OBJECTIVETo find the patterns of the rDNA ITS sequence variation in Gentiana, and establish the molecular biological method for the identification of the four kinds wild Gentiana from different regions in Gansu.

METHODThe ITS gene fragments were PCR amplified and sequenced. The rDNA ITS regions were analyzed by means of the software of Clustal X, MEGA3.

RESULTThe Complete ITS sequence of G. macrophylla, G. straminea, G. dahurica and G. officinale was 800 bp. The sequences of ITS1, ITS2 and 5.8S were 290, 340, 170 bp, respectively. Phylogenetic tree based on ITS1 and ITS2 sequences data was constrcuted by Neighbor-joining method.

CONCLUSIONITS sequence could be as the evidence for the molecular authentication of Gentiana.

DNA, Plant ; chemistry ; DNA, Ribosomal ; chemistry ; DNA, Ribosomal Spacer ; chemistry ; Gentiana ; genetics ; Phylogeny

OBJECTIVETo evaluate the genetic differentiation and phylogenetic relationship between Whitmania pigra Whitman and Hirudo nipponia Whitman.

METHODBy the sequences of the rDNA internal transcribed spacer (ITS) the molecular phylogenetic tree was constructed by MP method using software MEGA 4.0.

RESULTThe average length of ITS was 857.2-861.2 bp. The A, T, G and C contents in this fragment were 25.12%, 28.28%, 17.34%, 29.29%, respectively. The GC content was higher than the AT content. Little sequence variation was observed in ITS gene fragments with in species, and transition in only 45 loci was revealed in 14 populations. 14 W. pigra and H. nipponia populations were clustered into 2 groups by MP phylogenetic tree.

CONCLUSIONThe results also showed that the intraspecific variation was dominated in variation types of W. pigra and H. nipponia. The classification result of W. pigra and H. nipponia and its adulterants based on DNA sequences are not totally consistent with those based on classification. It showed that a little of mutation of base in ITS sequences had occurred in the process of evolution, such as transition cites, transvertion cites among base or base gap.

Animals ; Base Sequence ; DNA, Ribosomal Spacer ; chemistry ; Leeches ; classification ; genetics ; Molecular Sequence Data ; Phylogeny

OBJECTIVETo analyse a special kind of Schisandra chinensis with the white fruit using ITS2 barcode at molecular levels.

METHODITS2 regions were sequenced bidirectionally. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner, MEGA 5.0 software was used to align the sequences. The ITS2 secondary structure was predicted using ITS2 web server, BLAST 1 method was used to identify the S. chinensis with the white fruit.

RESULTThe length of the ITS2 sequence was 231 bp. And the sample was identified as S. chinensis using the method of BLAST 1. Their mean interspecific genetic distance (K2P distance) among the populations of the S. chinensis with the white fruit and S. chinensis was far lower than the mean interspecific genetic distance between the S. chinensis and S. sphenanthera.

CONCLUSIONBy using ITS2 the S. chinensis with the white fruit was identified as S. chinensis, and the ITS2 barcode could be used to identify S. chinensis and S. sphenanthera.

DNA, Plant ; chemistry ; genetics ; DNA, Ribosomal Spacer ; chemistry ; genetics ; Fruit ; chemistry ; classification ; genetics ; Molecular Sequence Data ; Nucleic Acid Conformation ; Schisandra ; chemistry ; classification ; genetics ; Sequence Analysis, DNA ; Software

Astragalus membranaceus var. mongholicus and Hedysarum polybotrys belong to different genera, but have similar drug efficacy in traditional Chinese medicine theory, and H. polybotrys was used as the legal A. membranaceus var. mongholicus previously. In this study, similarities and differences between them were analyzed via their ITS/ITS2 fragments information. The ITS (internal transcribed spacer) regions were amplified using polymerase chain reaction and then sequenced in two-way. The alignment lengths of ITS regions were 616 bp, in which 508 loci were consistent, and 103 loci were different, accounting for 82.47% and 16.72% of the total ITS nucleotides in length, respectively. As genotype determines phenotype, 1HNMR-based metabolomic approach was further used to reveal the chemical similarities and differences between them. Thirty-four metabolites were identified in the 1H NMR spectra, and twenty-seven metabolites were the common components. Amino acids, carbohydrates and other primary metabolites were similar, while a large difference existed in the flavonoids and astragalosides. This study suggests that A. membranaceus var. mongholicus and H. polybotrys show similarities and differences from molecular and chemical perspectives, which has laid a foundation for elucidating the effective material basis of drug with similar efficacy and resources utilization.
Astragalus membranaceus ; chemistry ; genetics ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Drugs, Chinese Herbal ; chemistry ; Fabaceae ; chemistry ; genetics ; Flavonoids ; chemistry ; Metabolome ; Metabolomics

OBJECTIVEThe study the patterns of rDNA ITS sequence variation of 9 medicinal species of genus Bupleurum in China to find the DNA molecular markers for identification of these crude drugs.

