Development of simple and accurate detection systems for Cannabis sativa using DNA chromatography

Accepted Manuscript Title: Development of simple and accurate detection systems for Cannabis sativa using DNA chromatography Authors: Tadashi Yamamuro, Shigehiko Miyamoto, Masashi Kitamura, Tomonori Muro, Yuko T. Iwata, Hiroki Segawa, Kenji Kuwayama, Kenji Tsujikawa, Tatsuyuki Kanamori, Hiroyuki Inoue PII: DOI: Reference:

S0379-0738(18)30492-4 https://doi.org/10.1016/j.forsciint.2018.08.006 FSI 9430

To appear in:

FSI

Received date: Accepted date:

13-7-2018 3-8-2018

Please cite this article as: Tadashi Yamamuro, Shigehiko Miyamoto, Masashi Kitamura, Tomonori Muro, Yuko T.Iwata, Hiroki Segawa, Kenji Kuwayama, Kenji Tsujikawa, Tatsuyuki Kanamori, Hiroyuki Inoue, Development of simple and accurate detection systems for Cannabis sativa using DNA chromatography, Forensic Science International https://doi.org/10.1016/j.forsciint.2018.08.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Development of simple and accurate detection systems for Cannabis sativa using DNA chromatography

Tadashi Yamamuroa,*, Shigehiko Miyamotob, Masashi Kitamurac, Tomonori Murod,

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Yuko T. Iwataa, Hiroki Segawaa, Kenji Kuwayamaa, Kenji Tsujikawaa, Tatsuyuki

a

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Kanamoria, Hiroyuki Inouea

National Research Institute of Police Science, 6-3-1 Kashiwanoha, Kashiwa, Chiba

277-0882, Japan

Medical Devices Solutions Vehicle, Kaneka Corporation, 1–8 Miyamaemachi,

Takasago-cho, Takasago, Hyogo 676-8688, Japan

Kanazawa, Ishikawa 920-8553, Japan

Criminal Investigation Laboratory, Shimane Prefectural Police Headquarters, 1751-15

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Forensic Science Laboratory, Ishikawa Prefectural Police Headquarters, 1-1 Kuratsuki,

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b

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Heisei-cho, Matsue, Shimane 690-0038, Japan

*Corresponding author: Tel.: +81-4-7135-8001, Fax: +81-4-7133-9173, E-mail address:

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Abstract

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[email protected]

In recent years, the need for analyzing cannabis DNA has increased in order to

accommodate the various types of cannabis samples encountered in forensic investigation.

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This study was aimed to establish a simple and accurate cannabis DNA detection system using DNA chromatography. Two chromatography chip systems with different features were successfully developed. One system (the “four-line version”) involves tetraplex PCR amplification, which could be used to detect cannabis DNA and distinguish between drug-type and fiber-type cannabis using the tetrahydrocannabinolic acid synthase gene 1

sequence. The other system was the “three-line version” with triplex amplification, which was specialized to distinguish cannabis from other plants, and had a sensitivity (10 fg DNA/reaction) that was 100 times greater than the four-line version. In both versions, no false positives were observed for 60 medicinal plants, and accurate detection could be

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performed for several simulated forensic samples such as cannabis leaves, buds, stems, roots, seeds, resin, and cannabis leaves blended 1/100 in tobacco. Detection could be

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performed by the naked eye and only a thermal cycler was required for operation. Thus, DNA chromatography systems for cannabis detection are expected to contribute to the

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analysis of cannabis DNA in forensic chemistry laboratories without extensive equipment.

