For research use

Loopamp TM

Legionella Detection Kit E[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 conditions 1) 2). (2) 4 primers recognizing 6 distinct
regions on the target achieve high specificity. (3) High amplification efficiency allows
amplification within a short time. (4) It produces tremendous amount of amplified products
which makes simple detection possible 3) 4) 5). Amplification of nucleic acids with this kit isconducted by the LAMP method using Legionella spp. 16S rRNA sequence as the primers.
The homology between the sequence for primers and that of non-Legionella spp. are very low,in contrast, high specificity in detection of the 11 species and 21 strains Legionella including
the 11 serotypes Legionella pneumophila when using LAMP method for amplification.
From whether the amplification occurred or not, the existence of Legionella spp. can bedetermined. By using the specifically designed Loopamp Realtime Turbidimeters, detection
does not need electrophoresis and all steps from amplification to detection are done within
one reaction tube. Comparing with conventional culture methods6), LAMP detection ofLegionella can be done much faster7)8).

[Contents of the kit]
48 tests
1.8 ml × 2 tubes(1) Extraction Solution for Legionella (EX Leg) *1
1.0ml × 1 tube
(2) 1M Tris-HCl:pH7.0(Tris)*1
1.0mL× 1 tube
(3) Reaction Mix. Leg(RM Leg) *1*1
(4) Bst DNA Polymerase(Bst DNA Polymerase)
60 l × 1tube*10.1 ml × 1 tube
(5) Positive Control Leg(PC Leg)*1: The notation on each reagent tube is shown in ( ).

[Intended Use]
Detection of Legionella spp. in environmental specimens (hot spring water, bath tub water,cooling tower water etc.) and concentrate it 100times by filtrate concentrating or coolingcentrifugation concentrating to detect Legionella spp6).

then remove the supernatant (about 1960µL) and leave around 40µL.
2) After spin down, add 50µL of Extraction Solution for Legionella (EX Leg) and mix it wellusing vortex mixer.
3) After spin down, incubate at 95 ºC for 15 minutes and cool it on ice immediately.
4) After spin down, add 8µL of 1M Tris-HCl: pH7.0 (Tris) and mix it well using vortex
mixer.
5) Centrifuge under 4 ºC, 13,000×g for 10 minutes, then keep it on ice, use the supernatantas sample solution (equivalent 2000 times concentrated sample water).
4. Operation procedure1) Mixing Master Mix with 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 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 Leg (PC Leg) as positive control and5 l of Extraction Solution for Legionella (EX Leg) for as negative control.
2) The amplification reaction and realtime turbidity detection(1) Loopamp Realtime Turbidimeters are applicable to this kit. Turn on the device about 20
minutes before use. 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 screenof the device. If the positive control’s: Positive Control Leg (PC Leg) turbidity rose andthe negative control’s: Extraction Solution for Legionella (EX Leg) turbidity didn’t, thenLAMP reaction proceeded properly (Fig.1). If not, there might have been error in theprocess. Restart from reagent preparation and check again.
(5) If the increase in turbidity is confirmed within 60 minutes, then it is judged as positive, ifnot, negative (Fig.2).
(6) The rising timing or turbidity of samples might be different from the positive control.
(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.
Amplification Curve Pattern
Positive Control

Negative Control

Positive Sample1

0.4

0.4

0.3

0.3

Turbidity

This kit uses LAMP method as assay principle. First, extract DNA from concentrated
environmental water specimen (concentrated sample water) with alkalis heat extraction
method and then neutralize the DNA solution, and use it as sample solution. Mix the sample
solution with Reaction Mix. Leg and Bst DNA Polymerase and incubate it. When Legionella
16S rRNA sequence can be recognized by the primer, its DNA will be amplified with theactivity of Bst DNA polymerase. The detection of the nucleic acid is done by detecting theturbidity change3)4) caused by the amplification by-product magnesium pyrophosphate (whiteprecipitate) and then determines whether there is Legionella spp.
For further details of the LAMP principle, refer to Eiken GENOME SITE (URL:
http://loopamp.eiken.co.jp/e/).

