TYLCV Detection Kit Ver.2

Instruction Manual
version 11.0.1

TYLCV Detection Kit Ver.2
Instruction Manual version 11. 0. 1
[ Read the following instructions before the test ]Thank you very much for purchasing TYLCV Detection Kit Ver.2. Before using the kit, pleaseconfirm the following matters.
Notices for use-

This kit is the product to detect TYLCV ( Tomato yellow curl leaf virus ) using LAMP method.
This kit must not be used for clinical diagnosis, therapeutic purpose and the test other than
TYLCV detection.

-

Concerning the storage procedure of the kit, please read section 2 “Notes” for yourreference. The unopened kit is stable at -20˚C for 6 months. It should be kept in dark. Avoid
repeated freezing and thawing.

-

Use this kit according to this instruction manual. Nippon Gene Co., Ltd. has no responsibility
for any trouble caused by the incorrect use and the different purpose from instructions.

-Concerning the secondary use of the assay result by the kit, please be notified that the user
must be responsible for all the consequential damage from the mishandling or misuse.
Nippon Gene Co., Ltd. has no responsibility for any trouble other than that caused by kit
defects.

-

Regular test is needed because there is a case virus is not detected depend on the infected
part, infection level, and virus concentration. This kit is a simple detection kit for auxiliary
testing, so definite diagnosis cannot be guaranteed.

-

Please avoid running electrophoresis, autoclaving of amplified sample after test andpositive control in order to keep the environment free from contaminants.

-

In case of using reagents that are not included in this kit, please follow the notices in thesafety instruction of the reagent that you are using. Please do not mix the foreign reagentswith the reagents in this kit. Refer to the Safety Data Sheet (SDS) about safe use of thisproduct. SDS is exhibited in the homepage of Nippon Gene Co., Ltd.
URL; https://nippongene-analysis.com/-

This kit is not food. Do not put the reagents into an eye or a mouth. During test, wear a labcoat or gloves and protect the body.

-

Eiken Chemical Co., Ltd. owns the patent right for execution of Loop-mediated isothermal
Amplification. Nippon Gene Co., Ltd. has been granted the license to develop, manufactureand sell the kit for TYLCV detection.

Table of contents
Page
1. About the kit ······················································································ 1
Product Overview
Diagnosis of TYLCV disease
About LAMP (Loop-mediated Isothermal Amplification) method
About The Synthetic Oligonucleotide Included in This Kit

2. Reagents provided with the kit····························································· 2
Notes: Reagents provided and storage condition

3. Equipments and Reagents not provided with the kit······························· 3
4. Instructions ······················································································· 5
Simple protocol ······························································································· 5
TYLCV detection protocol ·················································································· 7
Sample preparation
How to prick the leaf
Equipment preparation
Testing environment
Detailed protocol ····························································································· 9
Test reaction
Judgement

5. Troubleshooting ··············································································· 16
6. References ······················································································ 17
7. Supplementary data ········································································· 18
Quality control
Strains

Other methods for sample preparationMemo1. About the kit
Product Overview
TYLCV Detection Kit offers detection of TYLCV (TYLCV; Tomato yellow leaf curl virus) from plant samples
or silverleaf whitefly using Loop-mediated Isothermal Amplification (LAMP) method. LAMP method is a fastand easy DNA amplification method which is also used for the diagnosis of influenza virus and detection of
norovirus, Legionella sp., Salmonella sp., and Verotoxin-producing E. coli, exhibiting excellent specificity andsensitivity. In this kit, a part of TYLCV DNA amplifies using LAMP method, and infection of TYLCV can be
judged if the amplification occurs or not.
The operation needed for the detection is extremely easy; simply soak the tooth pick into the test solution(mixture of TYLCV Detection Solution, TYLCV Enzyme Solution, and Fluorescent Detection Solution) in asample tube after pricking tomato leaf, other plant sample or silverleaf whitefly, and keep the tube at 60-65
degree Celsius for 60 minutes. The existence of TYLCV in the sample can be judged from whether the
specific sequence amplified with LAMP primer set or not.
For detection of DNA amplification, this kit utilizes visual inspection of fluorescence emitted from thesolution after the whole reaction, which means that the DNA amplification and detection can be done in one
closed tube. Therefore, the amplification of TYLCV DNA can be detected safely in a short period of time.
Diagnosis of TYLCVTomato yellow leaf curl disease is caused by infection of TYLCV which has arisen in Japan since the middleof 1990s. TTLCV is divided into Geminiviridae family, begomovirus genus and has cyclic single stranded DNA
as genomic DNA. TYLCV infection to tomato causes reduction of yields because of insufficient growthsubsequently to chlorosis and dwarf of leaves. Today, it is difficult to prevent the extension of virus infection
completely since silverleaf whitefly, the vector, is such minute that discovery and destruction are troublesome
nevertheless various countermeasures against TYLCV has been taken. Furthermore, since 2009, silverleaf
whitefly-biotype Q has been spreading which has different pesticide resistance to silverleaf whitefly-biotype B,
the conventional vector, which makes the situation much more difficult.
Even though ELISA (Enzyme-Linked Immunosorbent Assay) and PCR (Polymerase Chain Reaction) has
been developed to detect TYLCV, immuno-chromatography such as ELISA needs antiserum and antibody,furthermore, its low sensitivity leaves a problem of false positive/negative. Presently, DNA amplification by PCR
is considered to be valid way to detect TYLCV, but it needs at least two steps of repeated temperature change
and needs electrophoresis to consult the result, which matters when applying such troublesome method into
diagnostic area.
About LAMP (Loop-mediated Isothermal Amplification) Method
LAMP (Loop-mediated Isothermal Amplification) method allows the whole reaction process, including
denaturing, to proceed at a constant temperature in an incubator. Thermal cycling machine is not needed forthis kit.
Please refer the homepage of Eiken Chemical Co., Ltd. about the detailed principle of LAMP method.
Eiken GENOME SITE; https://loopamp.eiken.co.jp/en/
About The Synthetic Oligonucleotide Included in This Kit
The primers included in this kit are all “Reliable & Traceable oligo”. “Reliable & Traceable oligo” is one of
the highly reliable oligonucleotide series manufactured under control of ISO 13485:2003 certification by
Nippon Gene Material Co., Ltd. The oligonucleotides are all produced under dedicated positive pressure
environment, with checklists to control process and ensure the full traceability of the production.
Please refer the homepage of Nippon Gene Material Co., Ltd. about the details of “Reliable & Traceable
oligo”.
Nippon Gene Material Co., Ltd.; https://www.nippongenematerial.com/

12. Reagents provided with the kit[ Kit components (for 10 tests) ]Reagent(tube label)
Instruction Manual
Test TubeTYLCV Detection Solution
TYLCV Enzyme Solution
Fluorescent Detection Solution
TYLCV Positive Control
Mineral Oil

Form(top label)
BookletBag
Red label
Yellow label
Purple label
Gray label
Blue label

Contents
10 tests
1 booklet
10 tubes
230.0 µl15.0 µl15.0 µl
5.0 µl400.0 µl

Storagetemperature
Room temperature
Room temperature-20˚C (avoid light)-20˚C (avoid light)-20˚C (avoid light)-20˚C (avoid light)-20˚C (avoid light)

Notes
This kit can set up the test reactions up to 10 samples by preparing test solution for 10 tests in one
tube. Please be noticed that the number of the test that you can do will be less than 10 if you prepare
the test solution over several times from one kit.
If the water drop is attached to the test tube or bag, completely dry it before you open and use.
Store all reagents other than Instruction Manual and Test tube at -20˚C. Protect them from light. Use
until 6 months upon arrival date.
The reagents should be thawed each time when used, and the remaining should be stored at -20˚Cagain. Repeated freeze-thaw may deteriorate the quality of this kit. Aliquot the contents to several
tubes if necessary.
TYLCV Enzyme Solution must be handled with extra care. Do not leave the solution at room
temperature or 4˚C for a long period of time. Do not freeze it. Under inappropriate temperature
condition, the enzyme may lose its activity.
TYLCV Positive Control is the solution of DNA fragment which contains a DNA sequence specific to
TYLCV genomic DNA. To avoid cross-contamination, do not spill the solution, and avoid the contactof the used filter tip to the clean equipment and reagents.
Consecutive dispensing of the reagent may cause cross-contamination. Use the filter tip as adisposable in every dispensing batch.
Mineral Oiｌ might belong to the hazardous substance depending on the local administration. Pleaserefer to the local regulation concerning hazardous substances.

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3. Equipment and reagents not provided with the kit[ Required equipment and reagent ]Micropipette(0.5-10 µl, 10-100 µl, 200-1,000 µl)

Incubator
Any equipment that can maintain 60-65˚C
for given time (e.g. water bath, heating block,thermal cycler, air incubator etc.)

Filtered pipette tip (sterilized)

Tooth pick made of wood

[ Equipment which may be useful ]Microtube for preparation of test solution(1.5 ml or 2.0 ml)

Use these equipment if necessary:
Microtube rack

Disposable gloves

3

Plate rack
Centrifuge for test tube

Vortex mixer

Float plate
Use when the sample needs to be heatedwith water bath.
UV transilluminator
Use when the sample is detected with
Fluorescent detection solution.
Transilluminator should output the light withwavelength of 240-260 nm or 350-370 nm.

Centrifuge for microtube

UV goggles or face shieldTweezers
Crushed ice
Vinyl pack for sampling
100 mM Tris-HCl (pH 8.0)
0.5 N Sodium hydroxide

44. Instructions[ Simple protocol ]For the detailed protocol of this kit, please refer to the instruction at page 7 and later.

Simplified Protocol
1. Prepare the test solution
Reagent
TYLCV Detection Solution
Fluorescent Detection Solution

1test
23.0 µl

4+1test *115.0 µl

8+1test*207.0 µl

1.0 µl

5.0 µl9.0 µlTYLCV Enzyme Solution
1.0 µl
5.0 µlTotal
25.0 µl125.0 µl
* Prepare the test solution for the number of the samples
that you test + 1 test, so that you have enough volume
for the test solution of all samples.

9.0 µl
225.0 µl

2. Transfer 23.0 µl aliquot of the test solution into the test tube
test solution
25.0 µl

On ice3. Prick the sample by tooth pick

Silverleaf whitefly
Tomato leaf
Plastic wrap or paraffin film

5

Simple protocol
4. Soak the tooth pick into test solution
Positive control

Negative control
Do not add samples

TYLCV Positive Control
1.0 µltest solution
5. Add 20 µl of Mineral Oil
Mineral Oil 20.0 µl

6. Incubate at 63˚C for 60 minutes (DNA amplification)
7. Incubate at 80˚C for 2 minutes (Enzyme inactivation)
8. Judge the result
UV transilluminator

Visual detection

Negative:
Red

Positive:Green

6

Negative

Positive

[ Preparation and precaution before detection ]Sample preparation
ControlThis kit contains TYLCV Positive Control to confirm the success or failure of the detection
results. To confirm that the results are successful, attention should be paid to the preparation of“positive control reaction solution” with TYLCV Positive Control added, and also the “negative
control reaction solution”.
Sample preparation
In case of using this kit in the field, prick a part of tomato or other plants (in case of tomato, pickup the terminal bud or second leaf from the top) which is suspected to be infected with TYLCV
and put the tooth pick in a plastic bag. In order to prevent contamination and false
positive/negative, keep the tooth pick separated each other, and when collecting samples, wear
disposable groves so that they can be disposed when touched by the edge of the tooth pick.
Sample is on the edge of the tooth pick and can be used for detection directly.
Detection with tooth pick can be done also by using samples from tomato or other plant’s leafor stem after collecting and putting into plastic bag. Although withered samples can be used,
please use fresh samples for stable results. Observe the leaves carefully and test from the most
suspicious one because even if there is no symptom, TYLCV can exist. Furthermore, virus canbe detected from TYLCV resistant varieties with no symptom.
When using silverleaf whitefly as a sample, catch one in a container which can be made airtight,
add appropriate amount of 100 mM Tris-HCl (pH 8.0) or sterilized water in order to prevent
dispersion, and then prick the sample using tooth pick. It is also possible to test with samples
attached on yellow adhesion board by pricking using tooth pick, however, please note that thetest solution can be coloring green regardless of TYLCV existence if too much adhesion liquid is
added.
In sample preparation, appropriate measures in each facility should be performed in order not
to diffuse TYLCV in other cultivation environment. Especially, be careful not to transport silverleaf
whiteflies between facilities by using insect proof net and insecticide, and by changing clothes. In
order to prevent false positive/negative, used tooth picks and vinyl packs, and samples after thetest should be in doubled plastic bag and dispose. Furthermore, please avoid autoclaving ofdisposed samples.

