(1) Dispense the appropriate amount into the separately sterilized tube under theproportion of Reaction mix. VT (RM VT) 20L and Bst DNA polymerase 1L per test(including positive and negative control tests).
(2) After dispensing mix the solution by gently tap the tubes a few times (hereinafterreferred to as tapping) or invert the tube or vortex them 3 times for 1 second, After
mixing well, spin down the tube and the mixture can be used as the master mix for thereaction.
Notice that too much mixing by the vortex mixer might inactive the polymerase, andassure that vortexing is conducted at 1 second × 3 times.
The prepared master mix. should be used as soon as possible.

For research use

LoopampTM

Verotoxin-producing Escherichia coli
Detection Kit[Characteristics]
LAMP (Loop-mediated Isothermal Amplification) method is a gene amplification
method capturing the following characteristics: ( 1 ) Only one enzyme is required and theamplification reaction proceeds under isothermal condition 1), 2), (2) 4 primers recognizing 6
distinct regions on the target achieve high specificity, ( 3 ) High amplification efficiency
allows amplification within a shorter time,(4)It produces tremendous amount of amplified
products which makes simple detection possible3), 4), 5),.
Amplification of nucleic acids with this kit is conducted by the LAMP method using
Verotoxin related gene sequence as the primers. From whether the amplification occurredor not, the existence of Verotoxin producing Escherichia coli (VTEC) in the foodspecimen can be determined.
By using the specifically designed Loopamp Realtime Turbidimeter, detection does notneed electrophoresis and all steps from amplification to detection are done within one
reaction tube. Simple and rapid detection of VTEC can be achieved6) 7).

4. Operation procedure1) Mixing of master mix. and sample solution (Operate on ice)
(1) Dispense 20L of Master Mix into each Loopamp Reaction Tube.
(2) Add 5L of sample solution to the master mix, and the volume of the solution
should be 25L in total. Mix the solution well by pipetting or tapping the tube withthe cap closed and then spin down. Be careful not to cause air bubbles when mixing.
(3) For control reactions, use 5L of Control DNA VT (Cont VT) as positive control,and 5L of Extraction Solution for Foods (EX F) as negative control instead ofsample solution.
2) Amplification reaction and real-time detection(1) Loopamp Realtime Turbidimeter is applicable to this kit. Set the parameter asfollows:[Temperature] ; Reaction Block: 65ºC, Hot Bonnet: 75 ºC[Measurement Time] ; 60 min[Inactivation]; 8 0 ºC, 2 min[Contents of the kit]
48 tests
1.8mL × 3 tubes*2（1）Extraction Solution for Foods（EX F）*1
（2）1M Tris-HCl:pH7.0（Tris）*1
1.0 mL × 1 tube*2（3）Reaction Mix. VT（RM VT）*1
1.0 mL × 1 tube
60 L × 1 tube
（4）Bst DNA Polymerase（Bst DNA Polymerase）*1
0.1 mL × 1 tube（5）Control DNA VT（Cont VT）*1(2) Confirm that the temperature has reached 65ºC.
(3) Set the prepared reaction tubes and immediately start reaction.
(4) Check whether the positive and negative controls turbidity rose from the display screen of
the device. If the turbidity of positive control rose and the turbidity of negative control
didn’t, then LAMP reaction proceeded properly (Fig.1). If not, there might have been
error in the process. Restart from reagent preparation and check again.
(5) Next, the judgment of each sample is conducted. If the increase in turbidity is confirmed
within 60 minutes, then it is judged as positive, if not, negative (Fig.2).
(6) The rising timing or turbidity of samples might be different from the positive control;
Control DNA VT (Cont VT).
(7) After enzyme inactivation (80ºC, 2 min), done automatically by Loopamp Realtime
Turbidimeter, is confirmed to have ended, remove the used reaction tubes and disposethem with caps closed.

