(4) After incubation, spin down the tube and add 10L of 1M Tris-HCl: pH7.0 (Tris).
(5) Close the caps and mix by vortex mixer. After centrifuge at 2,000×g for 60 seconds at

For research use

room temperature, keep it on ice and use the supernatant as sample solution (remainstable at 0~4C for 4 hours).
3. Preparing the reagents
1) Take out the reagents stored at -20C, and thaw them at room temperature.
Once the 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 2 × Reaction Mix. (RM) 12.5L, Primer Mix.Cam (PM Cam) 2.5L,Distilled Water (DW) 4L and Bst DNA Polymerase 1L per test (including positive andnegative control tests). For master mix preparation, it is recommended to prepare one test
amount extra to the amount that is needed for the number of tests (including positive andnegative control tests).
(2) After dispensing, mix the solution by gently tap the tubes a few times (hereinafter referredto as tapping), or invert the tube, or by vortex mixer at about 1 second×3 times. After
mixing well, spin down 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 the polymerase,
and assure that vortexing is conducted at 1 second×3 times.
The prepared master mix should be used as soon as possible.

Campylobacter 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
primers which recognize the sequences related to the Oxidoreductase genes 6 knows as theoxidase related gene from Campylobacter jejuni (hereinafter referred to as C.jejuni), and theAspartate kinase gene7 known as the amino-acid synthetase related gene from Campylobactercoli (hereinafter referred to as C.coli)*1. From whether the amplification occurred or not, theexistence of C.jejuni and C.coli can be determined. By using the specifically designedLoopamp Realtime Turbidimeters, detection does not need electrophoresis and all steps fromamplification to detection are done within one reaction tube. Simple and rapid detection ofC.jejuni and C.coli can be achieved6),7).
*1: This kit does not respectively identify C. jejuni or C. coli, but can detect the amplification
if either or both of the species exist.

[Contents of the kit]

4. Operation procedure1) Mixing 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 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 Positive Control Cam (PC Cam) for positive control,and 5L of Distilled Water (DW) for 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 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. 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 Cam (PC Cam).
(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.

48 tests(EX F)*2
1.8mL × 2 tubes
1.0mL × 1 tube
(Tris)*20.6mL × 1 tube
(RM)*20.12mL × 1 tube
(PM Cam)*22
1.0mL × 1 tube
(DW)*
60µL × 1 tube
(Bst DNA Polymerase)*22(PC Cam)*
0.1mL × 1 tube
*2: The notation on each reagent tube is shown in ( )(1) Extraction Solution for Foods(2) 1M Tris-HCl: pH7.0(3) 2× Reaction Mix.
(4) Primer Mix. Cam(5) Distilled Water(6) Bst DNA Polymerase(7) Positive Control Cam[Intended use]
Detection of C.jejuni and C.coli. in foods or environmental specimens.

[Assay 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 as sample. Mix the sample solution with 2×Reaction Mix.
(RM), Primer Mix. Cam (PM Cam), Distilled Water (DW) and Bst DNA polymerase, andthen incubate it. When C. jejuni Oxidoreductase gene sequence and C.coli Aspartate kinase
gene 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 are C.jejuni and C.coli8).
For information on LAMP reaction principle, refer to Eiken GENOME SITE(URL: http://loopamp.eiken.co.jp/e/).

Amplification curve pattern
Positive ControlTurbidity[How to use]
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 Tube
Aluminum rack for cooling tubes
Crushed ice and ice box
Loopamp Realtime TurbidimeterCentrifuge for microtubes
Centrifuge for 8-strip tube
High-speed centrifuge (centrifuge for micro-tubes with 2,000×g or above can also be used)

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
00

102030405060Time (min)Fig.1 The amplification curve pattern of controls*3

Positive sample 2

0.4

0.2

00

10

2030405060

Time (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.

