Genus Lophophora (Cactaceae) has two species: Lophophora williamsii Coulter, which is called peyote, and L. diffusa Bravo. Although it was reported that L. williamsii contained mescaline and L. diffusa did not, we found L. williamsii specimens that did not contain mescaline. This finding indicated that the two species could not be differentiated in terms of mescaline content. Moreover, the relationship between mescaline content and morphology of the two species is also unknown. In this study, we attempted to clarify the difference in morphology, mescaline content, and DNA alignment of the chloroplast trnL/trnF region between L. williamsii and L. diffusa. As a result, L. williamsii specimens were classified into two groups. Group 1 had small protuberances on the epidermis, contained mescaline, and the analyzed region on the trnL/trnF sequence was 881 base pairs (bp) long in all except one (877 bp). Group 2 had large protuberances on the epidermis, did not contain mescaline, and the analyzed region was 893 bp long. On the other hand, L. diffusa had medium-sized protuberances on the epidermis, did not contain mescaline, and the analyzed region was 903 bp long. Also investigated was the potential application of the PCR-restriction fragment length polymorphism (RFLP) method as a means of identification based on the trnL/trnF sequence. By applying the PCR-RFLP method, the two species could be distinguished and L. williamsii specimens could be differentiated into group 1 and group 2.

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.

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In Cannabis sativa L., tetrahydrocannabinol (THC) is the primary psychoactive compound and exists as the carboxylated form, tetrahydrocannabinolic acid (THCA). C. sativa is divided into two strains based on THCA content-THCA-rich (drug-type) strains and THCA-poor (fiber-type) strains. Both strains are prohibited by law in many countries including Japan, whereas the drug-type strains are regulated in Canada and some European countries. As the two strains cannot be discriminated by morphological analysis, a simple method for identifying the drug-type strains is required for quality control in legal cultivation and forensic investigation. We have developed a novel loop-mediated isothermal amplification (LAMP) assay for identifying the drug-type strains of C. sativa. We designed two selective LAMP primer sets for on-site or laboratory use, which target the drug-type THCA synthase gene. The LAMP assay was accomplished within approximately 40 min. The assay showed high specificity for the drug-type strains and its sensitivity was the same as or higher than that of conventional polymerase chain reaction. We also showed the effectiveness of melting curve analysis that was conducted after the LAMP assay. The melting temperature values of the drug-type strains corresponded to those of the cloned drug-type THCA synthase gene, and were clearly different from those of the cloned fiber-type THCA synthase gene. Moreover, the LAMP assay with simple sample preparation could be accomplished within 1 h from sample treatment to identification without the need for special devices or techniques. Our rapid, sensitive, specific, and simple assay is expected to be applicable to laboratory and on-site detection.

In plants, nucleotide alignment of specific DNA regions, such as nuclear ribosomal RNA or chloroplast DNA, has been used for the determination of phylogenetic relationships and species classification.1-3) Such DNA information has also been used to authenticate herbal medicine in pharmacognosy. 4,5) There have been attempts to use DNA polymorphism-based detection techniques-commonly employed in the life sciences-for the authentication of medicinal plants. Such techniques include amplification-refractory mutation system (ARMS), 6) polymerase chain reaction (PCR)-restriction fragment-length polymorphism (PCR-RFLP), 7) and TaqMan assay.8) However, not all techniques can be applied directly to plant materials because total DNA from plants contain many secondary metabolites and polysaccharides. In PCR amplification, difficulties are sometimes encountered when total DNA extracted from plant materials was used as template without preliminary preparation of the sample via purification or technical modification.9,10) Moreover, the sequence selectivity of oligonucleotides such as PCR primers is decreased in reaction solutions that contain impurities. As many herbal medicines are derived from underground parts having high polysaccharide content, we saw the need to develop a new method to overcome this problem.Loop-mediated isothermal amplification (LAMP) technique has been developed and used in the detection of microorganisms. [11][12][13] As LAMP employs 4 to 6 primers, it involves 4 to 6 reactive sites and 6 to 8 recognition regions, giving it increased sensitivity and specificity compared to typical PCR. Moreover, the isothermal reaction condition increases time efficiency compared to PCR, which requires three temperature steps. LAMP achieves detection in one to two hours. We have already observed that LAMP has good amplification ability in the detection and identification of Curcuma plants.14) LAMP was successful even though Curcuma rhizomes have high polysaccharide content, which made extraction of total DNA difficult.The genus Lophophora, Cactaceae, is distributed in Central and South America; L. williamsii, L. diffusa, and several other varieties are included in this genus. 20) In this manuscript, we have adopted the theory that these two species are different. L. williamsii is known by its common name, peyote. Because it contains mescaline, a psychedelic, its use is regulated in some countries. There should be the method to indentify this species but there is not because it is very difficult to differentiate those two species morphologically. The species-specific detection methods are all the more nonexistent. Thus, we have analyzed the nucleotide alignment of the chloroplast trnL intron region in L. williamsii and L. diffusa and clarified the differences. Three sequence patterns were observed in L. williamsii (DNA Data Bank of Japan (DDBJ) accession Nos. AB362488, AB362489, and AB362490), and one pattern was observed in L. diffusa (AB362491). We found that these two species have different sequences in the trnL int...

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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.

Identifying different species of the genus Atractylodes which are commonly used in Chinese and Japanese traditional medicine, using chromatographic approaches can be difficult. 1H NMR metabolic profiling of DNA-authenticated, archived rhizomes of the genus Atractylodes was performed for genetic and chemical evaluation. The ITS region of the nuclear rDNA was sequenced for five species, A. japonica, A. macrocephala, A. lancea, A. chinensis, and A. koreana. Our samples had nucleotide sequences as previously reported, except that part of the A. lancea cultivated in Japan had a type 5, hybrid DNA sequence. Principal component analysis (PCA) using 1H NMR spectra of extracts with two solvent systems (CD3OD, CDCl3) was performed. When CDCl3 extracts were utilized, the chemometric analysis enabled the identification and classification of Atractylodes species according to their composition of major sesquiterpene compounds. The 1H NMR spectra using CD3OD contained confounding sugar peaks. PCA removal of these peaks gave the same result as that obtained using CDCl3 and allowed species distinction. Such chemometric methods with multivariate analysis of NMR spectra will be useful for the discrimination of plant species, without specifying the index components and quantitative analysis on multi-components. Graphic abstract