2018.12
Code No. KN-T10C805
第1版

Instruction Manual
Kaneka Nucleic Acid Chromatography [Cannabis grass DNA Detection Kit]
Caution⚫

Keep the reagents in this kit in strict compliance with the storage conditions described in thisinstruction manual. Be careful of contamination when storing.

⚫

Please wear protective equipment (protective gloves, protective glasses, mask, etc.) before using thiskit.

⚫

The specifications of this kit are subject to change without notice.

⚫

For equipment, instruments, reagents, etc. used to prepare PCR solutions and others, follow theinstructions specified by the respective manufacturers and distributors.

⚫

It is the customer's responsibility to determine and use the results obtained with this kit. The Company
shall not be liable for any damage or loss resulting from the detection or use of the results, whether
direct or indirect.

⚫

The customer is responsible for verifying the operating procedures not described in this instruction
manual and the validity of the detection results for each sample.

1.

Kit Overview
This kit uses a multiplex PCR and a nucleic acid chromatographic test strip to detect cannabisderived DNA quickly and easily.

2.

Principles/Main Features
This kit is based on the principle of nucleic acid chromatography. Nucleic acid amplification isperformed by targeting a region common to green plants on the genome and two regions specific tocannabis grass on the chloroplast genome. The amplified product is spread on a test strip, and thecolor pattern of the line is used to determine whether the sample contains cannabis DNA.

【Main Features of this Kit】
Testing for Cannabis positivity is possible from samples (cannabis resin, roots, etc.) that do nothave the characteristic (bristles, etc.) of cannabis grass.

No device (gel imaging device, real-time PCR device, etc.) is required for determining results, andit can be determined visually.

It can perform DNA extraction to result determination within 3 hours.

－1 －３．Kit composition/Storage conditions
Component

Contents (20 tests)

Contents (100 tests)

Test strip

20 strips

100 strips

Chase buffer

3.2 mL

16 mL

DNA extraction Solution A

1 mL

5 mL

DNA extraction Solution B

20 mL

100 mLPCR mix

200 µL

1,000 µL

Primer mix

100 µL500 µL

Positive control DNA40 µL

200 µL

Storage Temp.

Room Temp.

Frozen（Below -20℃）

Name of test strip parts【Precautions】
The test strip may lose its detection performance if left for a long time in a humid environment.
After opening the test strip, keep the container lid tightly closed and store it carefully.

Do not use components from different lots together or use reagents other than those supplied withthis kit, as they may not produce proper results.

４．Expiration Date
Stated on the label of each content.
５．How to Use
5.1 Equipment and device required in addition to this Kit

Micropipette

Tip for micropipette

Thermal cycler (Recommended device: LifeECO Thermal Cycler (Bioer Technology))

1.5 mL tube



0.2 mL tube（For PCR）
－2 －

Small incubator (for 1.5 mL, capable of heating above 95℃)

Low speed centrifuge

5.2 Samples
In addition to various parts of cannabis plant, processed products containing cannabis plant can beused as samples of this kit.
 Cannabis grass leaves, spikes, stems, seeds, roots (dry or raw, juvenile
phase is acceptable) Cannabis resinSuitable Cannabis oil (including plant pieces) Cannabis and other plant mixed samples
 Extraction residue after inspection of contained components (use afterdrying after evaporation of solvent)

Suitable in someconditions

 Combustion residue
 Food (cannabis cookies, etc.)
 Combustion residue (ashed)

Not Suitable Cannabis oil (without plant fragments)
 Other samples in bad condition (septic, etc.)

5.3

Sample weight

Weigh a sample volume (generally 1-2 mg) suitable for testing with this kit into a 1.5 mL tube・Non-seed area: For dried samples, use 1-2 mg.
(In the undried state, the amount of DNA recovered per unit weight tends to decrease.)
・Seeds: We recommend crushing one grain and using the entire amount.
・Cannabis Resin: As it contains PCR inhibitors, we recommend a starting amount of 1 mg orless.
・Cannabis oil: It is recommended to use only the precipitate obtained by centrifuging andremoving the supernatant.
5.4 DNA Extraction
1) Set a small incubator (can be substituted with a thermal cycler) at 98°C and heat it in advance.
2) Add 50 µL of the extraction reagent Solution A to the 1.5 mL tube containing the weighed sample.
3) Heat the 1.5 mL tube containing the sample at 98 °C for 8 minutes.
4) After heating, add 1 mL of extraction reagent Solution B to a 1.5 mL tube at room temperature andmix.
5) Use the supernatant as a DNA extract*.
*The results may be improved by diluting the DNA extract 10 to 50 times.

－3 －5.5 Preparation of PCR solution
1)

Thaw the PCR mix and primer mix in advance.

