Autenticação de Ginseng brasileiro utilizando a técnica de High Resolution Melting (HRM)

Detalhes bibliográficos
Autor(a) principal: Almeida, Francine Alves Nogueira de
Data de Publicação: 2018
Tipo de documento: Dissertação
Idioma: por
Título da fonte: Repositório Institucional da Universidade Federal do Espírito Santo (riUfes)
Texto Completo: http://repositorio.ufes.br/handle/10/7113
Resumo: As the herbal industry grows, the authenticity of its products is a matter of consumer safety and cannot be neglected. Brazilian Ginseng, for example, refers to two distinct species, Pfaffia glomerata and Hebanthe eriantha. Despite the similarity between these two species, which hinders morphological identification, they have different chemical and pharmacological properties. In this project we proposed a High Resolution Melting technique (HRM) as a tool to discriminate and identify P. glomerata and H. eriantha species using a matK and rbcL gene region. For this purpose, six reference samples were obtained, three of each of the aforementioned species, and 60 (sixty) commercial samples, sold as Brazilian Ginseng, through physical or online purchase. The DNA is extracted by the CTAB method and kit. The primer designed were tested through amplification by conventional PCR and confirmed in polyacrylamide gel before standardization of the PCR amplificationHRM and finally the results were compared to sequencing. Three primers were developed for the matK gene, two for the HRM-matKD and HRM-matK, and one for the rbcL gene , HRM-rbcL . During the standardization, the influence of the annealing temperature and the concentration of the primer on the race result was noticed, however, the extraction method did not change the results. The analysis of HRM showed that the first HRM-matKD was the one that met the proposed objective presenting high sensitivity (92-93%) and specificity (100%) compared to the sequencing method. It was possible to validate the HRM technique using previously sequenced commercial samples, which allows us to state that the HRM technique can be used to discriminate H. eriantha and P. glomerata. HRM is a fast and low cost method, being a reliable tool for identification of Brazilian Ginseng species.
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spelling Paneto, Greiciane GaburroAlmeida, Francine Alves Nogueira deFerreira, Marcia Flores da SilvaMoura Neto, Rodrigo2018-08-01T21:35:03Z2018-08-012018-08-01T21:35:03Z2018-02-20As the herbal industry grows, the authenticity of its products is a matter of consumer safety and cannot be neglected. Brazilian Ginseng, for example, refers to two distinct species, Pfaffia glomerata and Hebanthe eriantha. Despite the similarity between these two species, which hinders morphological identification, they have different chemical and pharmacological properties. In this project we proposed a High Resolution Melting technique (HRM) as a tool to discriminate and identify P. glomerata and H. eriantha species using a matK and rbcL gene region. For this purpose, six reference samples were obtained, three of each of the aforementioned species, and 60 (sixty) commercial samples, sold as Brazilian Ginseng, through physical or online purchase. The DNA is extracted by the CTAB method and kit. The primer designed were tested through amplification by conventional PCR and confirmed in polyacrylamide gel before standardization of the PCR amplificationHRM and finally the results were compared to sequencing. Three primers were developed for the matK gene, two for the HRM-matKD and HRM-matK, and one for the rbcL gene , HRM-rbcL . During the standardization, the influence of the annealing temperature and the concentration of the primer on the race result was noticed, however, the extraction method did not change the results. The analysis of HRM showed that the first HRM-matKD was the one that met the proposed objective presenting high sensitivity (92-93%) and specificity (100%) compared to the sequencing method. It was possible to validate the HRM technique using previously sequenced commercial samples, which allows us to state that the HRM technique can be used to discriminate H. eriantha and P. glomerata. HRM is a fast and low cost method, being a reliable tool for identification of Brazilian Ginseng species.