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[ How to prick the leaf ]How to prick the leaf
When you prick a sample, do not penetrate the leaf. Too much juice of the leaf may
cause false positive.

Prick

Penetrate

Tomato leaf
In case if false positive is suspected, retest by pricking the leaf lightly on the hardtable with plastic wrap as following.
Important
In case that the leaf adheres to the tooth pick after picking, you should have picked
more lightly.

No adhesion

Prick

Plastic wrap

Plastic wrap
8Adhesion[ Preparation and precaution before detection ]Equipment preparation
Incubator
Turn on the power of an incubator and set the required temperature. In case of using a water
bath and a heat block, it might take time to reach the target temperature, so pre-heat the device
and confirm the temperature with a thermometer. In case of using an air incubator, thetemperature alters largely when the cabinet door is open. The door opening operation should bequickly done when the sample is set in an air incubator.
Other equipment
equipment
Micropipette
Microtube rack
Microtube
Filtered pipette tip(sterilized)
Writing materials
Disposable gloves
White robe

Instruction
Use micropipette exclusively in LAMP working area. Return to the original
location after nucleic acid removal operation in case if used in other area.
Use microtube rack exclusively in LAMP working area. Return to the original
location after nucleic acid removal operation in case if used in other area.
Select gamma-ray sterile, nucleic acid-free, and nuclease-free grade
microtube.
Select gamma-ray sterile, nucleic acid-free, and nuclease-free grade pipette
tip with hydrophobic filter, and unpack at working area. Consecutive
dispensing of the reagent with one tip may cause cross-contamination, use
the filter tip as expendables in every dispensing batch.
Use solely in each working area, and ensure a dedicated space for the
documents brought in the area.
Use gloves as expendables, change them when the contamination issuspected.
Use solely in each working area, be noticed of the contamination from thecuffs.

9Testing Environment
Because LAMP method is the DNA amplification technology with excellent sensitivities, it would
be difficult to make an accurate inspection if the testing environment is contaminated with TYLCV
positive control or amplified sample after test. For the handling of the sample, take extra care to
avoid the contact of the positive control and samples to the working white lobe and equipment.
Exchange of clothes is also strongly recommended. To prevent the false results after the test, theused tips, microtubes, and amplified sample after test should be packed together in doubled
plastic bags. Please avoid running electrophoresis, autoclaving of amplified sample after test andpositive control.
Working area
Assign a clean booth or working bench which has not used for nucleic acid extraction andamplification (which has not been contaminated with nucleic acid) as dedicated reagent
preparation area. Prepare the test solution only at the reagent preparation area. Do not use
TYLCV positive control, any solution or reagents that may become a template for LAMP method.
Separate the dedicated nucleic acid handling area from reagent preparation area. Addition ofsample and TYLCV positive control must be done only at nucleic acid handling area as the
dedicated working area.
Nucleic acid decontamination operation
Keep the equipment always clean. Wash the equipment with large amount of tap water todilute and wash off the nucleic acids on the surface if possible.
If it is suspected to have nucleic acids contamination on the surface of goods, especially after
handling highly concentrated nucleic acids, it is recommended to decontaminate the testing
environment from nucleic acids with 1% sodium hypochlorite aqueous solution. Sodium
hypochlorite generates chlorine gas and is corrosive on metals, so it is necessary to wipe
immediately the chlorine content from surface when used on metals. Sodium hypochlorite
aqueous solution can easily deteriorate under high temperature environment, so pay attention
to the days elapsed and storage temperature since 1% solution has been prepared.
<Protocol for nucleic acid decontamination using 1% sodium hypochlorite aqueous
solution>i) Wear disposable gloves on hands.
ii) Prepare 10,000 ppm (1%) sodium hypochlorite aqueous solution.
iii) Gently wipe the working bench and equipment with a paper towel moistened with sodium
hypochlorite aqueous solution followed by wiping with a paper towel moistened with 70%
ethanol.
iv) For non-metal equipment, soak the equipment in sodium hypochlorite aqueous solution for
more than one hour followed by rigorous rinsing with water and drying.
v) Keep working bench and equipment always clean, and perform wiping by sodium hypochlorite
aqueous solution regularly.