METHODThe ITS regions of these species were amplified by PCR and sequenced, and their sequences were analyzed by DNAssist Version 2.2.

RESULTSHomologous alignment indicated that the consanguinity of the ITS sequences between genus Bupleurum and outgroups was lower than 75%, and that within genus Bupleurum was higher than 88%. For the same species, the consanguinity was higher than 99%.

CONCLUSIONThe ITS sequences may serve as reliable molecular markers for identification of Radix Bupleuri.

Bupleurum ; classification ; genetics ; DNA, Plant ; chemistry ; genetics ; DNA, Ribosomal Spacer ; chemistry ; genetics ; Molecular Sequence Data ; Phylogeny ; Plant Roots ; genetics ; Sequence Analysis, DNA ; Species Specificity

OBJECTIVETo find the patterns of the rDNA ITS sequence variation of Crocus sativus, Chrysanthemum chanetii, Nelumbo nucifera, Zea mays and Garthamus tinctorius and to establish the molecular biological method for the identification of C. sativus and the others.

METHODAfter the total DNA of Crocus sativus, C. vernus-w and C. vernus-p were extracted, the ITS sequence was amplified by PCR with universal primer of ITS and PCR product was sequenced after purification and cloning. The ITS sequences of Chrysanthemrnum chanetii, Nelumbo nucifera, Zea mays and Garthamus tinctorius were obtained from GenBank.

RESULTThe complete ITS sequence of Crocus sativus, C. vernus-w and C. vernus-p, including ITSI rDNA, 5.8S rDNA, ITS2 rDNA were measured. The GenBank accession No. was DQ094185, DQ224363 and DQ224364 respectively. The similarity of ITS sequence between C. sativus and the two garden species of C. vernus was above 91%; the identity was 99.84% between C. vernus-w and C. vernus-p. The range of diversity between C. sativus and other herbs was above 46% based on ITS1 and above 41% based on ITS2.

CONCLUSIONC. sativus can be distinguished from misused substitutes by the ITS sequence. The ITS sequence is an available molecular marker for identification of the C. sativus.

Chrysanthemum ; genetics ; Crocus ; genetics ; DNA, Plant ; chemistry ; genetics ; DNA, Ribosomal ; chemistry ; genetics ; DNA, Ribosomal Spacer ; chemistry ; genetics ; Genetic Variation ; Molecular Sequence Data ; Nelumbo ; genetics ; Plants, Medicinal ; genetics ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Zea mays ; genetics

OBJECTIVETo analyze the nuclear ribosome DNA (nrDNA) internal transcribed spacer (ITS) sequences of 4 Leonurus species, and the possibility of using them for molecular authentication of the crude drugs from the genus.

METHODSThe nrDNA ITS sequence (including ITS1, 5.8S rDNA, ITS2, and partial 18S rDNA and 26S rDNA) of L. japonicus and its 3 adulterant species were amplified and sequenced, and CLUSTRAL X and MEGA software was employed for analysis.

RESULTSThe variation of ITS1 and ITS2 between L. japonicus and its adulterant species ranged between 7.2% and 18.8% and between 14.2% and 27%, respectively. The phylogenic tree derived from the dendrograms based on the ITS sequence data contained some discrepancy from the traditional classification.

CONCLUSIONThe nrDNA ITS sequences can be used potentially as efficient markers for identification of L. japonicus and its adulterants, and further study is needed for studying the phylogeny of Leonurus.