À medida que a indústria de plantas medicinais cresce, a autenticidade de seus produtos é uma questão de segurança do consumidor e não pode ser negligenciada. O Ginseng brasileiro, por exemplo, se refere a duas espécies distintas, Pfaffia glomerata e Hebanthe eriantha. Apesar da similaridade entre essas duas espécies, o que dificulta a identificação morfológica, elas possuem propriedades químicas e farmacológicas distintas. Foi proposto que a técnica de High Resolution Melting (HRM) como uma ferramenta para discriminar e identificar as espécies P. glomerata e H. eriantha utilizando uma região do gene matK e rbcL. Para tal, foram adquiridas seis amostras referência, três de cada uma das espécies citadas, e 60 amostras comerciais, vendidas como Ginseng brasileiro, por meio de compra física ou online. O DNA foi extraído pelos métodos CTAB e por Kit. Os primers desenhados foram testados através da amplificação por PCR convencional e confirmado em gel de poliacrilamida antes da padronização da amplificação por PCR-HRM e por fim os resultados foram comparados aos de sequenciamento. Foram desenvolvidos três primers dois para o gene matK, HRM-matKD e HRM-matK, e um para o gene rbcL, HRM-rbcL. Durante a padronização notou-se a influência da temperatura de anelamento e concentração do primer no resultado da corrida, entretanto o método de extração não alterou os resultados. A análise de HRM mostrou que o primer HRM-matKD foi o que atendeu ao objetivo proposto apresentando alta sensibilidade (92-93%) e especificidade (100%) comparado ao método de sequenciamento. Foi possível validar a técnica de HRM utilizando amostras comerciais previamente sequenciadas, o que nos permite afirmar que a técnica de HRM pode ser utilizada para discriminar H. eriantha e P. glomerata. HRM é um método rápido e de baixo custo, sendo uma ferramenta confiável para identificação de espécies de Ginseng brasileiro.Texthttp://repositorio.ufes.br/handle/10/7113porUniversidade Federal do Espírito SantoMestrado em BiotecnologiaPrograma de Pós-Graduação em BiotecnologiaUFESBRCentro de Ciências da SaúdeIdentificationMedicinal plantAdulterationPfaffiaHebantheIdentificaçãoPlanta medicinalAdulteraçãoGinseng-brasileiroPlantas medicinaisPlantas - Biologia molecularBiotecnologia61Autenticação de Ginseng brasileiro utilizando a técnica de High Resolution Melting (HRM)info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da Universidade Federal do Espírito Santo (riUfes)instname:Universidade Federal do Espírito Santo (UFES)instacron:UFESORIGINALtese_12002_Dissertação_Francine Alves Nogueira de Almeida.pdfapplication/pdf2009835http://repositorio.ufes.br/bitstreams/3947fc31-2337-41b3-a834-a8e6393e14c2/downloadd864a01c75bbc516074e2befb4c57c5cMD5110/71132024-07-16 17:06:09.239oai:repositorio.ufes.br:10/7113http://repositorio.ufes.brRepositório InstitucionalPUBhttp://repositorio.ufes.br/oai/requestopendoar:21082024-10-15T18:00:01.911709Repositório Institucional da Universidade Federal do Espírito Santo (riUfes) - Universidade Federal do Espírito Santo (UFES)false
dc.title.none.fl_str_mv Autenticação de Ginseng brasileiro utilizando a técnica de High Resolution Melting (HRM)
title Autenticação de Ginseng brasileiro utilizando a técnica de High Resolution Melting (HRM)
spellingShingle Autenticação de Ginseng brasileiro utilizando a técnica de High Resolution Melting (HRM)
Almeida, Francine Alves Nogueira de
Identification
Medicinal plant
Adulteration
Pfaffia
Hebanthe
Identificação
Planta medicinal
Adulteração
Biotecnologia
Ginseng-brasileiro
Plantas medicinais
Plantas - Biologia molecular
61
title_short Autenticação de Ginseng brasileiro utilizando a técnica de High Resolution Melting (HRM)
