OBJECTIVETo identify the differential genes in Porphyromonas gingivalis (P.gingivalis) highly toxic strain W83 and minimally toxic strain ATCC 33277.

METHODSUsing suppression subtractive hybridization (SSH) to compare P.gingivalis highly toxic strain W83 (tester) and minimally toxic strain ATCC 33277 (driver). The chromosomal DNAs were purified from P.gingivalis W83 and P.gingivalis ATCC 33277, and digested by restriction enzyme RsaI. The tester DNA samples were separated and ligated with adaptor 1 and adaptor 2R. Two subtractive hybridization and PCR profile were performed. Tester-specific DNAs also were selectively amplified. The mixture of subtracted DNA fragments were ligated with pMD-18T vector and transformed to competent cells E.coli JM109. The differential subtraction library was established. The positive clones were identified by PCR and then sequenced, and searched homologically.

RESULTSSubtractive library which had high subtractive efficiency was successfully set up and 36 positive clones were screened by SSH. The fragments from 88 bp to 372 bp were enriched in P.gingivalis highly toxic strain W83 sequences which were absent from P.gingivalis ATCC 33277. Through dot blot analysis confirmed that all these fragments were present in P.gingivalis W83 but absent from ATCC 33277. The GenBank homology search indicated that among them, several genes were associated with two paralogous regions of the chromosome; Some genes are associated with evasion of P.gingivalis W83; Another gene was related to antibiotic resistance and the products of some genes were virulence and acquisition of peptides.

CONCLUSIONSComparative whole-genome analysis of highly toxic and minimally toxic strains of P.gingivalis has identified the clustering of genes that are present in W83 but divergent in or absent from ATCC 33277. These genes may provide an important clue for studying the mechanism of occurrence and development of periodontal disease.

Genes, Bacterial ; Genome, Bacterial ; Nucleic Acid Hybridization ; Porphyromonas gingivalis ; genetics ; pathogenicity ; Sequence Analysis, DNA ; Virulence ; genetics

OBJECTIVETo differentiate closely related pathogenic bacteria via phylogenetic method on the basis of gyrB gene sequences.

METHODSGyrB sequences of 19 strains of E.coli, 13 Shigella spp. 2 Aeromonas caviae, 2 Aeromonas hydrophilia,1 Aeromonas veronii were determined and combined with sequences retrieved from public databases to construct phylogenetic trees. For each sequence tested, the identification deduced from the clustering relation of sequences was compared with the phenetic identification.

RESULTSAll the tested sequences, except those of Shigella boydii and Shigella dysenteriae, were corresponded with the 5 closest sequences on the tree at the species level. While the BLAST queries returned some other bacteria organisms or undetermined entries.

CONCLUSIONPhylogenetics displays good discriminative power in bacterial sequences differentiation.

Aeromonas ; classification ; genetics ; Bacteria ; classification ; genetics ; Bacterial Typing Techniques ; DNA Gyrase ; genetics ; DNA, Bacterial ; genetics ; Escherichia coli ; classification ; genetics ; Phylogeny ; Sequence Analysis, DNA ; Shigella ; classification ; genetics

OBJECTIVETo set up random amplified polymorphic DNAs (RAPD) method in genotyping Neisseria gonorrhoeae on DNA level, and to explore its use to trace the source of infection.

METHODSFour different pretreatments were used to extract the Neisseria gonorrhoeae genomic DNA with its advantages and disadvantages compared. Arbitrary sequence was then used to amplify the genomic DNA of Neisseria gonorrhoeae and RAPD fingerprint maps was applied to distinct the Neisseria gonorrhoeae strains. Finally, RAPD fingerprint of Neisseria gonorrhoeae strain between patient and his/her sexual partner was compared.

RESULTSCetyltrimethylammonium bromide method was classical in extracting genomic DNA, and could get integrated genomic DNA and good fingerprint maps, since main segments were common to all the Neisseria gonorrhoeae but some were different among strains so that the fingerprint of different Neisseria gonorrhoeae were distinctive. However, fingerprint maps of Neisseria gonorrhoeae collected from sex partners were quite similar.