Turbidity[Assay principle]

0.2

0.1

1. Materials required but not provided
Sterilized tubes for master mix preparation (0.5mL or 1.5mL)
Micropipette (0.5~10 L, 10~100 L, 100~1000 L)
Pipette tips with filter
Sterilized tubes (2.0mL)
Loopamp Reaction Tube
Aluminum rack for cooling tubes
Heat block (Capable of heating up to 95 ºC )Crushed ice and ice box
Loopamp Realtime Turbidimeter
Refrigerated centrifuge for microtube
Centrifuge for 8-strip tube
Vortex mixer
2. Preparing the reagent
1) Take out the reagents stored at -20ºC, and thaw them at room temperature. Once thereagents are thawed, keep them on ice immediately.
2) Preparation of master mix (Operate on ice)
(1) Dispense the appropriate amount according to the number of tests to be conducted into
the separately prepared sterilized tube under the proportion of Reaction Mix. Leg (RMLeg) 20µL and Bst DNA Polymerase 1µL per test (including positive control andnegative control tests).
(2) After dispensing, mix the solution well by gently tap the tube a few times (hereinafterreferred to as “tapping”), invert the tube, or vortex mixer at 1 second ×3 times. 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,and assure that vortexing is conducted at 1 second×3 times. The prepared master mix
should be used as soon as possible.
3. Preparing sample solution
DNA extraction solution can be prepared by using alkali heat extraction method9).
1) Put 2mL of concentrated sample water (100 times concentrated sample water6)) in theseparately prepared sterilized tube (2mL) and centrifuge at 4 ºC, 13,000×g for 10 minutes

Negative Sample

0.20.1000102030405060

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

[How to use]

Positive Sample20102030405060

Time (min)Fig.2 Samples amplification curve pattern.

This kit is not developed for the purpose of quantitative analysis, therefore, the copy
numbers do not necessarily correlate with turbidity increment time.

[Cautions for operation]
1. Sample handling
1) When removing the supernatant (about 1960 L) from 2mL of 100times concentrated water
sample, pipette carefully as to convolute the precipitate.
2) 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 andthawing should be avoided.
2. Reagent handling
1) The reagent kit should be stored at -20ºC. 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
the quality control test. But, in order to maintain the reagent performance, avoid
unnecessary freezing and thawing.
2) Thaw the reagent at room temperature, and keep them on ice for reagents preparation.
Before use, spin down the tubes to drop down the solution staying on the tube wall or on
the cap. Mix well the solution and spin down again before use. Notice that fierce mixing
should be avoided as it can inactivate the Bst DNA Polymerase.
3) Positive Control Leg (PC Leg) contains high number of target DNA. In order to prevent
Positive Control Leg from contaminating other samples or reagents, always spin down thetube, and open the cap as shortly as possible. Moreover, to avoid contamination, do not use
Positive Control Leg in any other way not written in this instruction.
4) Keep positive and suspected positive samples away from the reagent when handling5) If there is any reagent left, do not mix or use it kits even if they are in the same lot.

Protocol<Pre-treatment; DNA extraction solution> (Operate on ice)Put 2mL of sample water (100 times concentrated sample water6)) in separately preparedsterilized tubes.
4 ºC, 13,000×g, 10 minutes

7. Inhibitory substance
LAMP reaction will be inhibited if there is over 3µmol/L of Iron ion or 500µmol/L of
Manganese ion in the 25µL of reaction solution. When use hot spring water which contains alarge amount of metallic ion as sample water, be careful that it might be interfered LAMP
reaction.

[Performance]

Remove the supernatant (about 1960µL) and leave it around 40µL
Pipette carefully not to convolute the precipitate.
After spin down, add 50µL of Extraction Solution for Legionella (EX Leg)and mix by vortex mixer.

After spin down, incubate at 95 ºC for 15 minutes and cool it on ice immediately.

After spin down, add 8 L of 1M Tris-HCl:pH7.0 (Tris)
Mix well by vortex mixer and then centrifuge at 4 ºC, 13,000×g, 10 minutes
Use the supernatant as sample solution (on ice).

1. Specificity
The 11 spices 21 strain of Legionella spp. that contains 11serogroupe of Legionella
pneumophilla can be specifically detected. The 19 species 19 strain of non-Legionella
spp. that contains community acquired pneumonia pathogen cannot be reacted with thiskit7)8).
2. Sensitivity
Minimum detection limit: 60 CFU (Colony Forming Unit)/test*5*5: The sensitivity is determined by using preserved strains of Legionella pneumophilla.
When use actual environment water, it cannot be compared directly with theperformance and sensitivity of conventional culture method.
3. Correlation of culture method
49 water samples which showed the positive results with culture method are determined
all positive by LAMP method, and these results do not find discrepancy. In the 51 water
samples determined as negative with culture method showed 22 of them as positive
results by LAMP method11).