*1: The notation on each reagent tube is shown in ( ).
*2: For the extraction method of Japanese regulation.

[Intended use]
Detection of VTEC in foods or environmental specimen.

[ Principle]■Amplification curve patterns

This kit uses LAMP method as assay principle. First, conduct enrichment culture of thefood specimen and then after Alkalis heat extraction, the solution can be used as sample.
Mix the sample solution with Reaction Mix. VT (RM VT) and Bst DNA polymerase, andincubate it. When Verotoxin related gene sequence that can be recognized by the primers
exists, its DNA will be amplified with the activity of Bst DNA polymerase. The detectionof nucleic acid is done by detecting the turbidity change caused by the amplification
by-product magnesium pyrophosphate (white precipitate) and then determines whether
there is VTEC.
For further details of the LAMP reaction principle, refer to Eiken GENOME SITE (URL:
http://loopamp.eiken.co.jp/e/).

0.6

0.5

0.5

0.4

0.4

0.3

0.1
01. Materials required but not provided
Enrichment media (for pre-enrichment)
Stomacher bag with filter
Sterilized tubes for master mix preparation (0.5mL or 1.5mL)
Micropipette (0.5 ~ 10L, 10 ~ 100L, 100 ~ 1,000L)
Pipette tips with filter
Sterilized tubes for pre-treatment of specimen (0.5mL)
Heat block (Use at 95C)
Loopamp Reaction Tube
Aluminum rack for cooling tubes
Crushed ice and ice box
Loopamp Realtime Turbidimeter
Centrifuge for microtubes
Centrifuge for 8-strip tube
Vortex mixer

Stomacher treatment

PositiveSample2

Negative Sample

0.3
0.2
0.1

0102030405060

Time (min)Fig.1 The amplification curve pattern of controls

001020304050Time（min）

Fig.2 Samples amplification curve patternThis kit is not developed for the purpose of quantitative analysis, therefore,
the copy numbers does not necessarily with turbidity increment time.
[Cautions for operation]
1. Sample handling
1) When collecting pre-enrichment culture, pipette carefully as to not convolute theprecipitate.
2) Basically, the sample (DNA extraction solution) should be used immediately.
However, if it is to be stored for long period of time, it should be stored under -80ºC
and repeated freezing and thawing should be avoided.
2. Reagent handling
1) This reagent kit should be stored at -20C. To prevent the reagents fromdeterioration, only take out the necessary amount of reagents from the freezer before
use. No decline was observed in the kit performance even after repeated freezing
and thawing for 20 times in the quality control test. But, in order to maintain thereagents performance, avoid unnecessary freezing and thawing.
2) Thaw the reagents at room temperature and keep them on ice for reagents
preparation. Before use, spin down the tubes to drop down the reagents staying on
the tube wall or on the cap, mix well the reagents and spin down again. Notice thatfierce mixing should be avoided as it can inactivate the Bst DNA polymerase.
3) Extraction Solution for Foods (EX F) gradually deteriorates when exposed to air.
Opening and closing the cap of Extraction Solution for Foods should be limited asminimum as possible so that the time for exposing the solution to air can be limited
as minimum as possible. Please add Extraction Solution for Foods (EX F) as soon as
possible for pre-treatment. When storing Extraction Solution for Foods, keep its cap
tightly closed, and do not aliquot the solution.
4) Control DNA VT (Cont VT) contains high number of target DNA. In order toprevent Control DNA VT from contaminating other samples or reagents, always spin
down before opening the tube and open the cap of the tube as shortly as possible.
Also add into the reaction tubes under the following order from negative control(Extraction Solution for Foods (EX F)), sample solution (extracted DNA), and leave
the adding of Control DNA VT(Cont VT) to the last and make sure that all othertube caps are closed when adding it. Moreover, to avoid contamination, do not use

2. Sample solution preparation
In the case of using pre-enriched culture as specimen for the detection of VTEC in foods:(Incubation at 42ºC for 18~24 hours) 

