Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 9 de 9
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Biol Pharm Bull ; 41(8): 1303-1306, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30068883

RESUMO

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.


Assuntos
Cannabis/genética , DNA de Plantas/análise , Técnicas de Amplificação de Ácido Nucleico , Colorimetria , Estruturas Vegetais/genética
2.
Shokuhin Eiseigaku Zasshi ; 59(4): 174-182, 2018.
Artigo em Japonês | MEDLINE | ID: mdl-30158396

RESUMO

Colchicum autumnale is a perennial, toxic plant that originated in Europe and North Africa. Although inedible, it is occasionally consumed accidentally because it resembles the edible Allium victorialis and other related species. This misidentification has led to episodes of food poisoning in Japan. However, determining the causative agent of a food poisoning outbreak by observing the sample visually or analyzing the chemical composition is challenging when dealing with small samples. Therefore, we developed a novel set of PCR primers that anneal to the internal transcribed spacer (ITS) region of C. autumnale ribosomal DNA, designed to detect the presence of C. autumnale in small samples. These primers successfully detected C. autumnale in all samples in which it was present, and did not give a positive PCR band in the 48 other distinct crop species tested, in which it was not present. Further, our method could amplify DNA from samples of C. autumnale that had been heat-treated and digested using artificial gastric fluids. Thus, this PCR strategy is highly specific and can be used to distinguish C. autumnale simply and rapidly from various other crops.


Assuntos
Colchicum/classificação , DNA de Plantas/isolamento & purificação , Doenças Transmitidas por Alimentos/diagnóstico , Primers do DNA , DNA Espaçador Ribossômico/genética , Humanos , Japão , Reação em Cadeia da Polimerase
3.
Forensic Sci Int ; 275: 160-166, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28366623

RESUMO

Food poisoning is frequently caused by the accidental ingestion of toxic plants that possess strong morphological similarities to edible plants. False helleborine (Veratrum album) is one of the most common plants involved in such accidents. In cases of poisoning by toxic plants, rapid and accurate identification, usually based on the morphological or chemical analysis of plant parts, is required for appropriate medical treatment or forensic investigation. However, morphological examinations require experience in systematic botany because the samples are fragmentary, and chemical analysis of natural compounds can be difficult. In this study, we developed a TaqMan real-time PCR method using trnH-psbA and trnL-trnF that could be carried out in 30-60min. The lower detection limit was less than 10pg of DNA and the primer sets were specific to V. album and Veratrum stamineum. Mixed samples, cooked samples, and simulated gastric contents were successfully identified, and a multiplex assay of two regions was also possible. These results indicate that the TaqMan real-time PCR analysis is a very effective method to detect small samples of V. album and V. stamineum accurately and rapidly in poisoning cases.


Assuntos
Plantas Tóxicas/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Veratrum/genética , DNA de Plantas/genética , Doenças Transmitidas por Alimentos , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos
4.
Shokuhin Eiseigaku Zasshi ; 58(1): 32-35, 2017.
Artigo em Japonês | MEDLINE | ID: mdl-28260730

RESUMO

Kuwazuimo (Alocasia odora) and shimakuwazuimo (Alocasia cucullata) are evergreen perennial plants that originated in East Asia. Although inedible, they are occasionally eaten by mistake because they resemble satoimo (Colocasia esculenta), and this has caused food poisoning in Japan. It is not easy to determine the cause of a food poisoning outbreak from the shape or chemical composition when the available sample is small. Therefore, we developed a new primer pair for PCR to identify kuwazuimo and shimakuwazuimo in small samples, based on the internal transcribed spacer (ITS) region of ribosomal DNA. Using PCR with the developed primer pair, we detected all samples of kuwazuimo obtained from the market, while excluding 17 other kinds of crops. The samples were identified as shimakuwazuimo by DNA sequencing of the PCR products. The present PCR method showed high specificity and was confirmed to be applicable to the identification of kuwazuimo and shimakuwazuimo from various crops.


Assuntos
Alocasia/química , Alocasia/genética , Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Reação em Cadeia da Polimerase/métodos , Alocasia/envenenamento , DNA Ribossômico , Doenças Transmitidas por Alimentos/etiologia , Doenças Transmitidas por Alimentos/prevenção & controle , Sensibilidade e Especificidade , Análise de Sequência de DNA
5.
J Nat Med ; 71(1): 86-95, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27535292

RESUMO

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.


Assuntos
Técnicas Biossensoriais/métodos , Cannabis/química , Sequência de Aminoácidos
6.
Biol Pharm Bull ; 39(7): 1144-9, 2016 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-27118244

RESUMO

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.


Assuntos
Cannabis/genética , Oxirredutases Intramoleculares/genética , Técnicas de Amplificação de Ácido Nucleico , DNA de Plantas/análise , Folhas de Planta/genética
7.
J Nat Med ; 68(4): 677-85, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24952707

RESUMO

In May 2011, numerous poppy plants closely resembling Papaver bracteatum Lindl., a type of narcotic plant that is illegal in Japan, were distributed directly from several large flower shops or through online shopping throughout Japan, including the Tokyo Metropolitan area. In order to better identify the narcotic plants, the relative nuclear DNA content at the vegetative stage was measured by flow cytometric (FCM) analysis in 3 closely-related species of the genus Papaver section Oxytona, namely P. orientale, P. pseudo-orientale, and P. bracteatum, based on the difference between the chromosome numbers of these species. The results showed that the nuclear DNA content differed between these 3 species, and that most of the commercially distributed plants examined in this study could be identified as P. bracteatum. The remaining plants were P. pseudo-orientale, a non-narcotic plant. In addition, the FCM results for the identification of P. bracteatum completely agreed with the results obtained by the morphological analysis, the inter-genic spacer sequence of rpl16-rpl14 (PS-ID sequence) of chloroplast DNA, and the presence of thebaine. These results clearly indicate the usefulness of FCM analysis for the identification of P. bracteatum plants, including when they are in their vegetative stage.


Assuntos
Citometria de Fluxo , Papaver/classificação , DNA de Cloroplastos/química , Flores/química , Japão , Entorpecentes/análise , Papaver/anatomia & histologia , Papaver/química , Papaver/genética , Tebaína/análise
8.
J Nat Med ; 65(1): 103-10, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20890669

RESUMO

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.


Assuntos
Cactaceae/química , Cactaceae/genética , Mescalina/química , Cactaceae/ultraestrutura , Microscopia Eletrônica de Varredura , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição
9.
Biol Pharm Bull ; 32(5): 887-91, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19420759

RESUMO

We have developed a convenient method for the detection of Lophophora williamsii using loop-mediated isothermal amplification (LAMP). We designed six species-specific primers for L. williamsii, including two loop primers. This L. williamsii-specific primer set was used for LAMP of total DNA extracted from L. williamsii and from L. diffusa. Real-time monitoring of LAMP was achieved by measuring turbidity due to the formation of magnesium pyrophosphate. Amplification occurred in samples mixed with total DNA from L. williamsii, but not in those mixed with total DNA from L. diffusa. We could also visually observe L. williamsii by adding fluorescent detection reagent to the reaction tube and exposing it to UV light. This new method amplified L. williamsii selectively and is expected to be applicable to the detection of peyote.


Assuntos
Cactaceae/classificação , DNA de Plantas/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Sequência de Bases , Cactaceae/genética , Primers do DNA , DNA de Plantas/isolamento & purificação , Dados de Sequência Molecular , Nefelometria e Turbidimetria , Sensibilidade e Especificidade , Fatores de Tempo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA