Instruction Manual
version 2.0.0

Phytoplasma Universal Detection Kit
Instruction Manual Version 2.0.0[ Read the following instructions before the test ]Before using the kit, please confirm the following notices.
Notices for use-

This kit offers detection of all Phytoplasma in the class Mollicutes from plant samples using
LAMP, means Loop-mediated Isothermal Amplification. This kit must not be used forclinical diagnosis, therapeutic purpose and the test other than Phytoplasma 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 different use and the different purpose from
instructions.

-Concerning the secondary use of the assay result by the kit, please be notified that theuser 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.

-

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. MSDS is exhibited in the homepage of Nippon Gene Co., Ltd.
URL; http://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 Phytoplasma identification.

Table of contents
Page
1. About the kit ······················································································ 1
Product Overview
Diagnosis of Phytoplasma diseases
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
Phytoplasma detection protocol ·········································································· 7
Sample preparation
Equipment preparation
Testing environment
Detailed protocol ····························································································· 9
DNA sample extraction
Test reaction
Judgement
Tips for judgement

5. Troubleshooting ··············································································· 15
6. References ······················································································ 16
7. Supplementary data ········································································· 17
Quality control

Copy number of Phytoplasma Positive control
Memo

The primer set of this kit and the Phytoplasma detection technology using the primer set and
LAMP method are developed by The University of Tokyo, Plant Clinic.

1. About the kit
Product Overview
Phytoplasma Universal Detection Kit offers detection of all Phytoplasma in the class Mollicutes inclusively
from plant samples using Loop-mediated Isothermal Amplification (LAMP) method. LAMP method is fast andeasy 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
and sensitivity. In this kit, a part of Phytoplasma DNA amplifies using LAMP method, and infection of
Phytoplasma can be judged if the amplification occurs or not.
The operation needed for the detection is extremely easy, simply to mix the DNA extracted from plant
sample to the test solution (mixture of Phytoplasma Detection Solution, Phytoplasma Enzyme Solution, andFluorescent Detection Solution) in a sample tube, and keep the tube at 64 degree Celsius for 60 minutes.
The existence of Phytoplasma in the plant can be determined from whether the specific sequence amplifies
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 Phytoplasma DNA can be detected safely in a short period oftime.
The “List of Phytoplasma diseases in Japan” is available at our homepage for your information.
The kit may not detect the complete absence of Phytoplasma depending on the plant species andPhytoplasma species. If Phytoplasma infection is still suspected when not detected, please consult with The
University of Tokyo, Plant clinic.
Diagnosis of Phytoplasma Diseases
Phytoplasmas are plant pathogenic bacteria hosting on the phloem of plants, which were first discoveredin Japan, 1967. They infect over 700 plant species worldwide, cause numerous symptoms as yellows, stunt,dwarf, proliferation, phyllody, witches’ broom, and are transmitted by insect vectors to many economicallyimportant crops, fruit trees and ornamental plants.
In order to prevent the devastating damage to the plant, it is necessary to provide effective means forcontrolling Phytoplasma such as removal of infected plant. However, since Phytoplasma is difficult tocultivate, it has not been provided the convenient way to diagnose the Phytoplasma infection. This kit can
detect Phytoplasma inclusively, with no means of difficult operation such as conventional culture methods.
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; http://loopamp.eiken.co.jp/e/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.; http://www.nippongenematerial.com/
Refer

1

2. Reagents provided with the kit[ Kit components (for 48 tests) ]Reagent(tube label)
Instruction Manual
Test tube
Phytoplasma Detection Solution
Phytoplasma Enzyme Solution
Fluorescent Detection Solution
Phytoplasma Positive Control
Mineral Oil
Phytoplasma Extraction SolutionForm(top label)
BookletBag
Red label
Yellow label
Purple label
Gray label
Blue label
BottleContents
48 tests
1 booklet
50 tubes
1,050 µl
50 µl
50 µl
50 µl
1,000 µl
5,000 µl

Storage temperature
Room temperature
Room temperature-20˚C (avoid light)-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 48 samples by preparing test solution for 49 tests in one
tube. Prepare the test solution for the number of the samples that you test + 1 test, so that you havejust enough volume for the test solution of all samples. Please be noticed that the number of the test
that you can do will be less than 48 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. Repeating freeze and thaw may deteriorate the quality of this kit. Aliquot the contents toseveral tubes if necessary.
 Phytoplasma Enzyme Solution must be handled with extra care. Do not leave it at room temperaturesor 4˚C for a long period of time. Do not freeze it. Under inappropriate temperature condition, theenzyme may lose activity.
 Phytoplasma Positive Control is the solution of DNA fragment which contains a DNA sequencespecific to Phytoplasma genomic DNA. To avoid cross-contamination, do not spill the solution, andavoid the contact of 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 the local regulation concerning hazardous substances.