CONCLUSIONBased on genomic levels, effective fingerprint maps could be identified and to classify the Neisseria gonorrhoeae into different genotypes. RAPD fingerprint maps could be used to trace the source of infection.

DNA Fingerprinting ; DNA, Bacterial ; analysis ; Genotype ; Humans ; Neisseria gonorrhoeae ; classification ; genetics ; Random Amplified Polymorphic DNA Technique

Metagenomic sequencing provides a powerful tool for microbial research. However, traditional experimental DNA extraction process will inevitably mix with environmental microorganisms which float in the air. It is still unclear whether the mixed environmental microbial DNA will heavily affect the metagenomic results of samples with extremely low microbial content. In this study, we first collected environmental bacteria in the laboratory and quantified the mixed environmental microbial DNA content during DNA extraction based on a qPCR-based quantification assay. We then extracted DNA from pure water in order to determine the mixed microbial taxons during extraction under open environment. At last, we extracted total DNA from a skin sample in a Biosafety cabinet or under open laboratory environment, to assess the impact of the mixed environmental microorganisms on the metagenomic results. Our results showed that DNA extraction under open laboratory environment in Beijing region resulted in 28.9 pg contaminant, which may accout for 30% of total DNA amount from skin samples. Metagenomic analysis revealed that the main incorporated environmental taxons were Cutibacterium acnes and Escherichia coli. Tens of environmental bacteria were foisted in the skin DNA samples, which largely decreased the relative abundance of dominant species and thus deteriorated the result accuracy. Therefore, analyzing microbial composition of samples with extremely low DNA content should better performed under aseptic environment.
DNA ; DNA, Bacterial/genetics* ; Laboratories ; Metagenomics ; RNA, Ribosomal, 16S ; Sequence Analysis, DNA

Animals ; Borrelia burgdorferi ; genetics ; China ; DNA, Bacterial ; genetics ; isolation & purification ; Rodentia ; microbiology ; Sequence Analysis, DNA

The energy of interaction between DNA strands in promoters is of great functional importance. Visualization of the energy of DNA strands distribution in promoter sequences was achieved. The separation of promoters in groups by their energetic properties enables evaluation of the dependence of promoter strength on the energetic properties. The analysis of groups (clusters) of promoters distributed by the energy of DNA strands interaction in -55, -35, -10 and +6 sequences indicates their connection with the transcriptional activity.
DNA, Bacterial ; genetics ; Escherichia coli ; genetics ; Promoter Regions, Genetic ; Sequence Analysis, DNA

BACKGROUNDFew literatures pertain to the 16S ribosomal DNA (16S rDNA) analysis of bacteria contributing to primary and persistent endodontic lesions, with no information available for the Chinese population. As such, we investigated endodontic bacteria associated with primary and persistent endodontic lesions in adult Chinese patients living in Beijing, China using 16S rDNA gene sequencing techniques.

METHODSEndodontic microbial samples were obtained from fourteen adult Chinese patients and subjected to DNA extraction. Pllymerase chain reaction (PCR) products were cloned and 100 clones from each generated library were randomly selected. Purified plasmid DNA with 16S rDNA gene inserts was sequenced, and the sequences were searched against GenBank databases using the BLASTN algorithm. Only significant identification with the highest-scored BLAST result and 99% minimum similarity was considered for phylotyping.

RESULTSMore than 150 taxa were obtained. Primary endodontic infection was mainly associated with Burkholderia cepacia, Actinomyces, Aranicola spp. and Streptococcus sanguinis, whilst Burkholderia cepacia was predominant in the persistent endodontic infections.

CONCLUSIONThere is a difference in the species profile associated with endodontic infections of Chinese patients living in Beijing in comparison to other geographical or ethnic reports.