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[ Detailed protocol ]Test reaction
Thawing of the reagents
Thaw the following reagents at room temperature.
TYLCV Detection Solution
TYLCV Positive Control
Mineral Oil# Take out TYLCV Enzyme Solution and Fluorescent Detection
Solution from the freezer just before use because they do notfreeze at -20˚C.
Mix and spin down

Gently tap the tubes several times (hereinafter referred to as “tapping”), or vibrate the tubes 3
times for approximately 1 second on a vortex mixer. After the mixture becomes homogenous,
centrifuge the tubes to collect the solution in the bottom of the tubes (hereinafter referred to as“spin down”), and then put the reagents on ice.

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Preparation of Test Solution
To make Test Solution, add TYLCV Detection Solution,
Fluorescent Detection Solution, and TYLCV Enzyme Solution toa sterilized microtube (1.5 ml or 2.0 ml). Mix the reagents bytapping or vibrating 3 times for approximately 1 second on avortex mixer, and spin down the tube. Place the tube on ice.
Refer also to the following table.

[ Table of volume ]Reagent
TYLCV Detection Solution
Fluorescent Detection Solution
TYLCV Enzyme Solution
Test Solution Total

1test
23.0 µl
1.0 µl
1.0 µl25.0 µl

4+1 tests
115.0 µl
5.0 µl
5.0 µl125.0 µl

8+1 tests*
207.0 µl
9.0 µl9.0 µl
225.0 µl

* Prepare the test solution for the number of the samples that you test + 1 test, so that you haveenough volume for the test solution of all samples.

Important
For preparation of test solution, use another 1.5 ml microtube (rightside one of the photograph), and do not use the test tubes attached.
Prepare the test solution for the number of the samples that you
test + 1 test, so that you have enough volume for the test solutionof all samples. Consecutive dispensing of the reagent may cause
cross-contamination, use the filtered tip as a disposable in every
dispensing batch.
TYLCV Enzyme Solution is extremely viscous so be careful not
to stick too much amount of solution on the outer surface of thefiltered micro tip. Spin down prior to use.

12

Distribution of Test Solution
Pick a Test Tube out from the bag with a clean tweezer.
Dispense 23.0 µl of Test Solution into each Test Tube.

Important
Only use the specified Test Tube which is attached to this kit. Use of different volume, shape,and material may cause misjudgment.
Sample addition

Soak the tooth pick into the test solution after pricking thesample, and rub the bottom of the tube lightly. After that
immediately remove the tooth pick and dispose in plastic bag.
After adding the sample, add 20.0 µL of mineral oil in each tube
and close the cap.
In order to prepare control solution,
Add 20.0 μL of mineral oil and close the cap before adding
sample in negative control test tube.
ii)
Add the sample with tooth pick and then 20.0 μL of mineral
oil in each tube, and close the cap.
iii)
In the end, add 1.0µL of TYLCV positive control and then
20.0 μL of mineral oil in each tube, and close the cap.
i)Judgement will be difficult if the tooth pick is soaked in the test
solution for a while because the tooth pick adsorb the solution andthe amount of solution decrease. Do not leave the tooth pick inthe test solution after adding sample.
If mineral oil is not added, reaction efficiency will significantly
decrease because of condensation of the solution caused byevaporation. Please add mineral oil which is attached in this kit
when doing the test.

