reagents are thawed, keep them on ice.
2) Preparation of master mix (Operate on ice)
(1) Dispense the appropriate amount into the separately prepared sterilized tube under theproportion of the following per test and it can be used as master mix: 2 × Reaction Mix.
(RM) 12.5L, Primer Mix.O157 (PM O157) 2.5L, Distilled Water (DW) 4L, Bst
DNA Polymerase 1L.
For master mix preparation, it is recommended to prepare one test amount extra to theamount that is needed for the number of tests (including positive and negative controltests).
(2) After dispensing, mix the solution by gently tap the tubes a few times (hereinafter referred
to as tapping), or invert the tube, or by vortex mixer at about 1 second×3 times. Aftermixing well, centrifuge the tubes and the mixture can be used as the master mix for thereaction. Notice that too much mixing by the vortex mixer might inactivate thepolymerase, and assure that vortexing is conducted at 1 second×3 times.
The prepared master mix should be used as soon as possible.

For research use

LoopampTM

Escherichia coli O157 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 the amplification
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 thesequences related to Escherichia coli serotype O1576) as the primers. From whether theamplification occurred or not, the existence of Escherichia coli O157 can be determined.
By using the specifically designed Loopamp Realtime Turbidimeters, detection does notneed electrophoresis and all steps from amplification to detection are done within one
reaction tube. Simple and rapid detection of Escherichia coli O157 can be achieved7).
This kit cannot be used to determine the existence of Verotoxin-producing Escherichia coli.
Verotoxin-producing Escherichia coli should be separately detected.

(1)
(2)
(3)
(4)
(5)
(6)
(7)

48 tests
1.8mL × 2 tubes
1.0mL × 1 tube
0.6mL × 1 tube
60L× 1 tube
1.0mL × 1 tube
0.12mL × 1 tube
0.1mL × 1 tube

Extraction Solution for Foods (EX F)*1

1M Tris-HCl: pH7.0
2 × Reaction Mix.
Bst DNA Polymerase
Distilled Water
Primer Mix.O157
Positive Control O157

[Intended use]

(Tris)*1(RM)*1(Bst DNA Polymerase)*1(DW)*1(PM O157)*1(PC O157)*1*1: The notation on each reagent tube is shown in ().

Detection of Escherichia coli O157 in foods or environmental specimens.

[Principle]
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 2 × Reaction Mix. (RM), Bst DNA polymerase, Distilled Water(DW) and Primer Mix. O157 (PM O157), and incubate it. When Escherichia coli serotype
O157 that can be recognized by the primers exists, its DNA will be amplified with the activityof Bst DNA polymerase. The detection of 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 Escherichia coli O157.

Amplification curve pattern
Positive Control

For information on LAMP reaction principle, refer to Eiken GENOME SITE (URL:
http://loopamp.eiken.co.jp/e/).
Turbidity[How to use]
1. Materials required but not provided
Enrichment media
Stomacher back 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
Ice (crushed ice) and ice box
Loopamp Realtime Turbidimeter
Centrifuge for micro-tubes
Vortex mixer

0.5

0.5

0.4

0.4

0.3

Positive sample 2

Negative sample

0.3

0.2

0.2

0.1

0.1
00204060

Time (min)Fig.1 The amplification curve pattern of controls*20204060

Time (min)Fig.2 Samples amplification curve pattern*2: This kit is not developed for the purpose of quantitative analysis, therefore, the copy numbers
does not necessarily correlate with turbidity increment time.

[Caution for operation]
1. Sample handling
1) When collecting enrichment culture, pipette carefully as not to convolute the precipitate.
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 repeatedfreezing and thawing should be avoided.
2. Reagent handling
1) This reagent kit should be stored at -20C. To prevent the reagents from deterioration, only
take out the necessary amount of reagents from the freezer before use (No decline wasobserved in the kit performance even after repeated freezing and thawing for 20 times in thequality control test. But, in order to maintain the reagents performance, avoid unnecessaryfreezing 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 that fierce mixing should beavoided as it can inactivate the Bst DNA polymerase.
3) Extraction Solution for Foods (EX F) gradually deteriorates when exposed to air. Openingand 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 be limited as minimum as

In the case of using enriched culture as specimen for the detection of Escherichia coli O157

in foods:→Positive sample 10.602. Sample solution preparation

Food 25g +Novobiocin containing mEC media 225mL

Negative Control

0.6

Turbidity[Contents of the kit]

