ABSTRACT
Armillaria gallica is a symbiotic fungus in the cultivation process of Gastrodia elata and Polyporus.The rhizomorph of A. gallica invades the stalk of the G. elata or the Sclerotium of the Polyporus,and is digested and utilized by the latter,becoming their important source of nutrition. Different nature of A. gallica affects the growth of G. elata and Polyporus. The authors collected A. gallica from 13 commercially available regions and screened two A. gallica,A and B,at the genetic and metabolic levels,in order to distinguish between the two A. gallica market. We have established convenient and effective DNA molecular identification method.By comparing the sequence differences between the A. gallica type A and type B invertase genes,PCR-RFLP primers were designed based on differential fragment. Primer ZTM.F/ZTM.R can amplified A. gallica type A and B,producing a band of about 304 bp in length. The restriction endonuclease EcoR V could recognize the difference sequence of A and B types of A. gallica. The type B was digested to form two fragments,thereby specifically identifying the A. gallica as type B. The established methods of PCR-RFLP is an accurate identification method for A. gallica. Therefore,in the cultivation process of G. elata and Polyporus,suitable strains can be selected according to different needs of variety,growth stage and ecological environment,and the yield and quality can be improved according to local conditions.
Subject(s)
Armillaria , Classification , Gastrodia , Microbiology , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , PolyporusABSTRACT
To establish a robust and accuracy molecular method to identify Achyranthis Bidentatae Radix and Cyathulae Radix formula granules. ITS sequences of Achyranthes bidentata and Cyathula officinalis were aligned, specific SNPs (single nucleotide polymorphisms) were excavated, specific primers were designed and allele-specific PCR method was established. The genomic DNA was successfully extracted from the herbal medicine and its formula granules by using an improved CTAB (cetyltrimethyl ammonium bromide) method and then performed PCR with the designed primers. The 187 bp specific band could be amplified only in the presentation of C. officinalis and its granules when use of C. officinalis specific primers, whereas the 162 bp band could be amplified only in the presentation of A. bidentata and its granules when use of A. bidentata specific primers. This method was also successfully applied in the identification of commercial formula granules.
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Direct-PCR technology was using a 15 minutes heat-lysis step instead of DNA extraction to get DNA templates with small amount of plant materials followed by sensitive PCR process to amplify target genes. In order to facilitate DNA barcoding in medicinal herb identification with Direct-PCR, we collected different tissues from 80 medicinal plants as material to amplify the ITS fragments. Through optimizing the PCR reaction, ITS of 80 plant samples was all successfully amplified. PCR products were sequenced and to do Blast analysis. These results suggest that Direct-PCR would improve the efficiency of DNA barcoding in the application of medicinal herb molecular authentication.
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Rapid allele-specific PCR primer was designed base on Cytb 155 A/T single nucleotide polymorphism, DNA was extracted by alkaline lysis and the PCR reaction systems including denatured and annealing temperature and cycle numbers were optimized. The results were performed to authenticate Ranae Oviductus and its 4 adulterants. When 100×SYBR Green I was added in the PCR product at 90 ℃ denatured 3 s, 62 ℃ annealing 20 s and 32 cycle. Ranae Oviductus visualized strong green fluorescence under 365 nm UV lamp whereas adulterants appeared negative. The whole process can be completed in 40 minutes．The established method provides the technical support for authentication of the Ranae Oviductus.
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Traditional authentication method is hard to identify herb's authenticity of traditional Chinese medicine(TCM) formula granules because they have lost all their morphological characteristics. In this study, a new allele-specific PCR method was established for identifying the authentication of Jinyinhua formula granule (made from Lonicerae Japonicae Flos) based on an SNP site in trnL-trnF fragment. Genomic DNA was successfully extracted from Lonicerae Japonicae Flos and its formula granules by using an improved spin column method and then PCR was performed with the designed primer. Approximately 110 bp specific bands was obtained only in the authentic Lonicerae Japonicae Flos and its formula granules, while no bands were found in fake mixed products. In addition, the PCR product sequence was proved from Lonicerae Japonicae Flos trnL-trnF sequence by using BLAST method. Therefore, DNA molecular authentication method could make up the limitations of character identification method and microscopic identification, and quickly identify herb's authenticity of TCM formula granules, with enormous potential for market supervision and quality control.
