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

BACKGROUNDAccurate identification of bacterial isolates is an essential task in clinical microbiology. This study compared culturing to analyzing 16S rRNA gene sequences as methods to identify bacteria in clinical samples. We developed a key technique to directly identify bacteria in clinical samples via nucleic acid sequences, thus improving the ability to confirm pathogens.

METHODSWe obtained 225 samples from Beijing Tongren Hospital and examined them by conventional culture and 16S rDNA sequencing to identify pathogens. This study made use of a modified sample pre-treatment technique which came from our laboratory to extract DNA. 16S rDNA was amplified by PCR. The amplified product was sequenced on a CEQ8000 capillary sequencer. Sequences were uploaded to the GenBank BLAST database for comparison.

RESULTSAmong the positively cultivated bacterial strains, seven strains were identified differently by Vitek32 and by 16S rDNA sequencing. Twelve samples that were negative by standard culturing were determined to have pathogens by sequence analysis.

CONCLUSIONThe use of 16S rRNA gene sequencing can improve clinical microbiology by providing better identification of unidentified bacteria or providing reference identification of unusual strains.

Bacteria ; isolation & purification ; DNA, Ribosomal ; chemistry ; Humans ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S ; genetics ; Sequence Analysis, DNA ; methods

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

OBJECTIVETo confirm the existence of point mutations in the SSU rDNA variable regions of 5 Leishmania donovani (L.d.) isolates from different epidemic foci in China.

METHODSSpecific SSU rDNA fragments from nuclear DNA of 7 Leishmania species/isolates were amplified by PCR and then cloned into pGEM(R)-T Easy Vectors. After that, the specific fragments were sequenced by an automated DNA sequencer.

RESULTSSequence analysis showed that the amplified DNA fragments of 7 Leishmania species/isolates were all 392 bp in length. All 5 point mutations were located in two unique sequence blocks (UQ-I and UQ-II), and no insertions or deletions were found. The identities of comparison of Leishmania in GeneBank were more than 98%.

CONCLUSIONFive point mutations exist in the SSU rDNA variable region of 5 L.d. isolates from different epidemic foci of visceral leishmaniasis (VL) in China. Sequence differences of the SSU rDNA variable region exist among L.d. isolates from different foci.

Animals ; DNA, Protozoan ; chemistry ; DNA, Ribosomal ; chemistry ; Humans ; Leishmania donovani ; genetics ; Leishmaniasis, Visceral ; parasitology ; Point Mutation ; Polymerase Chain Reaction

OBJECTIVETo establish the specific 16S-23S rRNA gene spacer regions 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 from sixty-one strains of standard bacteria and corresponding clinical isolates representative of 20 genera and 26 species was amplified by PCR, and further analyzed by RFLP, DNA cloning and sequences analysis. Furthermore, all specimens were examined by bacterial culturing and PCR-RFLP analysis. The evaluation of these assays in practical clinic practice was also discussed.

RESULTSRestriction enzyme analysis revealed one, two or three bands or more observed among the 26 different standard strains. The sensitivity of PCR reached 2.5 colony-forming unit (CFU), and there was no cross reaction with human genomic DNA, fungus or virus. Fourteen species could be distinguished immediately by PCR, while another 10 species were further identified by Hinf I or Alu I digestion. The only difference between K.pneumoniae and E. durans was located at the site of the 779th nucleotide according to the sequence analysis and only XmaIII digestion could distinguish one from another. Of 42 specimens from septicemic neonates, 15 were identified as positive by blood culture at a rate of 35.7%. However, 27 specimens identified as positive by PCR, with a rate of 64.2%, a method significantly more effective than blood culture (P < 0.01). Of 6 cerebrospinal fluid (CSF) specimens, one tested positive for S.epidermidis was also positive by PCR, two culture negative were positive by PCR and diagnosed as S.epidermidis according to the DNA pattern. One positive for C.neoformans was negative by PCR. The other two specimens were negative by both PCR and culture.

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

Bacteria ; genetics ; isolation & purification ; DNA, Bacterial ; analysis ; chemistry ; DNA, Ribosomal ; analysis ; chemistry ; Genes, rRNA ; Humans ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; Sensitivity and Specificity ; Sequence Analysis, DNA

Marine Actinobacteria are emerging as new resources for bioactive natural products with promise in novel drug discovery. In recent years, the richness and diversity of marine Actinobacteria from the South China Sea and their ability in producing bioactive products have been investigated. The objective of this work is to isolate and identify bioactive secondary metabolites from a marine actinobacterium SCSIO 1934 derived from sediments of South China Sea. The strain was identified as a Streptomyces spieces by analyzing its 16S rDNA sequence. Streptomyces sp. SCSIO 1934 was fermented under optimized conditions and seven bioactive secondary metabolites were isolated and purified by chromatographic methods including colum chromatography over silica gel and Sephadex LH-20. Their structures were elucidated as 17-O-demethylgeldanamycin (1), lebstatin (2), 17-O-demethyllebstatin (3), nigericin (4), nigericin sodium salt (5), abierixin (6), respectively, by detailed NMR spectroscopic data (1H, 13C, COSY, HSQC and HMBC). This work provided a new marine actinobacterium Streptomyces sp. SCSIO 1934, capable of producing diverse bioactive natural products.
Anti-Bacterial Agents ; chemistry ; China ; DNA, Ribosomal ; chemistry ; genetics ; Geologic Sediments ; microbiology ; Oceans and Seas ; RNA, Ribosomal, 16S ; genetics ; Streptomyces ; chemistry ; classification ; genetics ; isolation & purification

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

Twenty-four endophytic actinomycetes strains were isolated from the Salvia przewalskii in Tibetan Plateau of China by tablet coating method. Fusarium moniliforme, Helminthosporium turcicum and Bipolaris maydis were selected as indicator fungi to test the antimicrobial activities of these endophytic actinomycetes by tablet confrontation method. The results showed that 21 strains can produce antimicrobial substances which accounts for 85.7% of the total separates number. Four strains of endogenous actinomyces have more obvious antifungi activity. According to results of morphology and culture properties and 16S rDNA sequences of endophytic actinomyces, it is concluded that all of the isolates were streptomycetes trains.
Actinomyces ; chemistry ; genetics ; China ; DNA, Ribosomal ; genetics ; Fusarium ; drug effects ; Helminthosporium ; drug effects ; Salvia ; microbiology

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

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