Improved On-Site Protocol for the DNA-Based Species Identification of <i>Cannabis sativa</i> by Loop-Mediated Isothermal Amplification

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  • Kitamura Masashi
    Laboratory of Molecular Pharmacognosy, Division of Pharmaceutical Sciences, Graduate School of Medical Sciences, Kanazawa University Forensic Science Laboratory, Ishikawa Prefectural Police H.Q.
  • Aragane Masako
    Medicinal Plant Garden, Tokyo Metropolitan Institute of Public Health
  • Nakamura Kou
    Medicinal Plant Garden, Tokyo Metropolitan Institute of Public Health
  • Adachi Tatsushi
    Kaneka Corporation
  • Watanabe Kazuhito
    Daiichi University of Pharmacy
  • Sasaki Yohei
    Laboratory of Molecular Pharmacognosy, Division of Pharmaceutical Sciences, Graduate School of Medical Sciences, Kanazawa University

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  • Improved On-Site Protocol for the DNA-Based Species Identification of Cannabis sativa by Loop-Mediated Isothermal Amplification
  • Improved on-site protocol for the DNA-based species identification of Cannabis sativa L. by loop-mediated isothermal amplification

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Abstract

<p>Cannabis sativa L. is cultivated worldwide for a variety of purposes, but its cultivation and possession are regulated by law in many countries, necessitating accurate detection methods. We previously reported a DNA-based C. sativa identification method using the loop-mediated isothermal amplification (LAMP) assay. Although the LAMP technique can be used for on-site detection, our previous protocol took about 90 min from sampling to detection. In this study, we report an on-site protocol that can be completed in 30 min for C. sativa identification based on a modified LAMP system. Under optimal conditions, the LAMP reaction started at approximately 10 min and was completed within 20 min at 63°C. It had high sensitivity (10 pg of purified DNA). Its specificity for C. sativa was confirmed by examining 20 strains of C. sativa and 50 other species samples. With a simple DNA extraction method, the entire procedure from DNA extraction to detection required only 30 min. Using the protocol, we were able to identify C. sativa from various plant parts, such as the leaf, stem, root, seed, and resin derived from C. sativa extracts. As the entire procedure was completed using a single portable device and the results could be evaluated by visual detection, the protocol could be used for on-site detection and is expected to contribute to the regulation of C. sativa.</p>

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