(5) Add 10L of 1M Tris-HCl: pH7.0 (Tris).
(6) Close the caps and mix by vortex mixer. After centrifuge at 2,000×g for 30 seconds at room

For research use

temperature, keep it on ice and use the supernatant as sample solution (remain stable at 0~4C

Loopamp

TMfor 4 hours).

3. Preparing the reagents
1) Take out the reagents stored at -20C, and thaw them at room temperature. Once thereagents are thawed, keep them on ice.
2) Preparation of master mix (Operate on ice)
(1) Dispense the appropriate amount according to the number of testes to be conducted into
the separately prepared sterilized tube under the proportion of Reaction Mix. Sal (RMSal) 20L and Bst DNA Polymerase 1L per test (including positive and negativecontrol tests).
(2) After dispensing, mix the solution by gently tap the tubes a few times (hereinafterreferred to as tapping), or invert the tube, or by vortex mixer at 1 second×3 times.
After mixing well, spin down the tubes and the mixture can be used as the master mix
for the reaction. 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.

Salmonella 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
Salmonella invasion gene (invA)6) sequence as the primers. From whether the amplification
occurred or not, the existence of Salmonella spp. 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 Salmonella spp. can be achieved7) 8)9).

[Contents of the kit]
(1)
(2)
(3)
(4)
(5)

Extraction Solution for Foods (EX F)*1
1M Tris-HCl: pH7.0(Tris)*1
Reaction Mix. Sal(RM Sal)*1
Bst DNA Polymerase(Bst DNA Polymerase)*1
Control DNA Sal(Cont Sal)*1
*1

1.8mL
1.0mL
1.0mL
60L
0.1mL

4. Operation procedure1) Mixing 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 be25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 Control DNA Sal (Cont Sal) as positive control, and5L of Extraction Solution for Foods (EX F) as negative control.
2) Amplification reaction and realtime turbidity 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 immediately 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 (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 Sal (Cont Sal).
(7) After enzyme inactivation (80ºC, 2min), done automatically by Loopamp Realtime
Turbidimeters, is confirmed to have ended, remove the used reaction tubes and disposethem with caps closed.

48 tests
× 3 tubes
× 1 tube× 1 tube× 1 tube× 1 tube: The notation on each reagent tube is shown in ().

[Intended use]
Detection of Salmonella spp. 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 Reaction Mix. Sal (RM Sal) and Bst DNA polymerase, and incubate
it. When Salmonella invasion gene (invA)6) sequence that can be recognized by the primers
exists, its DNA will be amplified with the activity of Bst DNA polymerase. The detection ofnucleic acid is done by detecting the turbidity change caused by the amplification by-productmagnesium pyrophosphate (white precipitate) and then determines whether there isSalmonella spp.
For further details of the LAMP reaction principle, refer to Eiken GENOME SITE
(URL: http://loopamp.eiken.co.jp/e/).

Amplification curve pattern[How to use]
Positive Control

Positive sample 1

0.7

0.7

0.6

0.6

0.5

0.5

0.4
0.3

Negative sample

0.3
0.2

0.1

0.1
00102030

405060Time (min)Fig.1 The amplification curve pattern of controls*3Positive sample 20.4

0.20Vortex mixer

0102030405060Time (min)Fig.2 Samples amplification curve pattern*3: This kit is not developed for the purpose of quantitative analysis, therefore, the copy
numbers does not necessarily correlate with turbidity increment time.

2. Sample solution preparation
In the case of using pre-enrichment culture as specimen for the detection of Salmonella[Caution for operation]

spp. in foods:

1. Sample handling

Food 25g + 225mL of Buffered Peptone Water (BPW) or EEM Bouillon Media*21) When conducting pre-enrichment, the stomacher bag with filter should be used.

→ Stomacher treatment →(Incubate at 36±1C for 20-24 hours) → Pre-enrichment culture*2

Negative Control

TurbidityTurbidity

1. 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 TubeAluminum rack for cooling tubes
Crushed ice and ice box
Loopamp Realtime Turbidimeter
Centrifuge for microtubes
Centrifuge for 8-strip tube
High-speed centrifuge (centrifuge for micro-tubes with 2,000×g or above can also be used)

2) When collecting pre-enrichment culture, pipette carefully as to not convolute the precipitate.

: For the pre-enrichment of liver related materials, EEM Bouillon Media should be used.

3) 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 thawing1) Specimen pre-treatment (Preparing sample solution)(1) Prepare necessary quantity of sterilized tubes for specimen pre-treatment, and dispense

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

50L of pre-enrichment culture into each tube.
(2)

Close the caps, centrifuge at 2,000×g for 5 minutes. Remove the supernatantthe necessary amount of reagents from the freezer before use. No decline was observed in the kitcarefully as to not convolute the precipitate. If there is no precipitate in theperformance even after repeated freezing and thawing for 20 times in the quality control test. But,in order to maintain the reagents performance, avoid unnecessary freezing and thawing.

solution, remove 40L of supernatant.

2) Thaw the reagents at room temperature and keep them on ice for reagents preparation. Before use,

(3) Add 80L of Extraction Solution for Foods (EX F).

spin down the tubes to drop down the reagents staying on the tube wall or on the cap, mix well the(4) Close the caps and mix by vortex mixer. Centrifuge for a few seconds (hereinafterreagents and spin down again. Notice that fierce mixing should be avoided as it can inactivate thereferred to as spin down) and incubate at 95C for 5 minutes.