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Keywords: Cannabis sativa; Drug-type cannabis; DNA analysis; DNA chromatography

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1. Introduction

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Cannabis is the most abused drug in the world and is controlled in many countries. Approximately 7,000 tons of cannabis herb and resin are seized worldwide every year,

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and it has been estimated that there are ~183 million cannabis users worldwide [1]. Beyond recreational drug use, cannabis has also been cultivated for thousands of years to

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obtain hemp fibers. The forensic identification of cannabis is generally performed by chemical analysis and microscopic observations [2, 3]. Detection of tetrahydrocannabinol

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(THC), a hallucinatory component of cannabis, is important in chemical analysis because “drug-type” cannabis strains have a high THC content, whereas industrial hempproducing “fiber-type” strains have been modified to produce less THC. Morphological

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examination is based on the characteristic structure of the plant Cannabis sativa, searching for trichomes on leaves [4]. However, THC detection and morphological examination cannot be used to test all suspected cannabis samples. For instance, the roots and seeds of cannabis contain little or no THC and have no trichomes. DNA-based testing seems to be an effective alternative detection approach in such samples. 2

There are several reports of cannabis identification by DNA analysis [5-17]. In addition, some reports have described sequence differences in the tetrahydrocannabinolic acid synthase (THCAS) gene, which could be used to identify drug-type and fiber-type cannabis strains [18-21]. However, many of them require expensive equipment such as a

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sequencer or a real-time PCR device. Gel electrophoresis-based methods of detecting amplicons are relatively easy to perform, but they require time, effort, some skill,

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dedicated reagents, and equipment for detection. The loop-mediated isothermal amplification (LAMP) assay has been reported as a simple and rapid technique for detecting cannabis DNA [15, 17, 21], but some precautions are necessary before

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beginning the procedure. For instance, the enormous degree of amplification by LAMP poses a high risk for contamination. Thus, it is difficult to perform DNA analysis for

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cannabis detection in a forensic chemistry laboratory, in contrast to other routine drug

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analyses.

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DNA chromatography is known as one of the simplest techniques for DNA testing. The technique involves the use of a paper-based chip to visually detect PCR amplification

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products and, thus, requires a simple procedure, inexpensive instrument, and low manufacturing cost. Detection is faster and more sensitive than agarose gel

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electrophoresis [22]. Different DNA chromatography systems were developed and studied by several groups, and have been applied for analysis of the oral microbiota [23],

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strawberry cultivar discrimination [22], causative plankton of paralytic shellfish poisons [24], carbapenemase genes in stool specimens [25], and herpes simplex virus [26]. The purpose of this study was to establish simple and accurate detection systems

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for cannabis DNA based on DNA chromatography that can be readily adopted in forensic chemistry laboratories.

2. Materials and methods

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2.1. Detection by DNA chromatography chip

2.1.1. Principle of DNA chromatography In this study, we adopted a DNA chromatography system developed by Kaneka's

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research group [24, 26, 27]. The principle of the system is shown in Fig. 1. DNA chromatography consists of PCR amplification and detection. Special primers with 5 tags

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are needed for amplification. The special primers have a main domain that anneals to the target sequence and a tag domain that hybridizes to a single-stranded DNA probe on the chip or gold nanoparticle. These domains are linked at sites that stop elongation by the

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DNA polymerase. Thus, PCR amplification with these primers results in amplicons with a different single-strand DNA tag at each end (Fig. 1A). Following PCR, an aliquot of the

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amplicons with a development buffer are applied to the chip and developed by capillarity.

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Fig. 1. Principle of the DNA chromatography systems. (A) Preparation of amplicons with

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single-stranded DNA tags by PCR, using special primers with tags. (B) Development of

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solutions and visual detection of target amplicons by DNA–DNA hybridization

During development, one single-stranded DNA tag of the amplicon hybridizes with a

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DNA probe that binds to the gold nanoparticle, thereby forming an amplicon–gold nanoparticle complex. Furthermore, as the complex moves on the chip, it interacts with the solid-phase DNA probe in the detection area, whose sequence is complementary to the DNA tag of the amplicon. Consequently, the amplicon–gold nanoparticle complex is trapped at the detection area, and signals originating from the gold nanoparticles can be visually detected as a colored band (Fig. 1B). Many target DNAs can be detected 5

simultaneously on a chip by multiplex PCR using different tags and optimized primer pairs.

2.1.2. Tagged-primer for cannabis detection

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The sequences of the PCR primers used in this study are shown in Table 1.

