The important role of intestinal microorganisms in human health has been widely confirmed. At present, most of the studies on intestinal microorganisms are based on amplification of the V3-V4 region of bacterial 16S rRNA gene, and little attention has been paid to archaea. In this study, a primer set which can amplify 16S rRNA gene of both bacteria and archaea at the same time was used. By comparing the community changes before and after probiotics intake, it showed that this primer set is suitable for analyzing the changes of human intestinal bacteria and archaea communities. The fecal samples of volunteers were collected, and the amplification and high-throughput sequencing were carried out by using bacterial primer set (B primer) and bacterial and archaeal universal primer (AB primer); several commonly used rRNA databases were used to determine the amplification ability of the primer set to bacteria and archaea. The results showed that AB primer could display the bacterial community amplified by B primer, and could obtain the sequence of common methanogenic archaea in intestinal tract. AB primer set can analyze the bacteria and archaea in the intestinal tract at the same time by only one amplification and sequencing, which can show the structure of intestinal microbial community more comprehensively, which is suitable for the research of intestinal microorganisms.
Archaea/genetics* ; Bacteria/genetics* ; DNA Primers ; DNA, Bacterial ; Humans ; Microbiota/genetics* ; Phylogeny ; RNA, Ribosomal, 16S/genetics*

The community structure and diversity of the gut microbiota are associated with human diseases. However, the analysis of different community structure might be influenced by experimental approaches such as the quality of DNA extraction. Therefore, evaluating the efficiency of different DNA extraction methods for specific intestinal species is a guideline for obtaining a comprehensive human gut microbial profile, which may assist the in-depth investigation into the structure of the gut microbial community. The aim of this study was to perform a comparative analysis of five different DNA extraction methods. With the aid of qPCR, the efficiency of five DNA extraction kits was evaluated in terms of the purity of the extracted DNA, the DNA concentration, and the abundance of genomic DNA extracted from specific intestinal species. The results showed that the kit Q gave the best extraction results, especially for Gram-positive bacteria such as Lactobacillus and Bifidobacterium. The average DNA concentration of the N kit was lower than that of the Q kit, but there was no significant difference between the two in terms of the purity. Compared to the other three commercial kits (M, PSP, TG), the efficiency of the N kit in extracting the genomic DNA of the specified microorganisms were the least different from those of the Q kit. In contrast, the DNA extracted by the M kit was of higher quality but of lower concentration, and was not very efficient for Gram-positive bacteria. The DNA extracted by the TG and PSP kits was inferior to the other validated kits in terms of the concentration, quality and bacterial abundance. These results provide a basis for the selection of genomic DNA extraction methods in microecological research experiments.
DNA/genetics* ; DNA, Bacterial/genetics* ; Feces/microbiology* ; Humans ; Microbiota/genetics* ; RNA, Ribosomal, 16S/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

Genome shuffling methods were explored for Bacillus subtilis strain molecular breeding. Recycling protoplast fusion, recycling transformation and recycling universal transduction were used for genome shuffling in B. subtilis. Four strains with different nutrition-deficiency markers were used as initial strains. After five rounds protoplast fusion, transformation or transduction, the descendant with 4 markers had not been detected, and the rate of descendant with 3 markers were 4.53 x 10(-4), 1.64 x 10(-4), 4.47 x 10(-3), respectively. A computer program was made to simulate the recycling fusion process. Based on simulation result and comparing the genome shuffling result of B. subtilis in this experiment and that of Streptomyces coelicolor reported in references, effective genome shuffling needs a high recombination rate of at least between 10(-3) and 10(-2).
Bacillus subtilis ; classification ; genetics ; DNA Shuffling ; Genetic Techniques ; Genome, Bacterial ; genetics ; Protein Engineering ; Transformation, Bacterial

OBJECTIVETo evaluate the application of different variable number tandem repeats (VNTR) locus in genotyping of Mycobacterium tuberculosis (M.tuberculosis) strains isolated from eight provinces in China, and to find the suitable locus-set of VNTR for epidemical strains in China.

METHODSAll 140 M.tuberculosis strains were randomly selected from 2800 M.tuberculosis strains isolated from eight provinces in China, 27 VNTR loci were used for typing all isolates. Discriminatory power (Hunter-Gaston Index, HGI) of every locus and different locus-set were analyzed by BioNumerics software. Meanwhile, Spoligotyping was used to identify Beijing family and non-Beijing family. Then the HGI of different locus-sets in two families was also evaluated.

RESULTSAll 140 isolates were clustered into Beijing kindred (112 strains, 80%) and non-Beijing kindred (28 strains, 20%) by Spoligotyping. The discriminatory power of Spoligotyping in 140 isolates was 0.4589. Every locus showed different polymorphism and HGI were from 0 to 0.809. The number of VNTR loci with HGI higher than 0.5 in all strains, Beijing family and non-Beijing family was 8, 7 and 14 respectively. 27 loci were combined into four groups which included 8, 12, 15 and 24 VNTR loci respectively. Four locus-sets showed different polymorphism, HGI of eight-locus, 12-locus, 15-locus, and 24-locus set in 140 strains was 0.9991, 0.9882, 0.9980 and 0.9986, and their discriminatory power were calculated in Beijing kindred (HGI: 0.9987, 0.9318, 0.9969 and 0.9975) and non-Beijing kindred (HGI: 1, 0.9894, 1 and 1).

CONCLUSIONDifferent VNTR locus and locus-set showed different discriminatory power in the selected M.tuberculosis strains isolated from China. Eight-locus set can be used in molecular epidemiological study of M.tuberculosis in China after standardization.

Bacterial Typing Techniques ; DNA, Bacterial ; genetics ; Mycobacterium tuberculosis ; classification ; genetics ; isolation & purification ; Tandem Repeat Sequences

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

A site-directed mutant DNA fragment was synthesized and transfected into clinical Neisseria gonorrhoeae (NG) stains to construct the transformants that contained the corresponding mutagenesis of regulation region of mtrR gene. According to the technique of gene splicing by overlap extension (SOEing), a DNA segment with specific mutagenesis was constructed by two-step polymerase chain reaction (PCR). The mutation fragments EF could be used for the next experiment in which the mutation NG strains were induced. By comparing the recombinant EF fragments to the corresponding DNA fragments of clinical NG strains, 2 of these were not compatible completely. The results of sequencing revealed that there was a 9 bp deletion between the 45 to 54 inverted repeat sequence localized within the mtrR promoter. It can be confirmed that the fragments EF are the specifically designed mutant fragments.
Bacterial Proteins/*genetics ; DNA Fragmentation ; DNA, Bacterial/genetics ; Mutagenesis, Site-Directed ; Neisseria gonorrhoeae/*genetics ; Neisseria gonorrhoeae/metabolism ; Recombination, Genetic ; Repressor Proteins/*genetics ; Sequence Deletion ; Transfection ; Transformation, Bacterial

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

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