revision : April 2015（Ver.1）