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Table 1. PCR primers used in this study Primer Target

Region

Primer name

Primer sequence (5–3)

Reference

ITS

ITS-A

tag(T1)-GGAAGGAGAAGTCGTAACAAGG

[28]

(nuclear DNA)

ITS-C

tag(Au)-GCAATTCACACCAAGTATCGC

[28]

Drug-type

THCA Synthase

THCAS-F

tag(Au)-AACATGCATTCGATCAAAATAGATG

This study

Cannabis

(nuclear DNA)

THCAS-R

tag(T2)-ATCAACATTGACTAAGTGTGCATCAATA

This study

trnL

cpCan

(chloroplast DNA)

CanRv

trnG-psbI

trnG-psbI-F

set

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Green plant

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#1

#4

tag(Au)-GAGTTGGCTGCGTTAATCCG

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Cannabis

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#3

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#2

Cannabis

trnG-psbI-R

tag(T3)-TGACATGTGGAATGGGACTC

This study

tag(T4)-TGTCAAGTCATATCCATATCCGTC

This study

tag(Au)-CCAAGAGGACTTAATGAGATTGAGATT

This study

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(chloroplast DNA)

[12]

For primer set #1, we selected previously described sequences that target the common

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internal transcribed spacer (ITS) region in green plants [28]. For set #2, new primers were designed to amplify the active THCAS gene of drug-type cannabis. For primer set #3, the trnL region on chloroplast DNA was selected for specific detection of cannabis. The

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forward primer sequence was previously described [12], although the reverse primer was optimized in this study to eliminate false positives with DNA from other species. For set #4, newly primers were designed to target the trnG–psbI region of cannabis chloroplast DNA. To detect amplicons by DNA chromatography, an oligonucleotide tag was added to the 5 end of each primer. Five tag sequences were generated in total. Four tags (tags 6

T1–T4) were complementary to the solid-phased probes, and one tag (tag Au) was complementary to the DNA probe fixed to the gold nanoparticle. The combined use of these tags and primers is illustrated schematically in Table 1. All tagged primers were supplied by Kaneka Corporation (Osaka, Japan) in the form of a “primer mixture,” which

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2.1.3. Structure of the two chips used for cannabis detection

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was prepared as a pre-mixed solution of primers at appropriate concentrations and ratios.

Two types of detection chips were used in this study. The structures of the chips

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are shown in Fig. 2.

Fig. 2. Structure of the detection chips used for cannabis detection. (A) Four-line version

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with the function of cannabis drug-type discrimination. (B) Three-line version specialized for sensitive cannabis identification

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One was the “four-line version” designed for discriminating between drug-type and fibertype cannabis. The chip had parallel four test lines (FT1–4) with solid-phased probes that could interact with tags T1–T4 at the detection area (Fig. 2A). The other detection chip was the “three-line version” designed for specific cannabis identification. At the detection area in the chip, one potential test line (TT2) had no probes and, thus, was never colored,

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so that only three test lines (TT1, 3, and 4) with probes complementary for tags T1, T3, and T4 were involved in detection (Fig. 2B). All detection chips were supplied by Kaneka.

2.1.4. PCR amplification

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PCR mixtures were prepared based on TaqHS Perfect Mix (Takara Bio Inc., Kusatsu, Japan), and consisted of 10 µL 2 TaqHS Mix, 5 µL primer mixture, 1 µL

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template DNA solution, and 4 µL sterilized water. Two types of primer mixtures were used, one containing primer sets #1–#4 for the four-line version and the other containing primer sets #1, #3, and #4 for the three-line version. Amplification was carried out by

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thermal cycler (Life ECO, BIOER Co. Ltd., Hangzhou, China). The tetraplex PCR conditions for the four-line version were as follows: preheating for 3 min at 95ºC; 40

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cycles of denaturation for 10 s at 94 °C, annealing for 10 s at 59 °C, and extension for 40

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s at 72 °C; and final extension for 5 min at 72 °C. The triplex PCR conditions for the

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three-line version were the same, except that the annealing temperature was 62 °C instead

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of 59 °C.

2.1.5. Visual detection of amplicons

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After PCR amplification, 160 µL of development buffer (Kaneka) was added to the amplicons. Then, the mixture was applied to the sample pad of the detection chip and

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developed on the chip. After 10 min, coloration of each line was confirmed visually.