<Preparation of master mix.> (Operate on ice)
Prepare the necessary volume of master mix. and mix well,(including positive, negative control)
Preparation of master mix. (per test)
Reaction Mix. Leg (RM Leg)
Bst DNA Polymerase
TotalMix well

<Procedure> (Operate on ice)

20 l
1 l
21 l*4and spin down the master mix, then keep it on ice

Dispense 20 l of master mix. into each reaction tube

Add 5 l of sample solution, or control (Total of 25 l as LAMP reaction)
Add 5 l of Positive Control Leg (PC Leg) for positive control and add 5 l ofExtraction Solution for Legionella (EX Leg) for negative control
Mix by tapping or pipetting then spin down
Be careful not to cause air bubbles<LAMP reaction>
Set the samples in turbidimeter reaction block and start reaction
65ºC, 60 min
Inactivation (80ºC, 2 min)(Loopamp Realtime Turbidimeter will perform inactivation automatically)
Turbidity detection, judgment*4:Mix by tapping or invert them several times or vortex them for 1 second × 3 times.

[Cautions for handling]
1. This kit is for the purpose of environment detection of Legionella spp., not for medical or
clinical diagnostic purposes on human or animal samples.
2. Although the test result is negative, this test is incapable of determining the complete
absence of Legionella spp. Combined interpretations with other test results arerecommended.
3. Abide by the necessary counter biohazard regulations for sample taking and handling6)10).
4. LAMP reaction is extremely sensitive and even the slightest amount of amplified products
tainted into the reaction might cause the 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.
5. Do not expose Loopamp Reaction Tube, master mix. preparation tubes to UV light. Achange in color or degeneration caused by ultraviolet lamp sometimes results in
misjudgment.
6. If there is DNase in the reaction solution, the reaction might cause the false result. Pay
attention to handle the operation under proper environment with clean equipments andinstruments.
7. If the operator does not have the knowledge or experience in genetic test, there is apossibility of false judgment. Therefore, be sure to use the kit under the supervision of thegenetic testing experienced and knowledgeable technicians.
8. EIKEN CHEMICAL CO., LTD. does not bear responsibility for false judgment or any
consequential damage from the misuse or mishandling.
9. Use the kit before the expiration date, which is labeled on the outer box (Exp. Date).
10. This kit should be stored at -20 ºC. Storing the kit at temperature lower than -20 ºC or repeatedfreezing and thawing might cause cracks on the tube of 1M Tris-HCl:pH7.0(Tris).

11. The reagent tube is made of polypropylene and the main material for kit case is paper.
The institution disposing the reagents, reagent tubes, container, and lab-ware and caseshould bear the responsibility and abide by the clinical waste disposal regulations, waterpollution prevention law, and any local regulation related.

[Unit, Storage, Expiration and code No.]
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 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 sample
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. Cautions 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 use it.
3) For judgment, check whether the turbidity of Positive Control Leg (PC Leg) 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 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 tube 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 might not be obtained until
the contamination is removed completely.
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 dispensing, do not conduct autoclavesterilization treatment for disposal.

ProductUnit

Storage

Expiration

Code No.

LoopampTM
Legionella Detection Kit E

48 tests-20°C

1 year

LMP661[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 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）The supervised by the Ministry of health, labour and welfare, Living environment: Theguideline for the prevent of Legionella disease (in Japanese, 2000).
7）Annaka T. et al.: J. Japanese society for Clin. Microbiol. 13, No.1, 19-25 (2003).
8）Annaka T. et al.: JARMAV, 14, No.1,25-30 (2003)
9) Beige, J. et al.: J. Clin. Microbiol. 33 : 90-95 (1995)
10) The guideline for the bio-safety and bio-hazard (by the Japanese Society forBacteriology) Japanese Journal of Bacteriology 54, No.3: 667-715 (1999)
11) Inoue H, et al.: SAAAJ, 32, No.10, 481-487 (2004)

Manufacturer8109

AMK

Rev. March. 2013