13Test Reaction
Close Test Tube and mix the reagents by tapping or vibrating on
a vortex mixer for about 1 second x 3 times, and spin down the
tube. Incubate Test Tube at 63˚C for 60 minutes in the incubator(Air incubator, heating block, or water bath).
In case of using water bath, use a float plate to prevent the Test
Tube tilted on the water surface.
Heat block

Float plate

14

Judgement
Judgement of success or failure of the test
No UV irradiation

Negative control

Positive control

UV irradiation

Negative Control

After 60 minutes incubation, terminate the reaction by heating for
2 minutes at 80˚C.
Fluorescence Detection Solution before reaction looks light red.
If TLCV exists, it turns to vivid green under UV irradiation.
In this procedure, UV transilluminator (wavelength: 240 - 260 nm
or 350 - 370 nm ), UV goggles, or a face shield is needed.
First, please confirm that Positive Control Solution emits
fluorescence and Negative Control Solution does not emit
fluorescence. In case which it does not satisfy this condition, theresult is invalid. Investigate the cause.

Positive control

Important
To avoid false result, judge the result immediately after the reaction is completed.
Judgement of the sample
After judgement of the control solutions having done and the test is judged as valid, do thejudgement of the results of sample. If Test Solution emits the fluorescence that is same levelwith the Positive Control Solution under UV irradiation, the sample may contain TYLCV. If thefluorescence is NOT detected under UV irradiation, the sample does not contain TYLCV.
[ Tips of judgement ]Test Solution emits fluorescence clearly
The plant may be infected with TYLCV. Even if the emission is weak, when difference can beobserved between negative control solution and sample solution, there might be infection.
Test Solution does not have any difference in fluorescence compared with Negative Control
Solution
The plant may not be infected with TYLCV.
However, there is a case fluorescent coloring is not recognized because of poor concentrationof virus at the beginning of infection. Retest the sample 10 days after in case the sample issuspected to be infected even if being judged “TYLCV negative” by this kit and there is no
symptom.

15

5. Troubleshooting
If you experience trouble with this kit, check the items below and try the solutions. Consult
Nippon Gene Co., Ltd. for further questions.
Problem
Test solution gives
green color andcannot judge theresultControl test solution
does not give theright coloring.

Irregular coloring offluorescent detection
solution
Test solution has
evaporated.

The judgement offluorescence isdifficult.

Possible cause and solution
A. Too much sample is added in test solution
Reaction of this kit is so sensitive that judgement can be donewith the tooth pick whose edge is not green. Too much carrierof sample may cause false positive.
A. Too much TYLCV Positive Control added to the test
solution
There are some cases that efficiency of test reaction decreases
when too much TYLCV Positive Control is added to the reaction.
Please follow the instruction for the correct amount of addition.
B. Reagents or testing environment are contaminated with
nucleic acid
In case of negative control testing solution gives coloring,
template DNA contamination is suspected. Contamination
monitoring of reagents and testing environment, and cleaning
procedure by 1% sodium hypochloride aqueous solution arerecommended to remove completely the contaminants. After theremoval, redo the test.
C. Chelete compounds or metal ion in the sample
The Fluorescent Detection Solution emits fluorescence whenchelate compounds such as EDTA exists in the reaction. On theother hands, if a lot of metal ion presents in reaction, thefluorescence is inhibited thus it would be difficult to judge theresult.
D. Reaction temperature and operating procedure notcorrect
Confirm that there is no problem on the test process.
A. Judgement not immediately after test reaction has
ended
Fluorescent Detection Solution irregularly gains or loses its
coloring when left in room temperature for long time. Follow the
instruction for storage and handling of the solution.
A. The reaction tube not heated homogeneously
Water bath, heat block may have not heated the test tube
homogeneously so that the test solution would be concentrated
because of evaporation. In such case the reactivity efficiency
goes down. Make sure that mineral oil to be added to the test
solution.
A. UV lamp wavelength not optimal.
UV lamp emitting light wavelength of 240-260 nm or 350-370
nm is necessary for the detection.
In case of the wavelength of the light is 320 nm, be notified thatnegative sample could emit fluorescence (false-negative).