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2.2. Plant materials Drug-type cannabis (leaves, buds, roots, stems, and corrupt fragments) and

cannabis resin were issued from the Narcotics Control Department (Kanto-Shin’etsu Regional Bureau, Ministry of Health, Labor and Welfare), according to formal procedures. The “corrupt fragments” were wet, scrap, and spoiled cannabis plants and selected as the representative of degraded samples. Fiber-type cannabis (male leaves, female leaves, and 8

roots) were provided with official permission from Prof. Kazuhito Watanabe (former professor of the Faculty of Pharmaceutical Sciences, Hokuriku University). Cannabis seeds (drug-type and fiber-type) were obtained from the Genebank Project (National Agriculture and Food Research Organization).

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Medicinal plants were cultivated at the Medicinal Plant Garden (Kanazawa University) and provided in the form of extracted DNA in solution. The names of all

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plants from which DNA was extracted are shown in Table 2.

Table 2. List of medicinal plant species tested for specificity evaluation Common name

Acer japonicum

Amur maple, Downy Japanese-maple

Aconitum carmichaeli Debx.

Chinese aconite, Carmichael's monkshood

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Botanical name

Aconitum japonicum Thunb.

Aconite, Oku-torikabuto (Japanese name) Desert false indigo, False indigo-bush

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Amorpha fruticosa

Hanasuge (Japanese name), zhi mu (Chinese name)

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Anemarrhena asphodeloides Anemone flaccida

Armoracia rusticana Artemisia absinthium

Absinthe, Wormwood Wild ginger

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Asarum sieboldii Miq.

Tōki (Japanese name) Horseradish

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Angelica acutiloba

Soft windflower

Mongolian milkvetch

Atractylodes japonica

Japanese atractylodes, Okera (Japanese name)

Atractylodes lancea

Hosoba-okera (Japanese name)

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Astragalus membranaceus

Kozo (Japanese name)

Callicarpa japonica

Japanese beautyberry

Cimicifuga simplex (DC.) Wormsk. ex Turcz.

Asian bugbane

Clematis chinensis

Sakishimabotanzuru (Japanese name)

Clinopodium gracile (Benth.) O. Kuntze

Slender wild basil

Cnidium officinale

Szechuan lovage

Cocculus orbiculatus

Queen coralbead

Coptis japonica (Thunb.) Makino var. major (Miq.) Satake

Coptis, Seriba-ouren (Japanese name)

Coptis trifoliolata

Coptis, Mitsubanobaika-ouren (Japanese name)

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Broussonetia kazinoki

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Saffron crocus, Autumn crocus

Foeniculum vulgare

Fennel

Fritillaria verticillata

(Lilly family) Amigasayuri (Japanese name)

Glechoma hederacea

Ground-ivy, Gill-over-the-ground

Glehnia littoralis

Beach silvertop, American silvertop

Glycyrrhiza glabra

Liquorice

Gynostemma pentaphyllum

Five-leaf ginseng, Poor man's ginseng

Hemerocallis fulva

Orange day-lily, Tawny daylily

Hibiscus cannabinus

Kenaf

Hordeum vulgare

Barley

Humulus japonicus

Japanese hop, Wild hop

Humulus lupulus

Hop

Lilium tenuifolium Fischer (Lilium pumilum)

(Lilly family) Itohayuri (Japanese name)

Lonicera japonica

Golden-and-silver honeysuckle, Japanese honeysuckle

Magnolia obovata

Houpu magnolia

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Matricaria chamomilla

Chamomile

Chinese mulberry

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Morus australis

Mōko-guwa (Japanese name)

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Morus mongolica Nicotiana tabacum Osmorhiza aristata

Panax ginseng

Yabu-ninjin (Japanese name)