2

3. Equipment and reagents not provided with the kit[ Required equipment and reagent ] Incubator
Any equipment that can maintain 64˚C for
given time (e.g. water bath, heating block,thermal cycler, air incubator etc.)

 Micropipette(0.5-10 µl, 10-100 µl, 200-1,000 µl)

 Filtered pipette tip (sterilized)
 Razor Paper towel Tweezers
 Crushed ice
 Microtube for preparation of test solution(1.5 ml or 2.0 ml)[ Equipment which may be useful ]Use these equipment if necessary: Microtube rack Disposable gloves3 Plate rack Centrifuge for test tube

 Vortex mixer Float plate
Use when the sample is heated with waterbath.
 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 shield44. Instructions[ Simple protocol ] Following is the simplified protocol described by way of example
utilizing Hydrangea as specimen.
For the detailed protocol of this kit, please refer the instruction at page 7 and later.

Simplified Protocol (In case of Hydrangea as specimen)
1.

Dispense 100.0 µl Pytoplasma Extraction Solution to a sample tube (1 tube persample)Phytoplasma Extraction Solution
100.0 µl2.

Cut a small piece of Hydrangea calyx or leaf vein and soak it to 100 µl
Phytoplasma Extraction Solution

2 x 5 mm

2 x 5 mm3.

Incubate the solution with sample piece at 95 ˚C for 10 minutes (this becomes
DNA sample)

4. Prepare the test solution
Reagent
Phytoplasma Detection Solution

1test
21.0 µl

8+1test*189.0 µl

48+1 test*
1029.0 µl

Fluorescent Detection Solution

1.0 µl9.0 µl49.0 µl

Phytoplasma Enzyme Solution
1.0 µl9.0 µl49.0 µl
Total
23.0 µl
207.0 µl1127.0 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 solutionof all samples.

5

Simple protocol
5. Aliquot 23.0 µl of the test solution to the test tube
6. Add 2.0 µl of DNA sample to the test solution
Negative control

Positive control

DNA sample

Phytoplasma
Extraction
Solution
2.0 µlDNA sample
2.0 µlPhytoplasma
Positive Control
2.0 µl7. Add 20 µl of Mineral Oil
Mineral Oil 20.0 µl

8. Incubate at 64˚C for 60 minutes (DNA amplification)
9. Incubate at 80˚C for 2 minutes (Enzyme inactivation)
10. Judge the result
UV transilluminator or lamp

Visual detection

Negative:
Red

Positive:Green

6

Negative

Positive

[ Preparation and precaution before detection ]Sample preparation
ControlThis kit contains Phytoplasma Positive Control to confirm the success or failure of thedetection results. To confirm the results are successful, attention should be taken to the
preparation of “positive control reaction solution” with Phytoplasma Positive Control added, andalso the “negative control reaction solution” with Phytoplasma Extraction Solution added.
Sample preparation

For detection of Phytoplasma, the kit utilizes DNA extracted from plant specimen. This kit
contains one-step DNA extraction buffer (Phytoplasma Extraction Solution) to extract DNA fromplant vascular bundle and a protocol for the extraction. Prior to detection, prepare the DNA with
Phytoplasma Extraction Solution.
Important
If you use DNA extraction kit available on market or CTAB method to extract the DNA, keep
the concentration of DNA sample 5 ng/µl or more.
Equipment preparation

Incubator

Turn on the power of incubator and set the required temperature. In case of using water bathand heat block, it might take time to reach the target temperature so pre-heat the device andconfirm the temperature with thermometer. In case of using air incubator, the temperature alters
largely when the cabinet door is open. The door opening operation should be quickly done when
the sample is set in air incubator.


Other equipment
equipment

Micropipette
Microtube rack
Microtube
Filtered pipette tip(sterilized)
Writing materials
Disposable gloves
White robe

Instruction
Monopolize micropipette for LAMP working area. Return to the original
location after nucleic acid removal operation in case if used in other area.
Monopolize microtube rack for 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, nuclease-free grade microtube.
Select gamma-ray sterile, nucleic acid-free, 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 a disposable 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 disposables, change them when the contamination issuspected.
Use solely in each working area, be noticed of the contamination from thecuffs.

