Master Mix VT2 in a same manner(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 mastermix for the reaction. Notice that too much mixing by the vortex mixer might
inactive the polymerase, and assure that vortexing is conducted at 1 second × 3times.
The prepared master mix. should be used as soon as possible.

For research use

LoopampTM

Verotoxin Typing Kit

4. Operation procedure1) Mixing of master mix. and sample solution (Operate on ice)
(1) Dispense 20L of Master Mix.VT1 or Master Mix.VT2 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Lin total. Mix the solution well by pipetting or tapping the tube
with the cap closed and then spin down. Be careful not to cause air bubble whenmixing.
(3) For control reactions, use 5L of Control DNA VT1 (or VT2) as positive
control and 5L of Extraction Solution for Foods as negative control instead ofsample solution.
2) Amplification reaction and real-time detection(1) Loopamp Real-time turbidimeter is applicable to this kit. Set the parameter asfollows:[Temperature] ; Reaction Block: 65C, Hot bonnet: 75C
[Measurement Time] ; 60 min[Inactivation] ; 80 C, 2 min(2) Confirm that the temperature has reached 65C.
(3) Set the prepared reaction tubes and immediately start reaction.
(4) Check whether the positive and negative controls turbidity rose from thedisplay screen of the device. If the turbidity of control rose and turbidity ofnegative 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 isconfirmed 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 VT1 or VT2 (Cont V1 or V2).
(7) After enzyme inactivation (80ºC, 2 min), done automatically by Loopamp
Realtime Turbidimeter, is confirmed to have ended, remove the used reaction
tubes and dispose them with caps closed.

[ Characteristics]
LAMP (Loop-mediated Isothermal Amplification) method is a gene amplification
method capturing the following characteristics: ( 1 ) Only one enzyme is required andthe amplification reaction proceeds under isothermal condition 1), 2), (2) 4 primers
recognizing 6 distinct regions on the target achieve high specificity, ( 3 ) Highamplification 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 thesequences related to Verotoxin (VT) as the primers, and VT related gene in thespecimen is typed.
By using the specifically designed Loopamp Real-time Turbidimeters, detection does
not need electrophoresis and all steps from amplification to detection are done within
one reaction tube. Simple and rapid typing of VT related gene can be achieved6)7)8).

[Contents of the kit]24 tests(1) Extraction Solution for Foods (EX F)*
1.8mL × 1 tube
(2) Reaction Mix.VT1(RM V1)*
0.5mL × 1 tube
(3) Reaction Mix.VT2(RM V2)*0.5mL × 1 tube
(4) Bst DNA Polymerase(Bst DNA Polymerase)*
60µL × 1 tube
(5) Control DNA VT1(Cont V1)*
0.1mL × 1 tube
(6) Control DNA VT2(Cont V2)*
0.1mL × 1 tube*: The notation on each reagent tube is shown in ( ).
[ Intended use]
Typing of Verotoxin I (VT1) and II (VT2) produced by Verotoxin producing
Escherichia coli.

[ Principle]
This kit uses LAMP method as assay principle. First, conduct enrichment culture of
the food specimen and then after Alkalis heat extraction, the solution can be used assample. Mix the sample solution with Reaction Mix.VT1 (RM VT1) or Reaction
Mix.VT2 (RM VT2) and Bst DNA polymerase, and then incubate them. When
Verotoxin I (VT1) and II (VT2) gene sequence that can be recognized by the primers
exists, its DNA will be amplified with the activity of Bst DNA polymerase. . Gene
amplification is detected using Loopamp Real-time Turbidimeter by monitoring
turbidity of white precipitates of magnesium pyrophosphate, which is a by-product ofamplification reaction2).Based on the amplification curve, the type of VT related gene in
the specimen is judged. For details of the LAMP method, refer to the Eiken
GENOME SITE (URL; http://loopamp.eiken.co.jp/e/)■Amplification curve patterns[ How to use]
1. Materials required but not provided
Distilled Water

Suspend in DW (McFarland No.18) )PositiveSample1

0.6

0.6

0.5

0.5

0.4

0.4

0.3
0.2
0.1

Sterilized tubes for master mix preparation (0.5mL or 1.5mL)
Micropipette (0.5~10µL,10〜100µL,100〜1,000µL)
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
2. Sample solution preparation
Suspension (3 × 106 CFU/mL) of bacteria suspected to be STEC by food microbe
testingMicrobe culture or the colony formed on the agar plateNegative Control

Turbidity

Turbidity

Positive Control

PositiveSample2

Negative Sample

0.3
0.2
0.1

00010203040506001020304050

Time（min）

Fig.1 The amplification curve pattern of controls

Fig.2 Samples amplification curve patternThis kit is not developed for the purpose of quantitative analysis, therefore, thecopy numbers does not necessarily with the 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 repeatedfreezing and thawing for 20 times in the quality control test. But, in order tomaintain the reagents 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. Noticethat fierce 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
as minimum as possible so that the time for exposing the solution to air can belimited 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 VT1 (Cont V1) and Control DNA VT2 (Cont V2) contains highnumber of target DNA. In order to prevent Control DNA VT1 and Control DNA
VT2 (Cont V2) from contaminating other samples or reagents, always spin down

Dilute 100 times.

