- 著者
- Masashi Kitamura Masako Aragane Kou Nakamura Tatsushi Adachi Kazuhito Watanabe Yohei Sasaki
- 出版者
- The Pharmaceutical Society of Japan
- 雑誌
- Biological and Pharmaceutical Bulletin (ISSN:09186158)
- 巻号頁・発行日
- vol.41, no.8, pp.1303-1306, 2018-08-01 (Released:2018-08-01)
- 参考文献数
- 15
- 被引用文献数
- 9
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.
- 著者
- Masashi Kitamura Masako Aragane Kou Nakamura Kazuhito Watanabe Yohei Sasaki
- 出版者
- 公益社団法人日本薬学会
- 雑誌
- Biological and Pharmaceutical Bulletin (ISSN:09186158)
- 巻号頁・発行日
- pp.b16-00090, (Released:2016-04-27)
- 参考文献数
- 31
- 被引用文献数
- 1 23
In many parts of the world, the possession and cultivation of Cannabis sativa L. are restricted by law. As chemical or morphological analyses cannot identify the plant in some cases, a simple yet accurate DNA-based method for identifying C. sativa is desired. We have developed a loop-mediated isothermal amplification (LAMP) assay for the rapid identification of C. sativa. By optimizing the conditions for the LAMP reaction that targets a highly conserved region of tetrahydrocannabinolic acid (THCA) synthase gene, C. sativa was identified within 50 min at 60-66 °C. The detection limit was the same as or higher than that of conventional PCR. The LAMP assay detected all 21 specimens of C. sativa, showing high specificity. Using a simple protocol, the identification of C. sativa could be accomplished within 90 min from sample treatment to detection without use of special equipment. A rapid, sensitive, highly specific, and convenient method for detecting and identifying C. sativa has been developed and is applicable to forensic investigations and industrial quality control.