7

Testing Environment
Because LAMP method is the DNA amplification technology with excellent sensitivities, itwould be difficult to make an accurate inspection if the testing environment is contaminated with
Phytoplasma positive control or amplified sample after test. For the handling of the sample, takeextra care to avoid the contact of the positive control and samples to the working white lobe andequipment. It is also strongly encourage thorough exchanging of the clothing. To prevent thefalse results after the test, the used tips, microtubes, and amplified sample after test should bepacked together in doubled plastic bags. Please avoid running electrophoresis, autoclaving of
amplified sample after test and positive control.
Working area

Assign a clean booth or working bench which has not used for nucleic acid extraction andamplification (which has not been contaminated by nucleic acid) as dedicated reagent
preparation area. Prepare the test solution only at the reagent preparation area. Do not use
Phytoplasma 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 Phytoplasma 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 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 take attentionof the expiratory date and storage temperature of the solution.
<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 paper towel moistened with sodium
hypochlorite aqueous solution followed by wiping with 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.

8

[ Detailed protocol ]DNA sample extraction (In case of Hydrangea phyllody Phytoplasma DNA)
Important
This kit contains one-step DNA extraction buffer (Phytoplasma Extraction Solution) to extract
Hydrangea phyllody Phytoplasma DNA from Hydrangea calyx or leaf vascular bundle and protocol
for the extraction.
If you use commercial DNA extraction kit or CTAB method to extract the DNA, keep theconcentration of DNA sample 5 ng/µl or more.
Thawing of the reagents
Completely Thaw the Phytoplasma Extraction Solution at room temperature. Keep the cap
closed and invert to mix upside down 10 times until homogeneity. Dispense 100.0 µl to a
microtube (1.5 or 2.0 mL, one tube per sample).

2 x 5 mm

Collection of plant vascular bundle sample
Collect the calyx or leaf from subjected Hydrangea trees,
separately for each tree in a clean polyethylene bags. After
collecting in the bags, handle the specimen with a tweezer. The
tip of tweezer must be burned with alcohol lamp or burner or
wiped with paper towel moistened with ethanol.
Use clean razor to cut the vascular bundle part of calyx or leaf
into a size of 2 x 5 mm (figures left).

In case of calyx

2 x 5 mm

DNA extraction
Soak completely the small piece of Hydrangea calyx or leaf
vein in Phytoplasma extraction solution in the microtube.
Incubate the microtube at 95˚C for 10 minutes. The solution
obtained is the DNA solution. The solution is ready to use and it
is not necessary to eliminate the Hydrangea piece from thesolution or to vortex the solution.

In case of leaf
ImportantPhytoplasma tends to localize at the phloem, so it is crucial to collect the part that containvascular bundle.
Take an extra care of Phytoplasma Extraction Solution not getting contaminated; since it would
be extremely difficult to make an accurate inspection once contaminated. In case of aliquot
unused Phytoplasma Extraction Solution to microtubes and store, dispense 100 µl of the solution
to each microtube and store at -20˚C under dark place.

9Test reaction
Thawing of the reagents
Thaw the following reagents at room temperature.
Phytoplasma Detection Solution
Phytoplasma Positive Control
Mineral Oil# For Phytoplasma Enzyme Solution and Fluorescent
Detection Solution, do not freeze 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 becomeshomogenous, centrifuge the tubes to collect the solution at thebottom of the tubes (hereinafter referred to as “spin down”).
The reagents, Phytoplasma Enzyme Solution and Fluorescent
Detection Solution, are placed on ice.

10

Preparation of Test Solution
To make Test Solution, add Phytoplasma Detection Solution,
Fluorescent Detection Solution, and Phytoplasma Enzyme
Solution to a sterilized microtube (1.5 ml or 2.0 ml). Mix thereagents by tapping or vibrating 3 times for approximately 1
second on a vortex mixer, and spin down the tube. Place thetube on ice.
Refer also to the following table.

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

1 test21.0 µl
1.0 µl
1.0 µl23.0 µl

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

48+1 tests*
1029.0 µl
49.0 µl49.0 µl1127.0 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
Consecutive dispensing of the reagent may cause cross-contamination, use the filtered tip as adisposable in every dispensing batch.
Phytoplasma Enzyme Solution is extremely viscous so be careful not to stick too much amountof solution on the outer surface of the filtered micro tip. Spin down prior to use.
Distribution of Test Solution
Pick a Test Tube out with a clean tweezer from the bag.
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.