Bst DNA polymerase.

1/2

equipment. 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 of samplesolution and 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
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 Control DNA Sal (Cont Sal) 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 thereaction started). There are cases where the turbidity of the sample starts rising later thanthe 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 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 until thecontamination 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.

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 be limited as minimum aspossible. Please add Extraction Solution for Foods (EX F) as soon as possible forpre-treatment. When storing Extraction Solution for Foods, keep its cap tightly closed, anddo not aliquot the solution.
4) Control DNA Sal (Cont Sal) contains high number of target DNA. In order to prevent
Control DNA Sal 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 thereaction tubes under the following order from negative control (Extraction Solution forFoods (EX F)), sample solution (extracted DNA), and leave the adding of positive control(Control DNA Sal (Cont Sal)) 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 Sal (Cont Sal)in any other way not written in this instruction (such as diluting the positive control oradding 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.

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 pre-enrichment culture into the separately prepared pre-treatment tube.
Centrifuge at 2,000×g for 5 minutes. Remove the supernatant with pipette*4.
Add 80L of Extraction Solution for Foods (EX F).
Mix by vortex mixer. Spin down and incubate at 95C for 5 minutes.

[Performance]

Add 10L of 1M Tris-HCl: pH7.0 (Tris)[Caution for handling]

Sensitivity: 60 CFU (Colony Forming Unit)/ test

1. LAMP reaction is very sensitive and even the slightest amount of amplified product tainted into thereaction might cause false result. Therefore, avoid this type of contamination by carrying out theMix by vortex mixer and centrifuge at 2,000×g for 30 seconds at room temperature.

sample and reagent preparation in different clean benches. Avoid electrophoresis or operations
that need to handle amplified products.

*5

Use the supernatant as sample solution (on ice) .

2. Rappaport Media, which are used for selective enrichment of Salmonella spp. should not be usedbecause they contain components that influence LAMP reaction.
3. About handling pre-enrichment media, refer to their instruction manuals. When handling the sample,always abide by the biohazard counter measures 10).

<Master mix. preparation> (Operate on ice)

4. Do not expose the Loopamp Reaction Tube, master mix preparation tubes to UV light. A change inPrepare the necessary volume of master mix (including positive and negative controls)

Master mix preparation (for 1 test)
Reaction Mix. Sal (RM Sal)
Bst DNA polymerase
Total

color or deterioration caused by ultraviolet lamp sometimes results in misjudgment.
5. This kit is for the purpose of foods and environment inspection, not for medical or clinical

20L
1L
21L

diagnostic 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 the supervisionof the experienced and knowledgeable technicians.

Mix thoroughly*6, then spin down and use as master mix (on ice).

7. This kit can detect Salmonella spp. through amplifying its gene, which is different from theconventional culture method that can only detect viable bacteria of Salmonella spp. Use this kit<Procedure>(Operate on ice)

as a part of self-imposed test.
8. The result of this kit might be different from that of the culture method.

Dispense 20L of master mix into each reaction tube.

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.

Add 5L of sample solution or control into the tube

10. Use the kit before the expiration date, which is labeled on the outer box (Exp. Date).

(Total of 25L as LAMP reaction solution.)

11. 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).

Add 5L of Control DNA Sal (Cont Sal) for positive control, and 5Lof Extraction Solution for Foods (EX F) for negative control.

12. The reagent tube is made of polypropylene and the main material for kit case is paper. The
institution disposing the reagent tube and case should bear the responsibility and abide by

Mix the solution well by pipetting or tapping the tube with the cap closed and then spin down.

the clinical waste disposal regulations, water pollution prevention law, and any otherregulation related.

(Be careful not to cause air bubbles when mixing.)[Unit, Storage, Expiration, Code No.]

<LAMP reaction>

Product Name

Set the reaction tubes into the reaction blocks of Loopamp Realtime Turbidimeter and start reaction.

LoopampTM Salmonella Detection
Kit

65C, 60 minutes
Enzyme inactivation (80C, 2min).

Unit

Storage

Expiration

Code No.

48 tests-20 C

1 year

LMP601[References](Done by Loopamp Realtime Turbidimeters automatically.)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 ofJapan (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 Societyof Japan (2003)
6) Galan J. E. et al.: J Bacteriol. 174, No.13: 4338-4349 (1992)
7) Yoshino M. et al.: Abstract for the 23rd Annual Meeting of Japanese Society of Food
Microbiology, P26 (2002)
8) Hara-Kudo Y. et al.: FEMS Microbiol. Lett. 253, No1: 155-161 (2005)9) Ohtsuka K. et al.: Appl. Environ. Microbiol. 71, No.11: 6730-6735 (2005)
10) 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)

Detection and judgment.
judgment*4: Remove the supernatant carefully as to not convolute the precipitate. If there is no
precipitate in the solution, remove 40 L of supernatant.
: Sample solution can remain stable at 0~4C for 4 hours.
*6: Mix by tapping, invert the tube or by vortex mixer at 1second×3 times.
*5

3. Handling the 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 orscratch on the tube might not only cause false judgment but also contaminate theManufacturer

2/28103

ＡＭＫ

Rev. Feb.2013