RESUMO
To establish an accurate, rapid and efficient method for authenticating Cuscutae Semen and Raphani Semen by using rapid PCR amplification. The samples of Cuscutae Semen, Raphani Semen and their adulterants were collected. The total DNA of the samples has been extracted, and ITS sequence from Cuscutae Semen, Raphani Semen and their adulterants was amplified by PCR and sequenced directionally. These sequences were aligned by using Clustal W. Specific primers were designed and amplified by two-steps PCR amplification method. The rapid PCR methods for authenticating Cuscutae Semen and Raphani Semen were established by optimizing the denatured and annealing temperature, cycle numbers, and etc. When 100 × SYBR Green I was added in the PCR product, strong green fluorescence was visualized under 365 nm UV lamp whereas adulterants showed no florescence. The results indicated that the rapid PCR method can identify Cuscutae Semen and Raphani Semen rapidly. This study provides the technical support for authentication of Chinese medicinal materials.
RESUMO
To explore a new method for identification of Mongolian patent medicine (MPM) by PCR amplification of specific alleles. Eight kinds of MPM were used to study the identification of "Digeda" raw materials. The total DNA of Lomatogonium rotatum and Corydalis bungeana samples were extracted through modified CTAB method, psbA-trnH sequence was amplified by PCR and sequenced directionally. Specific primer was designed. The DNA of 8 kinds of MPM also was extracted and purified by the commercial DNA purification kits. The rbcL and two pair of specific primers sequences were amplified. The specific amplified products were sequenced in forward directions. All specific sequences were aligned and were analyzed. The results indicated that L rotatum can be identified by specific primers from Digeda-4 Tang, Digeda-8 San, Digeda-4 San, and C. bungeana medicinal materials can be identified by specific primers from Li Dan Ba Wei San, Yi He Ha Ri-12 and A Ga Ri-35. PCR amplification of specific alleles can stably and accurately distinguish raw medicinal materials in MPM.
Assuntos
Alelos , Primers do DNA , Genética , DNA de Plantas , Genética , Medicina Tradicional da Mongólia , Dados de Sequência Molecular , Plantas Medicinais , Classificação , Genética , Reação em Cadeia da Polimerase , MétodosRESUMO
The sequences of ITS, matK, rbcL and psbA-trnH of 9 Gynostemma species or variety including 38 samples were compared and analyzed by molecular phylogeny method. Hemsleya macrosperma was designated as outgroup. The MP and NJ phylogenetic tree of Gynostemma was built based on ITS sequence, the results of PAUP phylogenetic analysis showed the following results: (1) The eight individuals of G. pentaphyllum var. pentaphyllum were not supported as monophyletic in the strict consensus trees and NJ trees. (2) It is suspected whether G. longipes and G. laxum should be classified as the independent species. (3)The classification of subgenus units of Gynostemma plants is supported.
Assuntos
Gynostemma , Classificação , Genética , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas , Genética , Análise de Sequência de DNARESUMO
To explore the new method of discriminating Cistanche deserticola, Cynomorium songaricum and Orobanche pycnostachya by using PCR amplification of specific alleles. 30 samples of the different C. deserticola, 21 samples of C. songaricum and O. pycnostachya were collected. The total DNA of the samples were extracted, the ITS sequences from C. deserticola, C. songaricum and O. pycnostachya were amplified by PCR and sequenced unidirectionally. These sequences were aligned by using ClustulW. Specific primer was designed according to the ITS sequences of specific alleles, and PCR reaction system was optimized. Additionally, compare with the identification of specific PCR method and DNA sequence analysis method. The result showed that the 331 bp identification band for C. deserticola and the adulterants not amplified bands by a single PCR reaction, which showed good identification ability to the three species. PCR amplification of specific alleles can be used to identify C. deserticola, C. songaricum and O. pycnostachya successfully.
Assuntos
Alelos , Cistanche , Classificação , Genética , Primers do DNA , Genética , DNA Intergênico , Genética , DNA de Plantas , Genética , Contaminação de Medicamentos , Filogenia , Reação em Cadeia da Polimerase , MétodosRESUMO
In this paper, Liuwei Dihuang pill was used to study the identification of Chinese patent medicine by fluorescence sequencing typing technology. The DNA of Paeonia suffruticosa was used as template to amplify by five pair of FAM fluorescence labeling primers. Then, the amplified products were sequenced. The sequencing results were analyzed by GeneMarker V1.80 to screen the best fluorescence labeling primers. As a result, psbA-trnH fluorescence labeling primer was used to identify the raw materials of Liuwei Dihuang pill. The results showed that three kinds of raw plant medicinal materials in Liuwei Dihuang pill were able to be correctly identified by psbA-trnH fluorescence labeling primer. The fluorescence sequencing typing technology can stably and accurately distinguish raw medicinal materials in Chinese patent medicine.