[Caution for operation]
1. Sample handling
1) When conducting pre-enrichment, the stomacher bag with filter should be used.
2) When collecting pre-enrichment culture, pipette carefully as to not convolute theprecipitate.
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 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 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

Vortex mixer

2. Sample solution preparation
In the case of using pre-enrichment culture as specimen for the detection of C.jejuni andC.coli in foods:
Food 25g + 100mL of Preston Media

Negative ControlTurbidityLoopampTM→ Stomacher treatment→(Incubate at 42C for 24 hours under microaerophilic condition) → Pre-enrichment culture1) Specimen pre-treatment (Preparing sample solution)(1) Prepare necessary quantity of sterilized tubes for specimen pre-treatment, and dispense
50L of pre-enrichment culture into each tube.
(2) Add 50L of Extraction Solution for Foods (EX F).
(3) Close the caps and mix by vortex mixer. Centrifuge for a few seconds (hereinafterreferred to as spin down) and incubate at 95C for 5 minutes.

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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 sample
solution 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 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 Positive Control Cam (PC Cam) has risen todetermine whether the reagent is performing properly (if the nucleic acid amplification
reaction is performed properly, the turbidity will start to rise around 30 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
but also 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.

the cap, mix well the reagents and spin down again. Notice that 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. 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
possible. 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) Positive Control Cam (PC Cam) contains high number of copies. In order to prevent
Positive Control Cam 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(Positive Control Cam (PC Cam)) to the last and make sure that all other tube caps areclosed when adding it. Moreover, to avoid contamination, do not use Positive Control Cam(PC Cam) in any other way not written in this instruction (such as diluting the positivecontrol 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.

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.

Add 50L of Extraction Solution for Foods (EX F).

[Performance]Mix by vortex mixer. Spin down and incubate at 95C for 5 minutes.

Sensitivity: 60 CFU (Colony Forming Unit)/ test

[Caution for handling]
After spin down, add 10L of 1M Tris-HCl: pH7.0 (Tris)

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 bycarrying out the sample and reagent preparation in different clean benches. Avoid
electrophoresis or operations that need to handle amplified products.
2. Rappaport Media, which are used for selective enrichment of C.jejuni and C.Coli should
not be used because they contain components that influence LAMP reaction.
3. About handling pre-enrichment media, refer to their instruction manuals. When handlingthe sample, always abide by the biohazard counter measures 9).
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 C. jejuni and C. Coli through amplifying its gene, which is different fromthe conventional culture method that can only detect viable bacteria of C .jejuni and C.
Coli. 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. 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).
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 andabide by the clinical waste disposal regulations, water pollution prevention law, and anyother regulations related.

Mix by vortex mixer and centrifuge at 2,000×g for 60 seconds at room temperature.
Use the supernatant as sample solution (on ice). *4<Master mix. preparation> (Operate on ice)

Prepare the necessary volume of master mix*5
Master mix preparation (for 1 test)
12.5L
2 × Reaction Mix. (RM)
2.5L
Primer Mix. Cam (PM Cam)
4L
Distilled Water(DW)
1LBst DNA polymeraseTotal20L
Mix thoroughly*6, then spin down and use as master mix (on ice).

<Procedure> (Operate 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 Cam (PC Cam) for positive control, and5L of Distilled Water (DW) 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>[Unit, Storage, Expiration, Code No.]

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

Product Name

65C, 60 minutes

LoopampTM Campyrobactor
Detection Kit

Enzyme inactivation (80C, 2 minutes)(Done by Loopamp Realtime Turbidimeters automatically.)

Unit

Storage

Expiration

Code No.

48 tests-20 C

1 year

LMP721[References]
Detection andjudgment

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) Wang R. F. et al.: J Rapid Methods Autom.Microbiol. 1: 101-108 (1992)
7) Linton D. et al.: J Clin. Microbiol. 35, No.10: 2568-2572 (1997)
8) Momoda T. et al.: Abstract for the 26th Annual Meeting of Japanese Society of Food
Microbiology, P82 (2005)
9) 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)

judgment*4: Sample solution can remain stable at 0~4C for 4 hours: It is recommended to prepare one test amount extra to the amount that is needed for thenumber of tests (including positive control and negative control).
*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 or
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

Manufacturer

2/2

8107

AMK

Rev. March .2013