11

DNA sample addition
Add 2.0 µl DNA sample to the Test Tube. Then add 20 µl of Mineral Oil to prevent
evaporation of a reagent and close the cap. (Be noticed that the test reaction efficiency
drastically decrease if the test solution gets concentrated). If a thermal cycler having the functionof hot bonnet heated lid is used as incubator, mineral oil addition is not necessary.
Important
When adding the DNA sample and Mineral Oil, be sure to operate with the following order.
1: Negative Control Sample
2: DNA sample
3: Positive Control Sample
Close the cap immediately after sample addition.
Further, add 2.0 µl Phytoplasma Extraction Solution and Mineral Oil to a Negative Control
sample and use 2.0 μl Phytoplasma Positive Control for a positive control sample.
Test Reaction
Close Test Tube and mix the reagents by tapping or vibrating on avortex mixer at about 1 second x 3 times, and spin down the tube.
Incubate Test Tube at 64˚C for 60 minutes in the incubator (Airincubator, heating block, or water bath).
In case of using water bath, use float plate to prevent the Test
Tube tilting horizontally.
Heat block

Float plate

12

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
Phytoplasma exists, it turns to vivid green under UV irradiation.
In this procedure, UV transilluminator (wavelength: 254 - 366 nm ),UV goggles, or a face shield is needed.
Normally, Positive Control Solution appears fluorescence and
Negative Control Solution does not appear fluorescence. If
Negative Control Solution appears fluorescence, there ispossibility that Phytoplasma Positive Control enters the test tube
by mistake. In this case, check the handling and try to test again.

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 Phytoplasma.
If the fluorescence is NOT detected under UV irradiation, the sample does not containPhytoplasma.
[ Tips of judgement ]Test Solution emits fluorescence clearly
The plant may be infected with Phytoplasma. Even if the emission is weak, when difference
can be observed between negative control solution and sample solution, there might be infection.
In such case, collect other sample from different part of the subjected plant and redo the test.
Test Solution does not have any difference in fluorescence as compared with Negative Control
Solution
The plant may not be infected with Phytoplasma. However, fluorescence may not appear
because of uneven distribution of Phytoplasma in plant body. Observe well the presence ofdisease symptom and if infection is suspected, collect several sample from different part of thesubjected plant and redo the test.

13

Important
If Phytoplasma infection is suspected regardless to the results given by this kit, please consult
with The University of Tokyo, Plant Clinic.

The University of Tokyo, Plant Clinic;
http://park.itc.u-tokyo.ac.jp/ae-b/hospital/
Place:TEL:
E-mail:Yayoi 1-1-1, Bunkyo-ku, Tokyo
03-5841-0567
byoin@todaiagri.jp

14

5. Troubleshooting
If you experience trouble with this kit, check the items on below and try the solutions. Consult
Nippon Gene Co. Ltd. for further questions.
ProblemControl test solution
does not give theright coloring.

Irregular coloring offluorescent detection
solution
Test solution has
evaporated.

The judgement offluorescence isdifficult.
There are not enoughreagents for testing.

Possible cause and solution
A. Too much Phytoplasma Positive Control added to thetest solution
There are some cases that efficiency of test reaction
decreases when too much Phytoplasma Positive Control isadded 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, 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
that negative sample could emit fluorescence (false-negative).
A. Reagent sticks on the inner tube surface.
Spin down the microtube before use.
B. Reagent evaporated during its storage
Completely close the cap after use.

15

6. Reference
1. Maejima K, Oshima K, Namba S. (2014) Exploring the phytoplasmas, plant pathogenic
bacteria. J Gen Plant Pathol. 80 (3) : 210
2. Mayama S, Namba S (ed) (2010) Plant Pathology, Bun-ei-do
3. Namba S (ed) (2008) Clinical Plant Science, Yoken-do
4. Namba S, Kato S, Iwanami S, Oyaizu H, Shiozawa H. (1993) Detection and differentiation ofplant pathogenic mycoplasmalike organisms using polymerase chain reaction.
Phytopathology. 83 786
5. PSJ NIAS (ed) (2015) Common names of plant diseases in Japan, 2nd edn (in Japanese)
6. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. (2000)
Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28 (12) : e63
7. Prince AM, Andrus L. (1992) PCR: how to kill unwanted DNA. Biotechniques. 12 (3) : 358

16

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 2.0 µl of Phytoplasma
Positive Control as template in 25.0 µl reaction volume under 64˚C temperature for 60 minutes.
[ Copy number of Phytoplasma Positive Control ]Phytoplasma Positive Control contains 107 copies of target sequence per 2.0 µl volume.

17

[ Memo ]18

[ Memo ]19

[ Memo ]

20





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“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 http://nippongene-analysis.com
E-mail support@nippongene-analysis.com
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