2)

Prepare a master mix by pipetting and mixing the required number plus 3 samples at a ratio ofPCR mix *10 µL and primer mix *5 µL into a 0.2 mL tube for preparing the master mix.

* Mix the PCR mix and primer mix thoroughly by pipetting before dispensing. If the PCR mix shows awhite precipitate after thawing, make sure to completely dissolve it before use.
PCR mix

10 µL

Primer mix

5 µLExample) 5 samples
Required quantity = 5 + 3 = 8 samples
Mix 80 µL of PCR mix and 40 µL of primer mix to make a master mix.
3) Add 5 μL of DNA extract to a new 0.2 mL tube.
4) As a positive control, prepare a 0.2 mL tube containing 5 μL of the positive control DNA includedin this kit.
5) As a negative control, prepare a PCR tube containing sterile distilled water or 5 μL of the extract
obtained by performing DNA extraction on a blank.
6) Add 15 µL of master mix to each sample and 0.2 mL tube of positive/negative control and mix bypipetting.
7) After adjustment, please keep on ice to prevent enzyme inactivation and non-specific amplification
reactions.
5.6 PCR
1)

Start the thermal cycler, set the 0.2 mL tube, and then perform the amplification reaction
with the following program.
＜PCR Program＞
Temp.（℃）

Time（Sec）

Cycle

25300

195

180

194

1062

1072

4072300

110∞

135

5.7. Test strip detection
1) Remove the 0.2 mL tube after the reaction, open the lid of the 0.2 mL tube, and add 160 µL ofchase buffer.
－4 －2) Insert the test strip insert into a 0.2 mL tube.
3) After 10 minutes, check the line coloring of the test strip.
【Precautions】
Determine by line coloring 10 minutes after insertion.

Wear gloves when handling the test strip and always hold the absorbent pad on the top. Be
careful not to touch the test strip insertion part (part with sample) and detection part with yourhands.

When inserting the test strip into the tube, be sure to insert it firmly until the part with the sample
hits the bottom of the 0.2 mL tube.

When inserting the test strip, be careful of contamination due to splashing of the reaction solution.

Pattern A: All three detection lines (1 to 3) are colored
Pattern B: In addition to the green plant common line (1), only one of the cannabis detection lines iscolored
Pattern C: Only the green plant common line (1)is colored
Pattern D: No coloring
Pattern E: Only the cannabis detection line is colored, and the common green plant line is not colored.
【Precautions】
If the positive control does not show pattern A (cannabis positive), the results are invalid because
the deterioration of the reagent is suspected.

Contamination is suspected if the negative control shows anything other than pattern D (no plantDNA). Decontaminate the equipment and the laboratory table and re-inspect.

If the sample shows pattern E (abnormal), it is suspected that the amount of DNA is insufficient, orimpurities impede PCR amplification. Please retest without determining the result.

Do not draw the final conclusion based on the results of this kit alone but make a comprehensive－5 －judgment together with the results of other detection methods.
６．Notes on using this kit<Contamination measures>
To prevent misjudgment due to contamination, replace the used micropipette tip every time. Also,use a micropipette tip with a filter.

We recommend that each step of DNA extraction, PCR solution preparation, and test strip detection
be performed in separate laboratories. If it is difficult to carry out in separate laboratories, separate
workbenches, or work areas within the same laboratory.

Before opening the tubes for reagents, extracted DNA, etc., spin them down with a low speed
centrifuge. (To prevent the contents from scattering and reagents attached to the lid from sticking
to fingers)Regularly clean micropipettes and other equipment and devices used with 0.1% sodium
hypochlorite and commercially available DNA removers.



If contamination occurs, decontaminate with 0.55% sodium hypochlorite (or a commerciallyavailable DNA remover) or UV irradiation.

＜Disposal precautions＞


When discarding the used test strip and amplification solution, be careful not to touch the detection
part and put them in a plastic bag, etc. before discarding.

When discarding the sample after DNA extraction, please take into consideration the sanitary andenvironmental aspects in accordance with the local regulations on waste and the regulations of thefacility.

＜Others＞
This kit is optimized for Bioer Technology's LifeECO Thermal Cycler. If you are using another model,
the result may be unclear.

Check the equipment and devices used for inspection according to the manufacturer's manual andmake sure to use properly operating devices.

７．Bibliography
1) Tadashi Yamamuro et. al, Development of simple and accurate detection systems for Cannabis sativa
using DNA chromatography, Forensic Science International, Volume 291, 2018, 68-75<Contact Information>
Kaneka Corporation Medical Solutions Vehicle
2-3-18, NAKANOSHIMA, KITA-KU, OSAKA, 530-8288, JAPANURL https://www.kaneka-labtest.com－6 －