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.

OBJECTIVE: To identify Dalbergiae Odoriferae Lignum and its adulterants using DNA barcoding. METHODS: ITS2 is one of the popular DNA barcoding in the identification of traditional Chinese medicine. In this paper, the ITS2 regions of Dalbergiae Odoriferae Lignum and its adulterants were amplified and sequenced bi-directional. The length and GC content of ITS2 sequence were analyzed through MEGA5.0 software. The genetic distances were computed by kimura 2-parameter (K2P) model. Dalbergiae Odoriferae Lignum and its adulterants have been identified through the species identification system for traditional Chinese medicine and neighbor-joining (NJ) phylogenetic tree. RESULTS: The sequence lengths of ITS2 of Dalbergiae Odoriferae Lignum were 216 bp, and the GC content was 68.5%. The minimum K2P interspecific genetic distances of Dalbergiae Odoriferae Lignum and its adulterants were 0.009, which was larger than that of the intraspecific genetic distances of Dalbergiae Odoriferae Lignum. The Dalbergiae Odoriferae Lignum and its adulterants can be obviously identified using the Species identification System and NJ phylogenetic trees. CONCLUSION: ITS2 Regions as DNA barcode can identify Dalbergiae Odoriferae Lignum and its adulterants accurately.

OBJECTIVE: To evaluate the accuracy and stability of ITS/ITS2 barcodes in identification of Zanthoxmli Pericarpium. METHODS: Total genomic DNAs from samples were extracted by using improved DNA extraction kits. ITS regions were amplified, and the ITS2 sequences were obtained by using the hidden Markov model (HMM) based on annotation method from the ITS sequences. The inter- and intra-specific variation of the Zanthoxmli Pericarpium and its adulterants were analyzed. Zanthoxmli Pericarpium was i-dentified through the Species identification System for Traditional Chinese Medicine and neighbor-joining (NJ) phylogenetic trees. RESULTS: The lengths of ITS/ITS2 sequence of Zanthoxmli Pericarpium were 618-620 bp and 224 bp. The intra-specific genetic distances were smaller than inter-specific ones in ITS/ITS2 regions of Z. schinifolium and Z. bungeanum. Zanthoxmli Pericarpium and its adulterants could be easily differentiated according to the nearest distance, the Species Identification System for Traditional Chinese Medicine and the NJ trees methods. CONCLUSION: ITS/ITS2 regions as DNA barcodes can stably and accurately distinguish Zanthoxmli Pericarpium and its adulterants.

In order to identify Peucedani Radix, Peucedani Decursivi Radix and their adulterants, the internal transcribed spacer 2 (ITS2) regions of Peucedani Radix, Peucedani Decursivi Radix and their adulterants were amplified and bidirectionally sequenced based on the Principles for Molecular Identification of Traditional Chinese Materia Medica Using DNA Barcoding, which has been promulgated by Chinese Pharmacopoeia Commission. Sequences were analyzed and assembled by Codon Code Aligner V3. 7.1. The relevant data were analyzed by MEGA 5. 0. Species identification analyses were performed by using the nearest distance methods and neighbor-joining (NJ) methods. The result showed that the ITS2 sequence lengths of Peucedani Radix were 229-230 bp and the average intra-specific genetic distances were 0.005. The ITS2 sequence lengths of Peucedani Decursivi Radix were 227 bp and the sequences contained no variation site. The average inter-specific K2P genetic distance of Peucedani Radix, Peucedani Decursivi Radix and their adulterants species were 0.044 and 0.065 respectively. The minimum inter-specific divergence is larger than the maximum intra-specific divergence of Peucedani Decursivi Radix. The nearest distance methods and NJ trees results indicated that Peucedani Radix, Peucedani Decursivi Radix and their adulterants species could be identification clearly. The ITS2 regions can stably and accurately distinguish Peucedani Radix, Peucedani Decursivi Radix and their adulterants.
Apiaceae ; classification ; genetics ; DNA Barcoding, Taxonomic ; methods ; DNA, Ribosomal Spacer ; Drug Contamination

OBJECTIVETo identify the Cynomorii Herba and its analogues species using DNA barcoding technique.

