3. Reagents preparation
1) Take out the reagents from its storage at -20C, thaw them at room temperature.
Right after they are thawed out, keep them on ice.
2) Preparation of master mix. (Operate on ice).

For research use

LoopampTM
Primer Set for Koi herpesvirus

Referring to the table below, dispense the necessary amount of the reagents into thesterilized tube for master mix. preparation. Primer Mix. KHV (PM KHV) iscontained in this reagent, and 2×Reaction Mix. (RM), Bst DNA Polymerase, andDistilled Water(DW) in Loopamp DNA Amplification Kit.

[Characteristics]
LAMP (Loop-mediated Isothermal Amplification) method is a gene amplification

Reagents

method captured the following main characteristics: ( 1 ) Only one enzyme is required

Amount: 1 testAmount: 10 tests

2 × Reaction Mix. (RM)

12.5 L125 L

Primer Mix. KHV (PM KHV)

2.5 L25 L

amplification efficiency allows amplification within a shorter time. (4) It produces

Bst DNA Polymerase

1.0 L10 L

tremendous amount of amplified products which makes simple detection possible3), 4), 5), 6).

Distilled Water (DW)

4.0 L40 L

20.0 L

200 Land the amplification reaction proceeds under isothermal condition1), 2). (2) Four primers

recognizing 6 distinct regions on the target achieve high specificity.

( 3 ) HighThis reagent consists of the primer set for the detection of the genomic DNA of koi

Total

herpesvirus; KHV 7), which is specifically designed for Loopamp DNA Amplification Kit8).

3) Fluorescent visual detection
Substitute Loopamp Fluorescent Detection Reagent (FD) for 1L of Distilled water in[Contents of the kit]
(1)(2)(3)(4)

48 tests
Extraction Solution for F(EX F) *1
1.8 mL × 3 tubes
1.0 mL × 1 tube
1M Tris-HCl: pH 7.0(Tris) *1
0.12 mL × 1 tube
Primer Mix. KHV(PM KHV)*1
0.1 mL × 1 tube
Positive Control KHV(PC KHV)*1*1:The notation on each reagent tube is shown in ( ).

master mix. Add 3L of Distilled Water (DW) and 1L of Fluorescent Detection
Reagent (FD) in the master mix and the total amount is 20L per 1 test.
4. Operation procedures (Follow the instruction of Loopamp DNA Amplification Kit).
1) Mixing master mix. solution and the sample solution (Operate on ice).
(1)Dispense 20L of the master mix. to each Loopamp Reaction Tube.

[How to use]

(2) Add 5L of sample solution, Positive Control KHV (PC KHV) or Negative Control

1. Materials required but not provided
1.5mL Homogenization Pestle for 1.5mL microtube(Distilled Water (DW)) to the master mix., and the volume of the solution is 25L intotal.

Sterilized tubes for sample preparation (1.5mL)

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

Sterilized tubes for master mix preparation (0.5mL or 1.5mL)* Be careful not to cause air-bubbles on mixing.

Micropipette (0.5~10 L, 10~100 L, 100~1000 L)* Check visually if proper amount of sample solution has been dispensed into thePipette tips with filter

reaction tube.

Aluminum rack for cooling tubes

2) Amplification and detection

Crushed ice and ice box(1) Set the temperature at 65C with Loopamp Realtime Turbidimeters or incubator

Heat block (Capable of heating up to 95 ºC )with hot bonnet (temperature accuracy within±0.5C). Confirm that the temperature

Centrifuge for microtube
Centrifuge for 8-strip microtube

has reached at 65 C.

Vortex mixer(2) Incubate the reaction tube at 65 C for 60min.

Loopamp Reaction Tube

(3) After the amplification reaction, inactivate the polymerase with heat block (80C for

Loopamp Realtime Turbidimeters

5min or 95C for 2min)and terminate the reaction.

TE buffer for simplified extraction method (10mM Tris-HCl,1mM EDTA:pH8.0)* Loopamp realtime turbidimeters perform enzyme inactivation automatically.

Commercially available DNA isolation reagent (DNeasy Tissue Kit: made byQIAGEN etc.)* Make sure that the enzyme is inactivated when detection process is performed after theLoopamp DNA Amplification Kit

amplification reaction as the separate step (the visual fluorescence detection).

2. Preparation of the specimen
1) For use commercially available DNA isolation reagent (for example, DNeasy Tissue Kit,

<Detection with Loopamp Realtime turbidimeter>(1) Loopamp Realtime Turbidimeters are applicable to this kit. Turn on the device aboutQIAGEN.)20 minutes before use.

(1) When carry out the extraction of DNA from the specimen using a commercially

Set the parameters for detection of KHV.

(2) Confirm that the temperature has reached 65ºC

available DNA isolation reagent, refer to the instruction manual in the kit.

(3) Set the prepared reaction tubes and immediately start reaction.