 Dilution (3 × 106 CFU/mL)
1) Pretreatment of testing specimen.
(1) Prepare necessary quantity of sterilized tube for pretreatment of specimen, andpour 50L each Extraction Solution for Foods (EX F) 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 andspin down with the centrifuge. Heat the tubes at 95C for 5 minutes. Thencentrifuge the tubes for 1 minute and place them on ice (Sample Solution).
The Sample Solution can be kept for 4 hours at 0~4C.
3. Reagents preparation
1) Take out the reagents stored at -20C, and thaw then at room temperature. Once thereagents are thawed, keep them on ice.
2) Preparation of master mix. (Operation on ice)
(1) Dispense the appropriate amount (including positive and negative control tests)
into the separately sterilized tube under the proportion of Reaction mix.VT1 (RMV1) 20L and Bst DNA polymerase 1L per test (Master Mix. VT 1). Prepare

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60Time（min）

before opening the tube and open the cap of the tube as shortly as possible. Alsoadd into the reaction tubes under the following order from negative control(Extraction Solution for Foods (EX F)), sample solution (extracted DNA), andleave the adding of Control DNA VT1(Cont V1) and Control DNA VT2 (Cont V2)
to the last and make sure that all other tube caps are closed when adding it.
Moreover, to avoid contamination, do not use Control DNA VT1 (Cont V1) andControl DNA VT2 (Cont V2) in any other way not written in this instruction (suchas diluting the positive control or adding it to samples).
5) Keep positive control and suspected positive samples away from the reagents
when handling.
6) If there is any reagent left, do not use it with other kits even if they are in thesame lot.

or V2) has risen to determine whether the reagent is performing properly (if thenucleic acid amplification reaction is performed properly, the turbidity will start torise around 20 minutes after the reaction started). There are cases where theturbidity of the sample starts rising later 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 to accidentally open the cap when talking the tubes out of the turbidimeter.
Contamination 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 disposed it according to
the relevant regulations and instructions by incineration or after double bagging itwith sealable vinyl bag. To prevent the amplified products from dispending, do not
conduct autoclave sterilization treatment for disposal.

■ Protocol

Pretreatment: DNA extraction solution preparation  (Operate on ice)
Put 50L Extraction Solution for food specimen into the pre-treatment tube.

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

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

[Caution for handling]

Spin down the tubes and heat-treat them at 95ºC for 5 minutes.

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
measure for the biohazard when the specimen is processed9).
4. Do not expose the Loopamp Reaction Tube, master mix preparation tubes to UV light.
A change 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 isused 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 thiskit 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 ispaper. The institution disposing the reagent tube and case should bear theresponsibility and abide by the clinical waste disposal regulations, water pollution
prevention law, and any other regulation related.

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. VT1 or VT220L
Bst DNA Polymerase
1LTotal21L

Mix well and spin down the master mix, then keep it on ice.

<Procedure> (Operate on 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 VT1 or VT2 (Cont V1or V2) for each 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.)Product nameLoopamp LAMP Reaction Set the reaction tube into the reaction block of Loopamp turbidimeter andstart the reaction.
65ºC, 60 minutes

TM

Verotoxin Typing Kit

UnitStorage

Expiration

Code No.

24 tests-20 C

1 yearLMP 641[Reference]
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)
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)
6) Nemoto J. et al.: the 23rd Japan Food microbiology Conference. Abstract p 26(2002)
7) Microbiology, Food sanitary testing guide, edited with Japan Food Sanitaryassociation, compiled under milk and meat sanitary sector, Life-sanitary department,
Minister of public welfare, Tokyo (1990), Addendum I(1993), Addendum II (1996)
8) Manual of Clinical Microbiology, fourth edition, p1095, American Society for
Microbiology, Washington, D.C. (1985)
9) Japan bacteriology, 54, No.3, 667-715 (1999)

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

Detection and Judgment* : Sample Solution remains active for up to 4 hours at 0-4ºC.
** : Invert tubes for mixing or vortex the tubes 3 times for 1 second.

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.

Crack or scratch on the tube might not only cause false judgment but also
contaminate the equipment. If the tubes are broken inside the reaction block of the
Loopamp Realtime Turbidimeter, the reaction mixture can spill inside theequipment 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.

Manufacturer
8105

5. Caution for detection and judgment
1) Use only Loopamp Realtime Turbidimeter and Loopamp Reaction Tube for theassay.
2) Start up Loopamp Realtime Turbidimeter at least about 20 minutes before using it.
3) For judgment, check whether the turbidity of Control DNA VT1 or VT2 (Cont V1

AMKRev. Feb.2013

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