Assuntos
Primers do DNA , Química , Genética , DNA de Plantas , Química , Genética , Medicamentos de Ervas Chinesas , Química , Padrões de Referência , Corantes Fluorescentes , Química , Plantas Medicinais , Química , Genética , Reação em Cadeia da Polimerase , Métodos , Controle de Qualidade , Coloração e RotulagemRESUMO
<p><b>OBJECTIVE</b>To explore the new method of discriminating Gelsemium elegans from Lonicera japonica and its close species by using specific PCR amplification.</p><p><b>METHOD</b>Thirteen samples of the different G. elegans materials and 58 samples of L. japonica, L. macranthoides and L. dasystyla were collected. The total DNA of the samples were extracted, and the DNA of G. elegans, L. japonica and L. macranthoides water extracts were extracted. PsbA-rnnH sequence from G. elegans was amplified by PCR and sequenced unidirectionally, ClustulW was used to align psbA-trnH sequences of the G. elegans and L. japonica and its close species from GenBank database.</p><p><b>RESULT</b>All samples were amplified by PCR with specific primer, DNA from G. elegans would be amplified 97 bp whereas PCR products from all of Lonicera samples had not bands.</p><p><b>CONCLUSION</b>Specific PCR amplification can be used to identify G. elegans from L. japonica and its close species successfully and is an efficient molecular marker for authentication of G. elegans and L. japonica and its close species.</p>
Assuntos
DNA de Plantas , Genética , Medicamentos de Ervas Chinesas , Gelsemium , Química , Genética , Lonicera , Química , Genética , Filogenia , Extratos Vegetais , Química , Genética , Reação em Cadeia da Polimerase , Água , QuímicaRESUMO
To evaluate the effect of PCR enhancer on molecular identification of Chinese herbal medicine, and select the optimal enhancers suitable for traditional Chinese medicine (TCM), genomic DNA from 180 kinds of Chinese herbal medicine was extract by CTAB method and alkaline lysis method, respectively. PCR success rate of five universal fragments (ITS2, psbA-trnH, rbcL, matK, trnL-trnF) was compared in a PCR system with and without enhancer. PCR efficiency of Real-time PCR was also compared in a PCR system with and without enhancer. Results showed that PCR success rate of ITS2,psbA-trnH, rbcL fragment was increased by using PVP and BSA. The PCR efficiency was decreased by PCR enhancer in Real-time PCR system. The results indicate that BSA and PVP as PCR enhancer can dramatically increase PCR success rate and genotyping accuracy in TCM molecular authentication.
Assuntos
DNA de Plantas , Genética , Medicamentos de Ervas Chinesas , Genótipo , Reação em Cadeia da Polimerase , Métodos , Controle de Qualidade , TemperaturaRESUMO
To explore the new method of discriminating Astragali Radix and Hedysari Radix by using PCR amplification of specific alleles, 30 samples of the different Astragali Radix materials and 28 samples of Hedysari Radix were collected. The total DNA of all samples were extracted, trnL-trnF sequence from Astragali Radix and Hedysari Radix was amplified by PCR and sequenced unidirectionally. These sequences were aligned by using Clustul W. Primer was designed and the PCR reaction systems including annealing temperature, dNTP, etc were optimized. All samples were amplified by PCR with specific primer, DNA from Astragali Radix would be amplified 136 bp, whereas PCR products from all of Hedysari Radix were 323 bp. This method can detect 10% of intentional Hedysari Radix DNA into Astragali Radix. PCR amplification of alleles can be used to identify Astragali Radix and Hedysari Radix successfully and is an efficient molecular marker for authentication of Astragali Radix and Hedysari Radix.
Assuntos
Alelos , Astrágalo , Classificação , Genética , Código de Barras de DNA Taxonômico , DNA de Plantas , Genética , Reação em Cadeia da PolimeraseRESUMO
Lian Qiao Bai Du Wan was used to study the identification of Chinese patent medicine by molecular marker technique. DNA was extracted through modified CTAB method. The psbA-trnH and rbcL sequences were gradient amplified, and PCR products were ligated with the pEASY-T5 vector and then transformed into Trans1-T1 cells, respectively. Clones were selected randomly and sequenced. All sequences were analyzed by BlastN and the neighbor-joining (NJ) phylogenetic tree was constructed by MEGA 4.0. The results showed that nine kinds of medicinal materials can be identified by psbA-trnH sequences, and six kinds of medicinal materials by rbcL sequences from Lian Qiao Bai Du Wan. Molecular marker technique can stably and accurately distinguish multi-origin medicinal materials in Chinese patent medicine.
Assuntos
Sequência de Bases , Cloroplastos , Genética , Análise por Conglomerados , Código de Barras de DNA Taxonômico , DNA de Cloroplastos , Genética , DNA Intergênico , Genética , DNA de Plantas , Genética , Medicamentos de Ervas Chinesas , Química , Forsythia , Química , Genética , Filogenia , Plantas Medicinais , Química , Genética , Reação em Cadeia da Polimerase , Ribulose-Bifosfato Carboxilase , Genética , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
<p><b>OBJECTIVE</b>To explore a new method for identification Astragali Radix from its adulterants by using ITS sequence.</p><p><b>METHOD</b>Thirteen samples of the different Astragali Radix materials and 6 samples of the adulterants of the roots of Hedysarum polybotrys, Medicago sativa and Althaea rosea were collected. ITS sequence was amplified by PCR and sequenced unidirectionally. The interspecific K-2-P distances of Astragali Radix and its adulterants were calculated, and NJ tree and UPGMA tree were constructed by MEGA 4.</p><p><b>RESULT</b>ITS sequences were obtained from 19 samples respectively, there were Astragali Radix 646-650 bp, H. polybotrys 664 bp, Medicago sativa 659 bp, Althaea rosea 728 bp, which were registered in the GenBank. Phylogeny trees reconstruction using NJ and UPGMA analysis based on ITS nucleotide sequences can effectively distinguish Astragali Radix from adulterants.</p><p><b>CONCLUSION</b>ITS sequence can be used to identify Astragali Radix from its adulterants successfully and is an efficient molecular marker for authentication of Astragali Radix and its adulterants.</p>