0.6

0.2

[ How to use]

Food 25g +225mL Novobiocin containing mEC media PositiveSample1

Negative Control

Turbidity

Turbidity

Positive ControlPre-enrichment culture1) Specimen pre-treatment (Preparing sample solution)(1) Prepare necessary quantity of sterilized tube for pretreatment of specimen, and pour50L each “Extraction Solution for Foods” in the tubes.
(2) Add 50L of testing specimen (pre-enrichment culture) into each tube.
(3) Close the cap of the tubes, invert them several times to mix thoroughly and spin
down with the centrifuge. Heat the tubes at 95ºC for 5 minutes. Then centrifuge
the tubes for 1 minute and place them on ice (Sample solution). The sample
solution can be kept for 4 hours at 0~4ºC.
3. Reagents preparation
1) Take out the reagents stored at -20 ºC, and thaw then at room temperature. Once thereagents are thawed, keep them on ice.
2) Preparation of master mix. (Operation on ice)
1/2

60

Control DNA VT (Cont VT) in any other way not written in this instruction (such asdiluting the positive control or adding it to samples).
5) Keep positive control and suspected positive samples away from the reagents whenhandling.
6) If there is any reagent left, do not use it with other kits even if they are in the same lot.

of amplified products on other samples may not only cause false judgment of the test
result but also pollute testing area. In this case, a correct test result may not be obtained
until the contamination is completely removed.
2) Keep the cap of the used tube completely closed and dispose it according to therelevant regulations and instructions by incineration or after double bagging it withsealable vinyl bag. To prevent the amplified products from dispersing, do not conduct
autoclave sterilization treatment for disposal.

■ Protocol

Pretreatment: DNA extraction solution preparation (Operate on ice)

[Performance]

Put 50L Extraction Solution for food specimen into the pre-treatment tube.

Minimum detection limitation: 60 CFU (Colony Forming Unit)/ test

[Caution for handling]

Add 50L specimen (pre-culture) to the tube and invert them for mixing.

1. LAMP reaction is very sensitive and even the slightest amount of amplified producttainted into the reaction might cause false result. Therefore, avoid this type ofcontamination by carrying out the sample and reagent preparation in different clean
benches. Avoid electrophoresis or operations that need to handle amplified products.
2. This kit is not available to the test for the food specimen consists of the liver, because
the LAMP reaction is susceptible to the liver ingredients.
3. The culture medium is handled according to each manual. Try the appropriate measurefor the biohazard when the specimen is processed8).
4. Do not expose the Loopamp Reaction Tube, master mix preparation tubes to UV light. Achange in color or deterioration caused by ultraviolet lamp sometimes results inmisjudgment.
5. This kit is for the purpose of foods and environment inspection, not for medical or
clinical diagnostic purposes on human or animal samples.
6. If the operator does not have the experience or knowledge in the field of nucleic acidtesting, there is a possibility of false judgment. Therefore, make sure that the kit is used
under the supervision of the experienced and knowledgeable technicians.
7. This kit can detect VTEC through amplifying its gene, which is different from theconventional culture method that can only detect viable bacteria of VTEC. Use this kit
as a part of self-imposed test.
8. The result of this kit might be different from that of the culture method.
9. 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.
10. Use the kit before the expiration date, which is labeled on the outer box (Exp. Date).
11. The reagent tube is made of polypropylene and the main material for kit case is paper.
12. The institution disposing the reagent tube and case should bear the responsibility andabide by the clinical waste disposal regulations, water pollution prevention law, and anyother regulation related.

Spin down the tubes and heat-treat them at 95ºC for 5 minutes.
Centrifuge the tubes at room temperature for 1 minute.
Place the tubes on ice (Sample Solution)*.
Preparation of Master Mix. (Operate on ice)
Prepare the necessary volumes of master mix and mix well.
(including the positive and negative control)**
Preparation of Master Mix. (per test)
Reaction Mix. VT
20L
Bst DNA Polymerase
1LTotal
21LMix well and spin down the master mix, then keep it on ice.
 Procedure (Operateon ice)
Dispense 20L of master mix into each reaction tube.