Chinese ginseng, Korean ginseng Japanese ginseng

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Panax japonicus

Tobacco

Chinese peony, Common garden peony

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Paeonia lactiflora

Patrinia scabiosifolia

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Crocus sativus

Patrinia Amur Cork tree

Platycodon grandiflorus

Platycodon, Balloon flower

Rosmarinus officinalis

Rosemary

Sanguisorba officinalis

Great burnet

Scutellaria baicalensis

Baikal skullcap

Staphylea pinnata

European bladdernut

Stauntonia hexaphylla

Japanese staunton vine

Styrax japonicus

Japanese snowbell

Symphytum officinale

Comfrey

Symplocos coreana (HLev.) Ohwi

Korean sweetleaf

Vaccinium corymbosum

Northern highbush blueberry

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Phellodendron amurense

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Valeriana fauriei

Japanese valerian

Vitex agnus-castus

Five-leaved chaste tree, Horseshoe vitex

Zanthoxylum piperitum

Japanese pepper

Herbal products, which contained synthetic cannabinoids, were obtained from retail shops

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prior to the introduction of sales regulations. Mitragyna speciosa, also known as Kratom, was provided from the Division of Pharmacognosy, Phytochemistry, and Narcotics

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(National Institute of Health Science).

2.3. DNA extraction

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Two commercially available kits were used for DNA extraction from plant

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materials. Extraction and purification were performed using a DNeasy Plant Mini Kit

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(DNeasy kit) purchased from Qiagen (Hilden, Germany), following the manufacturer’s instructions. Simple and rapid extraction was performed as described below using a

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Kaneka Easy DNA Extraction Kit, Version 2 (Kaneka kit). Briefly, 100 µL of alkaline

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“solution A” was added to the sample and incubated at 98ºC for 8 min. Then, the sample was neutralized with 14 µL of “solution B” followed by centrifugation at 6,000 × g for 5

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min to recover the supernatant. Nine volumes of sterilized water were added to obtain the

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final DNA solution.

2.4. Determination of the detection limit DNA was extracted from 10 mg of drug-type cannabis leaves using the DNeasy

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kit. The extracted DNA was quantified and serially diluted from 10 ng/µL to 1 fg/µL with sterilized water. PCR was performed with DNA at each concentration, and the detection patterns were examined by DNA chromatography. The colored band of each line was evaluated in four stages (++, +, ±, –), and the criteria for each symbol was as follows; ++: the line was clearly colored with a pink band, +: pink coloration of the line was thin, but

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could be observed, ±: coloration was unclear or was found only partially on the line, –: coloration was not observed. The experiments were repeated three times.

2.5. Specificity evaluation

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DNA extracted from 60 medicinal plants (Table 2) were utilized in specificity tests. The detection results of the DNA chromatography chip were evaluated in four stages, as

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described above. The experiment was repeated twice.

2.6. Detection test for simulated forensic samples

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Thirty conditions of simulated forensic samples were prepared for evaluation of practicality. DNA extraction of all samples were performed using the DNeasy kit or the

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Kaneka kit. For cannabis resin samples, the extracted DNA was diluted 10, 100, and 1000

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times for PCR. Mixed samples were prepared by blending dried drug-type cannabis

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leaves and commercial tobacco leaves at appropriate proportions (1: 1, 1: 9, and 1: 99). The DNA chromatography chip results were evaluated in four stages, as described above.

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The experiment was repeated twice.

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3. Results and Discussion

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3.1. Representative band patterns of the DNA chromatography chip Two detection systems based on DNA chromatography chips were designed in

this study and confirmed to work as intended. The representative band patterns for a

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variety of samples are shown in Fig. 3. In the four-line version of the system, four colored bands (FT1–4) were observed for drug-type cannabis samples, whereas three bands (FT1, 3, and 4) were detected for fiber-type samples (Fig. 3A; lanes 1, 2). In contrast, drug-type and fiber-type cannabis showed the same coloration patterns of three bands (TT1, 3, and 4) in the three-line version (Fig. 3B; lanes 1, 2). 12

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Fig. 3. Representative band patterns of the DNA chromatography chips. (A) Four-line version. (B) Three-line version. 1: Cannabis sativa (drug-type strain); 2: Cannabis

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sativa (fiber-type strain); 3: plant other than cannabis (Humulus lupulus); 4: negative control (sterilized water)