4. Operation procedure1) Mixing of master mix and sample solution (Operate on ice)
(1) Dispense 20L of the 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 with the cap closed
and then spin down. Be careful not to cause air−bubbles when mixing.
(3) For control reactions, use 5L of Positive Control O157 (PC O157) for positive control,and 5L of Extraction Solution for Foods (EX F) for negative control.
2) Amplification reaction and real-time detection(1) Loopamp Realtime Turbidimeters are applicable to this kit. Set the parameters as follows:[Temperature]: Reaction Block 65ºC, Hot Bonnet 75ºC
[Measurement Time]: 60 min[Inactivation]: 80ºC, 2 min(2) Confirm that the temperature has reached 65ºC.
(3) Set the prepared reaction tubes and start reaction.
(4) Check whether the positive and negative controls turbidity rose from the display screen ofthe device. If the turbidity of positive control rose and the turbidity of negative control
didn’t, then LAMP reaction proceeded properly. If not, there might have been error in
the process. Restart from reagent preparation and check again (Fig.1).
(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;
Positive Control O157 (PC O157).
(7) After enzyme inactivation (80ºC, 2 min), done automatically by Loopamp Realtime
Turbidimeters, is confirmed to have ended, remove the used reaction tubes and disposethem with caps closed.

Stomacher treatment→(Incubate at 42C for 18~24 hours)→ Enrichment culture1) Specimen pre-treatment (Preparing sample solution)(1) Prepare necessary quantity of sterilized tubes for specimen pre-treatment, and dispense
50L of Extraction Solution for Foods (EX F) to each tube.
(2) Add 50L of enrichment culture into the tube.
(3) Close the caps and mix thoroughly by inverting the tubes. Spin down for a few seconds
on centrifuge for micro-tubes and incubate at 95C for 5 minutes.
(4) After incubation, cool down on ice and then spin down. Add 10L of 1M Tris-HCl: pH7.0(Tris) and mix it using vortex mixer.
(5) After spin down for 1 minute at room temperature, keep it on ice and use the supernatantas sample solution (remain stable at 0~4C for 4 hours).
3. Reagents preparation
1) Take out the reagents stored at -20C, and thaw them at room temperature. Once the1/2

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 for the assay.
2) Start up Loopamp Realtime Turbidimeter at least about 20 minutes before use it.
3) For judgment, check whether the turbidity of Positive Control O157 (PC O157) 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 25 minutes after thereaction started). There are cases where the turbidity of the sample starts rising later thanthe positive control. Conduct the detection and the judgment right after the reaction finishes.
Measurement of the turbidity after 60 minutes of the start of reaction may obtain a lower
turbidity since magnesium pyrophosphate, the by-product of amplification reaction, may
sediment as precipitates at the bottom of the reaction tube.

possible. For the pre-treatment of specimen, dispense Extraction Solution for Foods justbefore the addition of specimen. When storing Extraction Solution for Foods, keep its cap
tightly closed, and do not aliquot the solution.
4) Positive Control O157 (PC O157) contains high number of copies. In order to prevent
Positive Control O157 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, leave theadding of positive control to the last and make sure that all other tube caps are closed whenadding it. Moreover, to avoid contamination, do not use Positive Control O157 in any otherway not written in this instruction (such as diluting the positive control or adding it tosamples).
5) Keep positive control and positive or suspected 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.

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 ofamplified products on other samples may not only cause false judgment of the test result butalso pollute testing area. In this case, a correct test result may not be obtained unlesspollution is completely removed.
2) Keep the cap of the used tube completely closed and dispose it according to the relevant
regulations and instructions by incineration or after double bagging it with sealable vinylbag. To prevent the amplified products from dispersing, do not conduct autoclavesterilization treatment for disposal.

Protocol< P re t re a t m e n t : D N A e xt ra c t i o n s o l u t i o n p re p a ra t i o n > ( O p er at e o n ic e )Dispense 50L of Extraction Solution for Foods (EX F) into the separately prepared pre-treatment tube.
Add 50L of enrichment culture into the tube. Close the caps and mix thoroughly by invert the tubes.

Spin down. Incubate at 95C for 5 minutes.

Cool down on ice. Add 10L of 1M Tris-HCl: pH7.0 (Tris) and mix with vortex mixer.

[Performance]
Sensitivity: 60 CFU (Colony Forming Unit) / test

Spin down at room temperature for 1 minute.