Bacteria ; classification ; genetics ; pathogenicity ; China ; DNA, Bacterial ; genetics ; DNA, Ribosomal ; genetics ; Female ; Humans ; Male ; Pulpitis ; microbiology ; Sequence Analysis, DNA ; methods

The aim of this study was to establish a fast and accurate method for developing specific DNA sequences and PCR primers for the detection of Staphylococcus aureus. An automatic C++ program for genomic comparison was used to identify specific DNA sequences from the genome of S. aureus MRSA 252. Four primer pairs were obtained from 9 specific target sequences by comparison of 2656 coding sequences with our local genome database, and 2 pairs of primers were confirmed to be specific to S. aureus by PCR evaluation against 137 bacterial strains, including 11 species of Staphylococcus. Furthermore, the DNA detection sensitivity of primer SA3 was 13.7 fg/microL and the cell sensitivity for this primer was 9.25 x 10(2) CFU/mL. This method has overcome the limitations of specific target mining in conventional assays, and it could be easily and widely used for other foodborne pathogens.
DNA Primers ; DNA, Bacterial ; analysis ; genetics ; Genome, Bacterial ; genetics ; Genomics ; methods ; Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA ; methods ; Software ; Staphylococcus aureus ; genetics ; isolation & purification

OBJECTIVETo provide further pathogenic evidence of Granulocytic ehrlichia infection in China.

METHODSSpecific primers derived from 444-Epank gene were used to amplify Granulocytic ehrlichia DNA from specimens of ticks, animals and human blood. PCR products of ticks were cloned and sequenced.

RESULTS444 bp specific DNA fragments were amplified from 2 of 62 pools of Ixodes persulcatus collected from Heilongjiang province and 1 of 129 blood specimens from forest workers in Inner Mongolia. Eight animal specimens were negative. PCR products from ticks were then cloned and sequenced. It differed at 23 positions in comparison to American strain (AF047897) with 94.9% homology. The homology of deduced ammonia was 88.44%.

CONCLUSIONOur findings further confirmed that Granulocytic ehrlichia infection did exist in China.

DNA, Bacterial ; analysis ; Ehrlichia ; classification ; genetics ; isolation & purification ; Ehrlichiosis ; microbiology ; Genes, Bacterial ; Humans ; Phylogeny ; Polymerase Chain Reaction ; Sequence Analysis, DNA

OBJECTIVETo analyze the genetic differences of Orientia tsutsugamushi (Ot) Sta56 gene between Shandong isolates and other strains deposited in GenBank.

METHODSPCR and restriction fragment length polymorphism (RFLP) were used to amplify the complete sequence of Ot-Sta56 gene. RFLP profiles of Ot were predicted by a computer program according to their complete sequences of Ot-Sta56 gene. PCR amplicon from XDM2 strain was sequenced and analyzed by Clustal X (1.8) and PHYLIP software.

RESULTSThe complete sequences (about 1.6 kbp) of Ot-Sta56 gene were amplified from B16 strain (isolated from patients), FXS2 strain (isolated from A. agrarius) and XDM2 strain. Four species of restriction endonucleases (Hha I, Hinf I, Hae III, Pst I) were used to digest the PCR amplicons from the 3 isolates. When comparing with the RFLP profiles of prototype Ot, the RFLP profiles of PCR amplicons from the 3 isolates were similar to those of Japan Kawasaki strain, but were quite different from the international reference strains Gilliam, Karp, Kato. Results from DNA sequence analysis showed that the complete sequence of Ot-Sta56 gene homology to Japan Kawasaki strain of XDM2 strain was 97%, and deduced amino acid sequence was 92%.

CONCLUSIONData from the complete sequence of Sta56 gene indicated that the genotypes of Ot isolates in Shandong province were similar, but with distinction from the Kawasaki strain.

Amplified Fragment Length Polymorphism Analysis ; Antigens, Bacterial ; genetics ; Bacterial Proteins ; genetics ; Bacterial Typing Techniques ; classification ; DNA, Bacterial ; genetics ; Genes, Bacterial ; Membrane Proteins ; genetics ; Orientia tsutsugamushi ; genetics ; isolation & purification ; Polymorphism, Restriction Fragment Length ; Sequence Analysis, DNA