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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.
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OBJECTIVE: To establish a new method for identification of Angelica sinensis from its adulterants by analyzing trnL-F and rpoC1 sequences. METHODS: The plastid trnL-F and rpoC1 of 25 samples of A. sinensis and its adulterants were amplified, sequenced, and analyzed. The K-2-P distances of A. sinensis and its adulterants were calculated, and the phylogenetic trees were constructed. RESULTS: The trnL-F sequence showed significantly larger length variation range, numbers of variable sites and informative sites, and evolutionary distance than rpoC1 sequence. There were significant differences between A. sinensis and the adulterants based on the trnL-F sequences. One SNP site and one repeated base A region could be used as specific authenticable sites. The distance of A. sinensis and its adulterants ranged from 0.002-0.231 as shown by trnL-F sequences. Phylogeny tree reconstruction using MP analysis based on trnL-F sequences could effectively distinguish A. sinensis from adulterants. However, analysis of the rpoC1 sequences could not identify A. sinensis and its adulterants. CONCLUSION: The rpoC1 sequence has poor capability for identification of A. sinensis and its adulterants. The trnL-F sequence could be used as an efficient molecular marker for authenticating A. sinensis from its adulterants.
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Authentications of Chinese herbal medicine have a critical effect in Chinese clinical medicine. DNA molecular marker, as an important component for true or false authentication, is more and more widely used in iden-tification of Chinese medicinal materials. At the same time, many new methods for authentication of Chinese medici-nal materials are continuously emerging. But the systematically comparative analysis of these new methods is lack. The present study taking Lonicera japonica as an example, systematically compared principles, characteristics, ex-periment methods, detection time and the application scope of express sequence tag-simple sequence repeat (EST-SSR), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), allele-specific PCR (AS-PCR), DNA barcoding and loop-mediated isothermal amplification (LAMP), and put forward corresponding improve-ment opinions. This study can help to screen appropriate approach for rapid authentication of L. japonica and offer demonstrating to other Chinese herbal medicines.
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Objective: To construct the phylogenetic relationship of Bidens pilosa and its relative species, and to accurately identify B. pilosa and its relative species. Methods: Total genomic DNA was isolated from B. pilosa and its relative species. Nuclear DNA internal transcibed spacer (ITS) and chloroplast gene psbA-trnH sequences were amplified and sequenced. The Kimura 2-parameter (K2P) distances were calculated. Authentication analyses were performed using Nearest Distance, BLAST1, and Neighbor-joining (NJ) methods. Results: The NJ trees (ITS and psbA-trnH data) indicated that the different populations of B. pilosa form a monophyletic clade [Bootstrap (BS) = 79% and 87%]. B. pilosa and B. pilosa var. radiata formed one monophyletic clade, and B. biternata was sister to B. bipinnata (ITS and combined data: BS = 100%). B. tripartita, B. cernua, and B. frondosa formed one monophyletic clade (ITS and combined data: BS = 100%; psbA-trnH data: BS = 99%). The inter-specific genetic distances (ITS and psbA-trnH data) between B. pilosa and its relative species were (0.00794-0.12880) and (0.005 18-0.074 52) which were far higher than intra-specific genetic distances of B. pilosa (0-0.00159) and (0-0.00252). Conclusion: The closest relative of B. pilosa is B. pilosa var. radiata. B. tripartita is closely relative to B. cernua and B. frondosa. The sister relationship between B. biternata and B. bipinnata is corroborated. ITS and psbA-trnH are two efficient barcodes for the authentication of B. pilosa and its relative species.
