Molecular Identification of Polygonum chinensis Using DNA Barcoding / 中国药学杂志

OBJECTIVE: To identify Polygonum chinensis and its adulterants by ITS2 sequences. METHODS: Total genomic DNA of P. chinensis was extracted using the plant genomic DNA kit. The internal transcribed spacer 2(ITS2) regions were amplified. The variable site of ITS2 regions were analysed through MEGA 6.0 software. The intra-versus inter-specific genetic distances of the ITS2 regions was calculated based on the kimura 2-parameter(K2P) model. Neighbor-Joining phylogenetic trees were constructed using MEGA6.0. RESULTS: The intraspecific variation of P. chinensis was small. However, the interspecific variation of P. chinensis and its adulterants was small distinct. The secondary structure of ITS2 of P. chinensis and its adulterants has significant difference. NJ trees can identify P. chinensis and its adulterants. CONCLUSION: ITS2 Regions can be used to authenticate P. chinensis and its adulterants which provide new METHODS for the identification of P. chinensis. The standard DNA barcodes of P. chinensis are establishment which would lay the foundation of identification and safety clinical drug application of P. chinensis.

Prevalence of infection and molecular confirmation by using ITS-2 region of Fasciola gigantica found in domestic cattle from Chiang Mai province, Thailand

OBJECTIVE@#To investigate the infection of Fasciola gigantica (F. gigantica) in domestic cattle from Chiang Mai province and molecular confirmation using ITS-2 region.@*METHODS@#The liver and gall bladder of Bubalus bubalis (B. bubalis) and Bos taurus (B. taurus) from slaughterhouses were examined adult worms and prevalence investigation. The species confirmation with phylogenetic analysis using ITS-2 sequences was performed by maximum likelihood and UPGMA methods.@*RESULTS@#The total prevalences of infection in B. bubalis and Bubalus taurus (B. taurus) were 67.27% and 52.94% respectively. The respective prevalence in both B. bubalis and B. taurus were acquired from Doi-Saket, Muang, and Sanpatong districts, with 81.25%, 62.50% and 60.00% for B. bubalis and 62.50%, 50.00% and 47.06% for Bos taurus respectively. The species confirmation of F. gigantica and some related species by basing on maximum likelihood and UPGMA methods used, 4 groups of trematodes were generated, first F. gigantica group including specimen of Chiang Mai, second 2 samples of F. hepatica, third group of 3 rumen flukes; Orthocoelium streptocoelium, F. elongatus and Paramphistomum epliclitum and fourth group of 3 minute intestinal flukes; Haplorchis taichui, Stellantchasmu falcatus, Haplorchoides sp. and liver fluke; Opisthorchis viverrini respectively.@*CONCLUSIONS@#These results can be confirmed the Giant liver fluke which mainly caused fascioliasis in Chiang Mai was identified as F. gigantica and specimens were the same as those of F. gigantica recorded in other different countries. Nucleotide sequence of ITS-2 region has been proven as effective diagnostic tool for the identification of F. gigantica.

DNA barcoding of authentic and substitute samples of herb of the family Asparagaceae and Asclepiadaceae based on the ITS2 region.

Background: Herbal drugs used to treat illness according to Ayurveda are often misidentifi ed or adulterated with similar plant materials. Objective: To aid taxonomical identifi cation, we used DNA barcoding to evaluate authentic and substitute samples of herb and phylogenetic relationship of four medicinal plants of family Asparagaceace and Asclepiadaceae. Materials and Methods: DNA extracted from dry root samples of two authentic and two substitutes of four specimens belonging to four species were subjected to polymerase chain reaction (PCR) and DNA sequencing. Primers for nuclear DNA (nu ITS2) and plastid DNA (matK and rpoC1) were used for PCR and sequence analysis was performed by Clustal W. The intraspecifi c variation and interspecifi c divergence were calculated using MEGA V 4.0. Statistical Analysis: Kimura’s two parameter model, neighbor joining and bootstrapping methods were used in this work. Results: The result indicates the effi ciency of amplifi cation for ITS2 candidate DNA barcodes was 100% for four species tested. The average interspecifi c divergence is 0.12 and intraspecifi c variation was 0.232 in the case of two Asparagaceae species. In two Asclepiadaceae species, average interspecifi c divergence and intraspecifi c variation were 0.178 and 0.004 respectively. Conclusions: Our fi ndings show that the ITS2 region can effectively discriminate Asparagus racemosus and Hemidesmus indicus from its substitute samples and hence can resolve species admixtures in raw samples. The ITS2 region may be used as one of the standard DNA barcodes to identify closely related species of family Asclepiadaceae but was noninformative for Asparagaceae species suggesting a need for the development of new markers for each family. More detailed studies involving more species and substitutes are warranted.

Molecular Tool for Identification of Closely Related Species of Trichogramma (Hymenoptera: Trichogrammatidae): T. rojasi Nagaraja & Nagarkatti and T. lasallei Pinto / Técnica Molecular para a Identificação de Espécies Próximas de Trichogramma (Hymenoptera: Trichogrammatidae): T. rojasi Nagarkatti & Nagaraja e T. lasallei Pinto

O sequenciamento da região ITS2 (espaço interno transcrito) do rDNA foi utilizado para identificar duas espécies próximas de Trichogramma: T. rojasi e T. lasallei, esta última recentemente constatada no Brasil. O DNA das amostras foi extraído utilizando-se Chelex 100. O produto de PCR foi ligado ao vetor pGEM-T ® e ambos foram então acondicionados em placas de Petri para o crescimento de colônias brancas, o que constata a ligação correta entre o vetor e o DNA. A partir dessas colônias, uma pequena quantidade (10æl) foi purificada utilizando material específico e seqüenciada. As seqüências foram alinhadas pelo programa ESEE 3.0. Os primers específicos utilizados para a amplificação do rDNA da região ITS2 das espécies de Trichogramma estudadas foram eficientes. O sequenciamento das duas espécies diferiu em comprimento e posição dos nucleotídeos, mostrando que são distintas. Os resultados mostraram que esta é uma boa técnica para identificação de espécies crípticas de Trichogramma, difíceis de identificar quando são utilizados apenas caracteres morfológicos.

The sequence of the ITS2 (internally transcribed spacer 2) region of the rDNA was used to identify two closely related Trichogramma species: T. rojasi and T. lasallei, the second species recently reported in Brazil. The DNA of the samples was extracted using Chelex 100. The PCR product was linked to a pGEM-T® vector and both were then placed into Petri dishes to grow white colonies, which demonstrate the correct ligation between the vector and the DNA. From these colonies, 10æl were purified and sequenced using a special kit, and the sequences aligned using the ESEE 3.0 software. Specific primers were efficient to amplify the ITS2 rDNA region of the Brazilian Trichogramma. The sequence of each species differed in length and in nucleotide position, showing that they are distinct. Thus, the results show that this technique is a good tool to identify Trichogramma cryptic species, otherwise difficult to identify when using only morphological characters.