METHODTotal genomic DNA extracted from all materials using the DNA extraction kit. The internal transcribed spacer 2 (ITS2) regions were amplified using polymerase chain reaction (PCR), and purified PCR products were sequenced bi-directionally. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner 3.7.1. The Kimura 2-Parameter (K2P) distances and GC content were computed using MEGA 5. 0. Species identification analyses were conducted through the species identification system for traditional Chinese medicine and neighbor-joining (NJ) trees.

RESULTThe ITS2 sequence lengths of Cynomorii Herba were 229 bp. The average intra-specific genetic distances of Cynomorii Herba were 0.003. The average inter-specific genetic distances between Cynomorii Herba and its adulterants species were 0.760. The results showed that the minimum inter-specific divergence is larger than the maximum intra-specific divergence. The species identification system for traditional Chinese medicine and NJ trees results indicated that Cynomorii Herba and its adulterants species can be easily identification.

CONCLUSIONThe ITS2 region is an efficient barcode for identification of Cynomorii Herba, which provide a new technique to ensure clinical safety in utilization of traditional Chinese medicine.

Cynomorium ; classification ; genetics ; DNA Barcoding, Taxonomic ; DNA, Intergenic ; genetics ; DNA, Plant ; genetics ; Polymerase Chain Reaction

UNLABELLED: The DNA barcoding method was used to accurately and rapidly identify Corni Fructus and its adulterants. METHODS: Genomic DNA extracted from Corni Fructus and its adulterants were used as templates. The ITS (internal trascribed spacer) regions were amplified using polymerase chain reaction. Sequence assembly was performed using CodonCode Aligner V 3.5.4. Genetic distances were computed using MEGA V 5.0. Species identification was conducted using neighbor-joining (NJ) trees. RESULTS: The ITS sequence length of Corni Fructus was 659 bp. The average intra-specific genetic distance of Corni Fructus was 0.005, markedly lower than the inter-specific genetic distance between Corni Fructus and its adulterants (0.357). The ITS2 sequence length of Corni Fructus was 250 bp. No variation was found among the different samples. The interspecific genetic distance of ITS2 between Corni Fructus and its adulterants was 0.571. NJ trees and BLAST results indicated that Corni Fructus and its adulterants can be easily differentiated with monophyly. CONCLUSION ITS/ITS2 regions can accurately and efficiently distinguish Corni Fructus and its adulterants. In addition, the results not only established the foundation for the clinical safety in the utilization of Corni Fructus, but also provided reference for molecular identification of other Chinese herbal medicine and Chinese herbal pieces.
Base Sequence ; Cornus ; classification ; genetics ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Drug Contamination ; Molecular Sequence Data ; Molecular Typing ; methods ; Phylogeny ; Species Specificity

Since the research of molecular identification of Chinese Materia Medica (CMM) using DNA barcode is rapidly developing and popularizing, the principle of this method is approved to be listed in the Supplement of the Pharmacopoeia of the People's Republic of China. Based on the study on comprehensive samples, the DNA barcoding systems have been established to identify CMM, i.e. ITS2 as a core barcode and psbA-trnH as a complementary locus for identification of planta medica, and COI as a core barcode and ITS2 as a complementary locus for identification of animal medica. This article introduced the principle of molecular identification of CMM using DNA barcoding and its drafting instructions. Furthermore, its application perspective was discussed.
Animals ; China ; DNA ; genetics ; DNA Barcoding, Taxonomic ; methods ; DNA, Ribosomal Spacer ; genetics ; Drugs, Chinese Herbal ; classification ; isolation & purification ; Electron Transport Complex IV ; genetics ; Materia Medica ; classification ; isolation & purification ; Medicine, Chinese Traditional ; Plant Proteins ; genetics ; Plants, Medicinal