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Aims: To develop Sequence Characterized Amplified Region (SCAR) marker for identification of Bacopa monnieri (L.) Wettst. Study design: Molecular biology tools for authentic identification of Bacopa monnieri. Methodology: RAPD-based SCAR marker was developed to identify Bacopa monnieri from its adulterant candidates namely Centella asiatica, Eclipta alba and Malva rotundifolia. 50 random primers were used for initial screening of different accessions of Bacopa monnieri, Eclipta alba and Malva rotundifolia. A putative 589 bp marker specific to Bacopa monnieri was identified using RAPD technique. This RAPD-amplicon was then sequenced and cloned. Based on the information of cloned sequences a pair of SCAR primers was designed. SCAR primers were then used for authentication of DNA samples of Bacopa monnieri and its adulterants. Market samples of Bacopa monnieri and Centella asiatica collected under the name of Brahmi was put to test with these primers. Results: Out of 50 random primers, only 14 primers were able to amplify the above plants. A 589 bp polymorphic band obtained with OPAA-3 primer which was specific to Bacopa monnieri accessions and not found in other adulterant candidates was selected. This band was eluted, cloned and further sequenced. A pair of SCAR primers (Bac F & Bac R) between 406 bp of 589 bp sequence of RAPD amplicon was designed. A single, bright, distinct band was obtained in Bacopa monnieri and not in the adulterants. Further validation was also done in the market samples. Conclusion: In essence, the study was to develop a RAPD-based SCAR marker for authentication of Bacopa monnieri. The SCAR marker was found to be useful for preventing the adulteration of other plants in Brahmi and also for screening of crude drug samples intended for export and domestic uses.
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Traditionally, the authentication of the traditional Chinese medicines (TCM), Angelica sinensis, is based on slightly different morphological characters and complex compounds. Usually, those methods are simultaneously affected by several factors, leading to subtle and ambiguous results. In this study, the internal transcribed spacer (ITS) regions of A. sinensis and seven other Angelica species used as adulterants were sequenced. A pair of specific primers was designed from the polymorphic ITS regions to distinguish A. sinensis from the adulterants and regional substitutes. These ITS-derived primers amplified approximately 520 bp specific fragments from the adulterants, whereas no products was amplified with the DNA of A. sinensis. We tested eight commercially crude materials purchased in the market by using these specific primers. The result showed that there were four samples adulterating A. sinensis with regional substitutes. This indicated that A. sinensis could be accurately distinguished from the adulterants and regional substitutes. Therefore, the method of molecular authentication based on the ITS sequences may be contributed to raw material production and quality control of A. sinensis.
Subject(s)
DNA, Ribosomal/analysis , DNA, Ribosomal/genetics , Angelica sinensis/genetics , Angelica sinensis/ultrastructure , Cytogenetic Analysis/methods , Chromosomes, Plant , Genome, Plant/genetics , Medicine, Chinese Traditional/methods , Polymerase Chain Reaction/methods , Sequence Analysis, DNAABSTRACT
Objective: To study rDNA ITS (internal transcribed spacers) sequences variation from di erent population of Aquilaria sinensis in main habitat of China. Methods: The rDNA ITS regions of various A. sinensis were ampli ed by PCR method and sequenced, and they were analyzed by means of the software of CLUSTAL and MEGA. Results: The sequences of rDNA ITS region of A. sinensis were reported for the rst time, and the sequences of ITS region were 680bp (ITS1 246bp, 5.8S 163bp, ITS2 271bp). There were 6 variable sites among populations and the genetic distance were 0.0% to 1.1%,which indicated the intraspecific genetic variation was low of A. sinensis. Conclusion: The variation of rDNA ITS sequences can be used to authenticate A.sinensis from di erent geographical regions and their adulterants.
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Object In order to identify the medicine at the molecular level, the internal transcribed spacers (ITS) of Saussurea medusa Maxim and its easily confusable species were sequenced Methods The double stranded DNA was amplified using PCR systems 9 600 kits and sequenced on an ABI 377 automated sequencer from both directions Results The ITS sequences of S medusa of different populations showed no variation, but there existed distinct variation between S medusa and its confusable species Conclusion ITS sequences can be used for the molecular authentication between S medusa and its confusable species