Add 5L of sample solution to the tubes or control into the tube (Total of 25L
as LAMP reaction solution).
Add 5L of Control DNA VT (Cont VT) for positive control
and add 5L of Extraction solution for Foods (EX F) for negative control.

[ Unit, Storage, Expiration, code No. ]Mix the solution well by tapping or pipetting the tube with the cap closed
and then spin down
(Be careful not to cause air bubbles.)Products Name

Unit

Storage

Expiration

Code No.

48tests-20 ºC

1 year

LMP621

LoopampTM
Verotoxin producing E.Coli Detection kit

 LAMP Reaction Set the reaction tube into the reaction block of Loopamp turbidimeter andstart the reaction.
65ºC, 60 minutes[ References ]1) Notomi T. et al.: Nucleic Acids Research 28 , No.12, e63 (2000)
2) Nagamine K. et al.: Clin. Chem. 47, No 9: 1742-1743 (2001)
3) Mori Y. et al.: Abstract for the 23rd Annual Meeting of the Molecular Biology Society of Japan (2000)

Enzyme inactivation (80 ºC, 2 minutes)(Done by Loopamp Realtime Turbidimeter automatically)

4) Mori Y. et al.: Biochem. Biophys. Res. Commun. 289, No.1, 150 - 154 (2001)
5) Tomita N. et al.: Abstract for the 26th Annual Meeting of the Molecular Biology Society of Japan(2003)

Detection and Judgment

6) Nemoto J. et al.: the 23rd Japan Food microbiology Conference. Abstract p.40 (2002)* : Sample Solution remains active for up to 4 hours at 0-4ºC.

7) Hara-Kudo Y. et al.: FEMS Microbiol. Lett. 253, No1: 155-161 (2005)** : Invert tubes for mixing or vortex the tubes 3 times for 1 second.

8) Japan bacteriology, 54, No.3, 667-715 (1999)

3. Handling reaction tube
1) Only use the specified Loopamp Reaction Tube for turbidity detection. Other reaction
tubes might have different optical transparency and can cause misjudgment.
2) Take full care when handling reaction tubes, as they are vulnerable to scratches ordamages.
3) Check carefully to see if the reaction tubes have any crack or scratch before use. Crackor scratch on the tube might not only cause false judgment but also contaminate theequipment. If the tubes are broken inside the reaction block of the Loopamp Realtime
Turbidimeter, the reaction mixture can spill inside the equipment and cause
unrecoverable contamination and malfunction.
4) By comparing the solution volume in all tubes, check visually if proper amount ofsample solution and master mix has been dispensed into the reaction tube.
4. Cautions for amplification reaction
Since bubbles in the solution will interfere the turbidity measurement and cause false
judgment, try not to cause any bubble when mixing the master mix and the sample
solution. If bubbles appear, spin down to remove them.
5. Caution for detection and judgment
1) Use only Loopamp Realtime Turbidimeter and Loopamp Reaction Tube for the assay.
2) Start up Loopamp Realtime Turbidimeter at least about 20 minutes before using it.
3) For judgment, check whether the turbidity of Control DNA VT (Cont VT) has risen to
determine whether the reagent is performing properly (if the nucleic acid amplification
reaction is performed properly, the turbidity will start to rise around 20 minutes after
the reaction started). There are cases where the turbidity of the sample starts risinglater than the positive control.
6. Handling reaction tubes after use
1) The caps of the used reaction tubes should not be opened. Pay special attention not toaccidentally open the cap when taking the tubes out of the turbidimeter. Contamination

Manufacturer
8104 AMK
Rev.Feb.2013

2/2