title_full Autenticação de Ginseng brasileiro utilizando a técnica de High Resolution Melting (HRM)
title_fullStr Autenticação de Ginseng brasileiro utilizando a técnica de High Resolution Melting (HRM)
title_full_unstemmed Autenticação de Ginseng brasileiro utilizando a técnica de High Resolution Melting (HRM)
title_sort Autenticação de Ginseng brasileiro utilizando a técnica de High Resolution Melting (HRM)
author Almeida, Francine Alves Nogueira de
author_facet Almeida, Francine Alves Nogueira de
author_role author
dc.contributor.advisor1.fl_str_mv Paneto, Greiciane Gaburro
dc.contributor.author.fl_str_mv Almeida, Francine Alves Nogueira de
dc.contributor.referee1.fl_str_mv Ferreira, Marcia Flores da Silva
dc.contributor.referee2.fl_str_mv Moura Neto, Rodrigo
contributor_str_mv Paneto, Greiciane Gaburro
Ferreira, Marcia Flores da Silva
Moura Neto, Rodrigo
dc.subject.eng.fl_str_mv Identification
Medicinal plant
Adulteration
topic Identification
Medicinal plant
Adulteration
Pfaffia
Hebanthe
Identificação
Planta medicinal
Adulteração
Biotecnologia
Ginseng-brasileiro
Plantas medicinais
Plantas - Biologia molecular
61
dc.subject.por.fl_str_mv Pfaffia
Hebanthe
Identificação
Planta medicinal
Adulteração
dc.subject.cnpq.fl_str_mv Biotecnologia
dc.subject.br-rjbn.none.fl_str_mv Ginseng-brasileiro
Plantas medicinais
Plantas - Biologia molecular
dc.subject.udc.none.fl_str_mv 61
description As the herbal industry grows, the authenticity of its products is a matter of consumer safety and cannot be neglected. Brazilian Ginseng, for example, refers to two distinct species, Pfaffia glomerata and Hebanthe eriantha. Despite the similarity between these two species, which hinders morphological identification, they have different chemical and pharmacological properties. In this project we proposed a High Resolution Melting technique (HRM) as a tool to discriminate and identify P. glomerata and H. eriantha species using a matK and rbcL gene region. For this purpose, six reference samples were obtained, three of each of the aforementioned species, and 60 (sixty) commercial samples, sold as Brazilian Ginseng, through physical or online purchase. The DNA is extracted by the CTAB method and kit. The primer designed were tested through amplification by conventional PCR and confirmed in polyacrylamide gel before standardization of the PCR amplificationHRM and finally the results were compared to sequencing. Three primers were developed for the matK gene, two for the HRM-matKD and HRM-matK, and one for the rbcL gene , HRM-rbcL . During the standardization, the influence of the annealing temperature and the concentration of the primer on the race result was noticed, however, the extraction method did not change the results. The analysis of HRM showed that the first HRM-matKD was the one that met the proposed objective presenting high sensitivity (92-93%) and specificity (100%) compared to the sequencing method. It was possible to validate the HRM technique using previously sequenced commercial samples, which allows us to state that the HRM technique can be used to discriminate H. eriantha and P. glomerata. HRM is a fast and low cost method, being a reliable tool for identification of Brazilian Ginseng species.
publishDate 2018
dc.date.accessioned.fl_str_mv 2018-08-01T21:35:03Z
dc.date.available.fl_str_mv 2018-08-01
2018-08-01T21:35:03Z
dc.date.issued.fl_str_mv 2018-02-20
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dc.format.none.fl_str_mv Text
dc.publisher.none.fl_str_mv Universidade Federal do Espírito Santo
Mestrado em Biotecnologia
dc.publisher.program.fl_str_mv Programa de Pós-Graduação em Biotecnologia
dc.publisher.initials.fl_str_mv UFES
dc.publisher.country.fl_str_mv BR
dc.publisher.department.fl_str_mv Centro de Ciências da Saúde
publisher.none.fl_str_mv Universidade Federal do Espírito Santo
Mestrado em Biotecnologia
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