[Caution for handling]
Use the supernatant as sample solution (Operate on ice) *3.

1. LAMP reaction is very sensitive and even the slightest amount of amplified product tainted
into the reaction might cause false result. Therefore, avoid this type of contamination andcarry out the sample and reagent preparation in different clean benches. Avoid
electrophoresis or operations that need to handle amplified products.
2. Liver related materials should not be used since they contain components that influence
LAMP reaction.
3. About handling enrichment media, refer to their instruction manuals. When handling thesample, always abide by the biohazard counter measures 8).
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 clinicaldiagnostic purposes on human or animal samples.
6. If the operator does not have the experience or knowledge in the field of nucleic acid testing,there is a possibility of false judgment. Therefore, make sure that the kit is used under thesupervision of the experienced and knowledgeable technicians.
7. This kit can detect Escherichia coli O157 through amplifying its gene, which is different
from the conventional culture method that can only detect viable bacteria of Escherichia
coli O157. Use this kit as a part of self-imposed test.
8. This kit cannot be used to detect Verotoxin-producing Escherichia coli. For the detection ofVerotoxin-producing Escherichia coli, use the Loopamp Verotoxin-producing Escherichia
coli Detection Kit or Loopamp Verotoxin Typing Kit.
9. The result of this kit might be different from that of the culture method.
10. 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.
11. Use the kit before the expiration date, which is labeled on the outer box (Exp. Date).
12. This kit should be stored at -20C. Storing the kit at temperature lower than -20C or
repeated freezing and thawing might cause cracks on the tube of 1M Tris-HCl: pH7.0 (Tris).
13. The reagent tube is made of polypropylene and the main material for the kit case is paper.
The institution disposing the reagent tube and case should bear responsibility and abide by
the clinical waste disposal regulations, water pollution protection low, and any otherregulation related.

<Master mix. preparation> (Operate on ice)
Prepare the necessary volume*4of master mix and mix well*5.
Master mix preparation (for 1 test)
2 × Reaction Mix. (RM)
Primer Mix.O157 (PM O157)
Distilled Water (DW)
Bst DNA polymerase

12.5L
2.5L
4L
1LTotal20L

Spin down and use as master mix (on ice).

<Procedure> (on ice)
Dispense 20L of master mix into each reaction tube.

Add 5L of sample solution or control into the tube.
(Total of 25L as LAMP reaction solution.)
Add 5L of Positive Control O157 (PC O157) for positive control, and5L of Extraction Solution for Foods (EX F) for negative control.
Mix the solution well by pipetting or tapping the tube with the cap closed and then spin down.
(Be careful not to cause air bubbles when mixing.)<LAMP reaction>
Set the reaction tubes into the reaction blocks of Loopamp Realtime Turbidimeter and start reaction.

65C, 60min
Enzyme inactivation (80C, 2min).
(Done by Loopamp Realtime Turbidimeters automatically.)
Detection and judgment.

[Unit, Storage, Expiration, Code No.]*3

: Sample solution can remain stable at 0~4C for 4 hours.
*4: It is recommended to prepare one test amount extra to the amount that is needed for thenumber of tests (including positive and negative control tests).
*5: Mix by invert the tube or mix by vortex mixer at 1second×3 times.

Product NameUnit

Storage

Expiration

Code No.

LoopampTM Escherichia coli O157
Detection Kit

48 tests-20 C

1 year

LMP631[References]

3. Handling reaction tubes
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 or damages.
3) Check carefully to see if the reaction tubes have any crack or scratch before use. Crack orscratch 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, thereaction mixture can spill inside the equipment and cause unrecoverable contamination andmalfunction.
4) By comparing the solution volume in all tubes, check visually if proper amount of sample
solution/master mix. has been dispensed into the reaction tube.
4. Caution for amplification reaction
Since bubbles in the solution will interfere the turbidity measurement and cause false

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) Wang L. et al.: Infect. Immun. 66, No 8, 3545-3551 (1998)
7) Sunada A. et al.: Abstract for the 14th Conference of The Japanese Society for ClinicalMicrobiology, 193 (2003)
8) The guideline for the bio-safety and bio-hazard (by the Japanese Society for Bacteriology)
Japanese Journal of Bacteriology 54, No.3, 667-715 (1999)

Manufacturer
2/28106 AMK
Rev. Feb.2013