(2) After extraction, incubate at 95 ºC for 5min then keep it on ice.

(4) Check whether the positive and negative controls’ turbidity rose from the display

2) For use simplified extraction method for LAMP

screen of the device. If the positive control’s (Positive Control KHV (PC KHV))(1) Put 5~10mg of specimens taken from infected fishes in the sterilized tubes forturbidity rose and the negative control’s (Distilled Water (DW)) turbidity didn’t, thensample preparation, and add 100 L of TE buffer, then homogenize the specimen with

LAMP reaction proceeded properly (Fig.1). If not, there might have been error in theHomogenization Pestle.

process. Restart from reagent preparation and check again.

(2) Add 100 L of Extraction Solution for F (EX F), and close the cap, then mix it by(5) If the increase in turbidity is confirmed within 60 minutes, then it is judged asvortex mixer (1 second × 3 times).

positive, if not, negative.

(3) Centrifuge for a few second (hereinafter referred to as “spin down”), then incubate(6) After enzyme inactivation (80ºC, 2 min), is confirmed to have ended, doneat 95 ºC for 5min.

automatically by LoopampRealtime Turbidimeter, remove the used reaction tubes(4) Add 10 L of 1M Tris-HCl: pH7.0 (Tris), then mix by vortex mixer.

and dispose them with caps closed.

(5) After mixing, centrifuge at over 2,000×g (room temperature) for 30 seconds, then

* The rising timing or turbidity of samples might be different from the positive

keep it on ice. Use the supernatant as sample solution.

control.

* If the simplified extraction method is used, its sensitivity might decrease incomparison with commercially available DNA isolation reagent.
* When handling the sample, adopt appropriate measures to avoid spreading of KHV toenvironment and prevent contaminations of the DNA to the sample.

1/2Amplification Curve Pattern
DW4) Tomita N. et al. : Abstract for the 73rd Annual Meeting of the Japanese Biochemical

PC KHVSociety (2000)0.65) Mori Y. et al. : Abstract for the 23rd Annual Meeting of the Molecular Biology Society

0.5of Japan (2000)

Turbidity0.46) Tomita N. et al. : Abstract for the 26th Annual Meeting of the Molecular Biology
Society of Japan (2003)

0.37) Gilad O. et al. : Dis. Aquat. Organ. 11, No.48, 101-108 (2002)

0.28) Yoshino M. et al. : Fish Pathology, 41, No.1, 19-27 (2006)

0.100102030405060Manufacturer

Time ( min )Fig.1 The real-time amplification of Positive Control KHV (PC KHV)*2*2: There is no correlation between the initial template number and the8028

turbidity increment time.

AMKRev. March.2013<Fluorescence visual detection>(1) After the enzyme inactivation, irradiate the reaction tube with UV lamp (wavelength240~260nm or 350~370nm) from the bottom and observe the fluorescent light fromthe side of the tube. When green fluorescent light similar to positive control isobserved, the sample is judged as positive. When no fluorescence like negative
control is observed, the sample is judged as negative.
(2) When observing the reaction tube, wear protective goggles that can efficiently block
ultraviolet light.
(3) If necessary, save the detection result as picture format using digital camera.
* The copy number does not necessarily correlate with the fluorescent intensity.
* If the infected fish which were bred in a large amount of metallic contained groundwater is tested, there is a possibility of interfering LAMP reaction.
* If the judgment with UV lamp does not make clear, use the following methods,
1) observe using commercially available black light from the side of the tube, 2) observe
under the light by naked eye, 3) observe the detection result as picture format using digitalcamera.
* For examples of detection cases using this reagent, refer to the products information
page in EIKEN Genome Site (URL; http://loopamp.eiken.co.jp/e/) .

[Caution for Handling]
1. This reagent is designed for research use only.
2. If the operator does not have the experience or knowledge in the field of the nucleic
acid testing, there’s a possibility of false judgment. Therefore, please make sure that thereagent is used under the supervision of the experienced and knowledgeable technician.
3. If any false judgment problem that is not related to capability of the reagent itself shalloccur (operation error for instances), Eiken Chemical Co., Ltd. does not bear anyresponsibility.
4. Please use the reagent before the expiration date, which is labeled on the outer box (Exp.
Date).
5. 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,water pollution prevention law, and any local regulation related.

[Unit, Storage, Expiration, Code No.]
Product Name
LoopampTM

Primer Set for

Koi herpesvirus

Unit

Storage

Expiration

Code No.

48 tests-20C

1 yearPM0003[References]
1) Notomi T. et al. : Nucleic Acids Research 28, No.12, e 63 (2000)
2) Nagamine K. et al. : Clin. Chem. 47, No. 9, 1742-1743 (2001)
3) Mori Y. et al. : Biochem. Biophys. Res. Commun. 289, No.1, 150-154 (2001)

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