16

6. Reference
1. Fukuta S, Kato S, Yoshida K, Mizukami Y, Ishida A, Ueda J, Kanbe M, Ishimoto Y. (2003)
Detection of tomato yellow leaf curl virus by loop-mediated isothermal amplification reaction. J
Virol Methods. 112 (1-2), 35
2. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. (2000) Loopmediated isothermal amplification of DNA. Nucleic Acids Res. 28 (12): e63
3. Prince AM, Andrus L. (1992) PCR: how to kill unwanted DNA. Biotechniques. 12 (3): 358

17

7. Supplementary data
[ Quality control ]It has been confirmed that the emission of fluorescence was detected when DNA amplification
reaction was done with attached Fluorescent Detection Solution, and 1.0 µl of TYLCV Positive
Control as template in 25.0 µl reaction volume under 63˚C temperature for 60 minutes.

[ Phyletic line ]LAMP method needs 4 to 6 primers. The primers contained in this kit is designed based on wellpreserved region in the strains below included in TYLCV genomic DNA.
GenBank
AccessionNo.
AB014347
AB116633
AB014346
AB110218
AB116632
AB116635
AB116636
AB116634
AB110217
AB116630
AB116629
AB116631
AB192965
AB192966
AY530931
NC_004005
AJ489258
AJ223505
AF024715
AY594174
AJ132711
AF105975
AY134494
AJ865337
AF071228
X61153

Strain NameTYLCV-Aichi (Aichi Prefecture)
TYLCV-Atu (Aichi Prefecture)
TYLCV-Shizuoka (Shizuoka Prefecture)
TYLCV-Israel Sz (Shizuoka Prefecture)
TYLCV-SzY (Shizuoka Prefecture)
TYLCV-SzD (Shizuoka Prefecture)
TYLCV-SzOs (Shizuoka Prefecture)
TYLCV-Kis (Mie Prefecture)
TYLCV-Israel Ng (Nagasaki Prefecture)
TYLCV-Omu (Nagasaki Prefecture)
TYLCV-Miy (Miyazaki Prefecture)
TYLCV-Mis (Kumamoto Prefecture)
TYLCV-Tos (Kochi Prefecture)
TYLCV-Tos (H) (Kochi Prefecture)
TYLCV-Florida
TYLCV-Alm
TYLCV-Almeria
TYLCV-Cuba
TYLCV-Dominican
TYLCV-Egypt
TYLCV-Iran
TYLCV-Portugal
TYLCV-Puerto Rico
TYLCV-Reunion
TYLCV-Spain7297
TYLCV-Sardinia V18

【Other methods for sample preparation】
Alkaline grinding method

Tooth pick suspension method
Add 100 mM Tris-HCl (pH 8.0)

Add 0.5 N sodium hydroxide

100 mM Tris-HCl (pH 8.0)
50.0-500.0 µl

Add 100.0 µl of
0.5 N sodium hydroxide

Suspend the tooth pick which pricked tomato
leaf

Add tomato leaf
50 mg of tomato leaf

100 mM Tris-HCl (pH 8.0)
Grind with pestle

Vector grinding method
Drop 100 mM Tris-HCl (pH 8.0) or sterilized
water
10.0 µl of 100 mM Tris-HCl (pH 8.0)
or
10.0 µl of sterilized waterTransfer grinded solution to new tube
Plastic wrap
orparaffin film

Put silverleaf whitefly and grind with pestle

silverleaf whitefly

Neutralize with 100 mM Tris-HCl (pH 8.0)
495.0 µl of
100 mM Tris-HCl (pH 8.0)

19

[ Memo ]20
19

It may change without a preliminary announcement about the written contents of the description, productspecification, and a price.
“Nippon Gene” is the registered trademarks of Nippon Gene Co., Ltd. in Japan.
Other proper nouns are the registered trademarks or trademarks of each company.

Information
NIPPON GENE CO., LTD.
Address: 2-7-18, Toiya-machi, Toyama
930-0834 Japan
TEL: +81-76-451-6548 FAX: +81-76-451-6547
URL: https://nippongene-analysis.com/en/
E-mail: support@nippongene-analysis.com

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