DNA, Plant ; chemistry ; genetics ; DNA, Ribosomal Spacer ; chemistry ; genetics ; Leonurus ; classification ; genetics ; Molecular Sequence Data ; Phylogeny ; Sequence Analysis, DNA ; Species Specificity

The ITS2 barcode was used to accurately identify Albiziae Cortex, Albiziae Flos and their adulterants in this study. A total of46 samples from Albiziae Cortex, Albiziae Flos and their adulterants were collected. The ITS2 regions were amplified and sequenced. Sequences were assembled using the CodonCode Aligner. The genetic distances of ITS2 region were calculated using MEGA 5.0. BLAST1, nearest distance and phylogenetic tree (NJ-tree) methods were used to assess the identification efficiency of the ITS2 barcode. The results revealed that the intraspecific genetic distances of Albizia julibrissin were lower than the interspecific genetic distances between A. julibrissin and its adulterants. The identification efficiency of ITS2 barcode using BLAST1 was 100%. The NJ-tree showed that A. julibrissin and their adulterants can be easily differentiated according to their monophyly. The ITS2 barcode is suitable to be as a barcode to identify Albiziae Cortex, Albiziae Flos and their adulterants.
Albizzia ; classification ; genetics ; DNA Barcoding, Taxonomic ; methods ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Drugs, Chinese Herbal ; chemistry ; classification ; Flowers ; classification ; genetics ; Molecular Sequence Data ; Phylogeny ; Quality Control

In this study, ITS2 barcode was used to identify Bupleurum chinense and B. longiradiatum. The ITS2 regions of 48 samples were amplified and sequenced. The sequences obtained above were aligned and the K2P distances were calculated. We used three methods, BLAST1, nearest distance and phylogenetic tree (NJ-tree), to test the identification ability. The results showed that the maximum intraspecific genetic distance of B. chinense was 0.013, and the minimum interspecific genetic distance between B. chinense and B. longiradiatum was 0.049. The NJ-tree can easily identify B. chinense and B. longiradiatum. Therefore, the ITS2 barcode is suitable to identify B. chinense and B. longiradiatum.
Bupleurum ; classification ; genetics ; DNA Barcoding, Taxonomic ; methods ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Drugs, Chinese Herbal ; chemistry ; classification ; Molecular Sequence Data ; Phylogeny ; Quality Control

OBJECTIVETo establish the specific 16S-23S rRNA gene spacer regions pattern in different bacteria using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequences analysis.

METHODSA pair of primers were selected from highly conserved sequences adjacent to the 16S-23S rRNA spacer region. Bacterial DNA of sixty-one strains of standard bacteria and corresponding clinical isolates representative of 20 genera and 27 species was amplified by PCR, and further studied by RFLP, DNA cloning and sequences analysis. Meanwhile, all specimens were examined by bacterial culturing and PCR-RFLP analysis.

RESULTSThe 27 different standard strains showed one, two, three or more than three bands. The sensitivity of PCR reached 2.5 colony-forming unit (CFU), and there was no cross reaction to the human, fungal or viral genomic DNAs. Fifteen species could be distinguished immediately by PCR, while another 10 species were further identified by Hinf I or Alu I digestion. Klebsiella pneumoniae (Kp) and Enterococcus durans (Ed) could not be differentiated from each other by Alu I or Hinf I digestion. The spacer sequences of the Kp and Ed were 908 bp and 909 bp, respectively, and they differed only at the site of the 779th nucleotide. The former was G, and the latter was A. The 760 - 790 bp sequence of Kp was as follows: CGACTGCACCGCCTCCTAC / GGCCGCGTATTC. The 760 - 790 bp sequence of Ed was as follows: CGACTGCAC CGCCTCCTAC / AGCCGCGTATTC. Only one enzyme XmaIII, could discriminate the two. The cleaving site of XmaIII is C downward arrow GGCCG. Kp DNA was cleaved into 778 bp and 130 bp fragments, while E. durans was not. Of 42 specimens with suspected septicemia, 15 were positive (35.7%) on blood culture, and 27 on PCR (64.29%). The positive rate of PCR was significantly higher than that of blood culture (P < 0.01). Of the six CSF specimens, one was positive for Staphylococcus epidermidis (Se) on culture as well as by PCR, while two specimens which were negative on cultures were positive by PCR and were diagnosed as Se according to its DNA pattern. One specimen was culture-positive for Cryptococcus neoformans (Cn) but was negative by PCR. The other two specimens were negative by both PCR and culture. Fifteen blood samples from healthy children were negative by both blood culture and PCR.

CONCLUSIONSThe method of detecting bacterial 16S-23S rRNA spacer regions using PCR-RFLP techniques was specific, sensitive, rapid and accurate in detecting pathogens in clinical bacterial infections.

Bacterial Infections ; diagnosis ; microbiology ; DNA, Bacterial ; chemistry ; genetics ; DNA, Ribosomal Spacer ; genetics ; Humans ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; Sequence Analysis, DNA