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For other plants, a band was detected only at FT1 in the four-line version, and at TT1 in the three-line version; no coloration was observed for the negative control in both versions (Fig. 3A, B; lanes 3, 4). These results indicate that the four-line version reflected amplification of the ITS, THCAS, trnL, and trnG–psbI regions, as intended. Thus, it was possible to determine whether the cannabis samples were drug-type or fiber-type, 13

depending on the coloration at FT2. Similarly, we confirmed that the three-line version reflected amplification of the ITS, trnL, and trnG–psbI regions, and could not distinguish between drug-type and fiber-type cannabis, but could distinguish cannabis from other

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plants.

3.2. Detection limit of the DNA chromatography chip

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Fig. 4 shows representative results of the detection limit of drug-type cannabis

DNA using DNA chromatography. In the four-line version, coloration was observed in all lines for 1 pg of DNA or more (Fig. 4A; lanes 1–5). At 100 fg of DNA, the band of FT2

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was almost invisible, and the bands for FT1 and FT4 were thin, so that the differences in the tint of each band were remarkable (Fig. 4A; lane 6). At 10 fg of DNA or less, little or

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the limit for accurately determination was 1 pg.

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no coloration was observed at each line (Fig. 4A; lanes 7, 8). These results indicated that

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In the three-line version, three colored lines were observed for 10 fg of DNA or more (Fig. 4B; lanes 1–7). At 1 fg of DNA, little or no coloration was observed in each

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line (Fig. 4B; lane 8). The detection limit with the three-line version was shown to be 10 fg, or approximately 100 times less than that of the four-line version. Multiplex PCR

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experiments revealed that the increased number of regions that were amplified

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simultaneously inhibited amplification via competition for the reaction components.

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Fig. 4. Detection limit of the DNA chromatography chips. (A) Four-line version. (B) Three-line version. Template DNA = drug-type cannabis. 1: 10 ng/test; 2: 1 ng/test;

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3: 100 pg/test; 4: 10 pg/test; 5: 1 pg/test; 6: 100 fg/test; 7: 10 fg/test; 8: 1 fg/test

The DNA chromatography systems for cannabis detection were shown to be

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important for the following uses: the four-line version was suitable for obtaining information about whether cannabis is the drug-type or fiber-type, and the three-line version was useful if the absolute quantity of the plant sample was small and sensitive cannabis identification is required.

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3.3. Specificity evaluation DNA chromatography was performed on the 60 medicinal plants shown in Table 2 to determine the specificity of cannabis detection. The results are shown in Table 3. In both versions, coloration enabling detection of the ITS region of green plants was clearly

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observed, and no false positives were detected with any of the tested plants. The coloration in FT1 and TT1 for ITS detection indicated that the amount of template DNA

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extracted from plants was sufficient, and that PCR amplification was performed

successfully. Among the examined plants, Humulus lupulus and Humulus japonicus are classified as members of the Cannabaceae family and are genetically closely related to

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cannabis. Hibiscus cannabinus and Vitex agnus-castus resemble cannabis in terms of their leaf shape. Glycyrrhiza glabra was reported to be used in blended herbal products

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including synthetic cannabinoids as designer drugs [29]. These results confirmed that

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both versions of the DNA chromatography chip have sufficiently high specificity for

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cannabis detection.

chromatography chips

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Table 3. Detection-specificity results for 60 medicinal plants by the DNA

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Three-line version

ITS

THCAS

trnL

trnG–psbI

ITS

trnL

trnG–psbI

++

60

0

0

0

60

0

0

+

0

0

0

0

0

0

0

±

0

0

0

0

0

0

0

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Detection

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Four-line version

16

–

0

60

60

60

0

60

60

3.4. Detection of simulated forensic samples Detection experiments were conducted using 30 different conditions of simulated

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forensic samples to evaluate the practicality of the DNA chromatography chip. The details

of the conditions used and the detection results are shown in Table 4. In the case of drug-

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type cannabis, coloration was detected in all lines with both versions for four tissues (leaves, buds, roots, and stems). For cannabis seed detection, all lines were positive when

DNA extraction was performed using whole seeds, but coloration failed in some lines

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when only part of the seed was used. These results suggest that multiplex PCR resulted

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in insufficient amplification due to the small amount of DNA contained in the seeds and/or inadequate extraction. Similarly, for the corrupt fragments, thin colored bands were found

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in the four-line version, which showed that the DNA extraction method or starting amount

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should be optimized depending on tissues or conditions used.

chromatography chips

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Table 4. Detection results obtained using 30 conditions for simulated samples by the DNA

Four-line version

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DNA extraction

Sample

Three-line version

(dilution ratio)

ITS THCAS trnL trnG–psbI

ITS

trnL trnG–psbI

Leaves (10 mg)

Kaneka kit

++

++

+

++

++

++

++

Leaves (1 mg)

Kaneka kit

++

++

++

++

++

++

++

Buds (5 mg)

Kaneka kit

++

++

++

++

++

++

++

Root (5 mg)

Kaneka kit

++

++

++

++

++

++

++

Stem (5 mg)

Kaneka kit

++

++

++

++

++

++

++

Seed A (partial use) (10 mg)

Kaneka kit

++

–

++

++

±

++

++

Seed B (whole seed) (21 mg)

Kaneka kit

++

++

++

++

++

++

++

Seed C (whole seed) (32 mg)

Kaneka kit

++

++

++

++

++

++

++

Corrupt fragments (3 mg)

Kaneka kit

++

+

++

+

++

++

++

A

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Drug-type cannabis

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Kaneka kit

++

±

±

++

++

+

++

Seized resin A (10 mg)

Kaneka kit (1/10)

++

+

+

++

++

++

++

Seized resin A (10 mg)

Kaneka kit (1/100)

++

++

++

++

++

++

++

Seized resin A (10 mg)

Kaneka kit (1/1000)

++

+

++

+

++

++

++

Seized resin A (1 mg)

Kaneka kit

++

++

++

++

++

++

++

Seized resin B (3 mg)

Kaneka kit

++

+

++

+

++

++

++

Seized resin C (3 mg)

Kaneka kit

++

+

++

+

++

++

++

Male leaves (10 mg)

DNeasy kit

++

–

++

++

++

++

++

Male leaves (3 mg)

Kaneka kit

++

–

+

++

++

++

++

Female leaves (10 mg)

DNeasy kit

++

–

++

++

++

++

++

Female leaves (3 mg)

Kaneka kit

++

–

+

++

++

++

++

Root (10 mg)

DNeasy kit

++

–

+

+

++

++

++

Seed D (partial use) (10 mg)

DNeasy kit

++

–

++

++

++

++

++

Seed E (whole seed) (23 mg)

Kaneka kit

++

–

++

++

++

++

++

Seed F (whole seed) (33 mg)

Kaneka kit

++

++

++

++

++

++

Cannabis : tobacco = 1 : 1 (10 mg)

DNeasy kit

Cannabis : tobacco = 1 : 9 (10 mg) Cannabis : tobacco = 1 : 99 (10 mg)

N –

A

Mixed sample

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Fiber-type cannabis

IP T

Seized resin A (10 mg)

U

Cannabis resin

++

+

++

++

++

++

DNeasy kit

++

++

+

++

++

++

++

DNeasy kit

++

+

+

+

++

++

++

DNeasy kit

++

–

–

–

++

–

–

DNeasy kit

++

–

–

–

++

–

–

Mitragyna speciosa(Kratom) (10 mg) DNeasy kit

++

–

–

–

++

–

–

Herbal product A (10 mg)

CC E

PT

Herbal product B (10 mg)

ED

Other samples

M

++

For cannabis resin, defects or reduction of coloration were observed in several

lines for DNA extracted with the Kaneka kit from 10-mg samples. Detection patterns

A

were improved by diluting the DNA solution or decreasing the sampling amount to 1 mg. Previous evidence suggested that the difficulty of detecting cannabis resin was caused by PCR inhibitors [13], which was consistent with another report showing that diluting the template DNA was effective in improving the amplification activity [30]. In the case of fiber-type cannabis, using the three-line version, coloration was 18

detected with the same pattern as found with drug-type cannabis. Using the four-line version, the lack of coloration at FT2 for THCAS clearly distinguished fiber-type from drug-type cannabis. In addition, accurate detection was possible regardless of whether male or female cannabis was tested, because the PCR target regions were not located on

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sex chromosomes. When testing mixed samples, coloration was detectable even at a 1/100 ratio of

SC R

cannabis leaves blended in tobacco leaves, for both versions. Because cannabis leaves are frequently mixed in cigarettes when smoked by abusers, the ability of detecting a small amount of cannabis contamination shows the utility of DNA chromatography for

U

investigating actual cases.

With other simulated samples, two herbal products including synthetic

N

cannabinoids and one of abused plant Mitragyna speciosa were tested. No bands other

A

than ITS were detected in any of the samples, so that they were identified as “not cannabis”

M

in both versions. The DNA chromatography systems showed no false positives for noncannabis plants that could be the targets of drug analysis.

ED

From these results, if the purified DNA was prepared in sufficient quantity, the developed DNA chromatography chip systems enabled accurate cannabis detection of

PT

various types of cannabis samples. The operating procedure of the system is simple and easy; thus, DNA chromatography could be carried out even for those who were not

CC E

accustomed to molecular biological experiments. For further improvement of the reliability, it will be important to test positive and negative controls simultaneously with samples to indicate that no problems occurred with the DNA extraction and the PCR

A

amplification, or that DNA contamination did not occurred in a series of operations.

4. Conclusions The aim of this study was to establish an accurate and simple cannabis DNA detection system. As a result, two systems were developed using a DNA chromatography 19

chip. The four-line version of the system could not only detect cannabis DNA, but also distinguish drug-type and fiber-type cannabis using the sequence of active THCA synthase gene. The three-line version of the system was specialized to identify cannabis and was 100 times more sensitive than the four-line version. In both systems, detection

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was performed with the naked eye, and the only instrument required during the operation was a thermal cycler. DNA extraction from plant samples can easily be performed using

SC R

a commercial kit, so that the DNA chromatography chip system enables cannabis DNA analysis in forensic chemistry laboratories. The developed detection systems have high accuracy, credibility, and robustness because they use results of multiple lines to

U

demonstrate the presence of cannabis. In addition, even if a plant species with crossreactivity for any lines is found, the DNA chromatography chip systems can be improved

N

simply by altering the sequences of the tagged primers. Thus, these systems for cannabis

A

DNA detection, the four-line version and the three-line version, are expected to make a

M

significant contribution to the forensic examination of cannabis.

ED

Author Contribution Statement:

Tadashi Yamamuro: Conceptualization, Methodology, Validation, Investigation, Writing

PT

– Original Draft

Shigehiko Miyamoto: Conceptualization, Methodology, Validation

CC E

Masashi Kitamura: Methodology, Validation, Resources Tomonori Muro: Methodology, Validation Yuko T. Iwata: Validation, Resources, Writing – Review & Editing, Supervision

A

Hiroki Segawa: Validation Kenji Kuwayama: Validation Kenji Tsujikawa: Validation Tatsuyuki Kanamori: Validation Hiroyuki Inoue: Writing – Review & Editing, Supervision, Project Administration 20

Declarations of interest: S. M. is an employee of Kaneka Corporation. T. Y. and S. M. have two pending patents relevant to this work. The other authors declare no conflicts of

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interest regarding this work.

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Acknowledgements

We thank Prof. Kazuhito Wanatabe (currently affiliated with Daiichi University of Pharmacy) and the members of the Genebank Project (National Agriculture and Food

U

Research Organization) for providing cannabis samples. We also thank the members of the Medicinal Plant Garden (Kanazawa University) and Division of Pharmacognosy,

N

Phytochemistry, and Narcotics (National Institute of Health Science) for providing plant

A

(DNA) samples. We would like to thank Editage (www.editage.jp) for English language

M

editing. This work was partially supported by JSPS KAKENHI Grant Number

ED

JP17K15475.

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