Brucella ; genetics ; isolation & purification ; DNA, Bacterial ; blood ; isolation & purification ; Polymerase Chain Reaction ; methods

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 develop a real-time polymerase chain reaction(PCR) based on TaqMan technology by using a new MGB probe for detecting enterotoxigenic Escherichia coli (ETEC) in paper.

METHODSPrimers and MGB probe were designed in the ecoding region of heat-stable toxin of ETEC. Real-time PCR detected ETEC by using the exterior standard method with protracting standard curves. The specificity, sensitivity, accuracy, stability of real-time PCR system was evaluated. An internal negative antithesis was added to the real-time PCR system in order to get rid of the false positive of system. Using UNG enzyme expelled the contamination of PCR reaction.

RESULTSPrimers and MGB probe were suited to the Real-time PCR. The assay showed that the method was quick, special, sensitive and stable. The real-time PCR system could detect ETEC in a large scale. The assay might be finished in two hour.

CONCLUSIONThese observations suggested that real-time PCR based on MGB probe should be an excellent candidate for a standard ETEC detection method.

Bacterial Toxins ; isolation & purification ; DNA Primers ; DNA Probes ; DNA, Bacterial ; Enterotoxigenic Escherichia coli ; isolation & purification ; Molecular Probe Techniques ; Polymerase Chain Reaction ; methods ; Taq Polymerase

OBJECTIVETo analyze the ribotyping fingerprint of Enterobacter sakazakii (E. sakazakii) isolated from food and its typing power.

METHODSTwo standard strains and twenty-eight isolates of E.sakazakii were analyzed by the DuPont Riboprinter(TM) microbial characterization system. The relevant database was established and the fingerprint patterns were analyzed with BioNumerics software.

RESULTSThis system grouped two standard strains and twenty-eight E.sakazakii isolates into 26 ribotypes, and four ribotypes included two strains respectively, the other twenty-two strains showed different ribotypes. The lowest similarity was 31.58%. The number of bands by ribotyping was approximately ten and the molecular weight of these bands ranged from 1 to 50 kb. By the clustering program in BioNumerics, these isolates could be grouped into four clusters.

CONCLUSIONThe automatic ribotyping method is convenient and fast in E.sakazakii typing.

Cronobacter sakazakii ; classification ; genetics ; isolation & purification ; DNA, Bacterial ; DNA, Ribosomal ; Food Microbiology ; Ribotyping ; methods

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

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 develop a rapid and definite diagnostic test of bacterial enteritis caused by pathogenic enterobacteria, the most frequent etiologic agent of infectious enteritis in the world.

METHODSA set of conventional PCR assays were applied to detect and identify salmonella, shigella, and E. coli O157:H7 directly from pure culture and fecal samples. The general primers of pathogenic enterobacteria were located on the uidA gene, which were found not only in E. coli nuclear acid, but also in shigella and salmonella genes. Shigella primer was from ipaH gene whose coded invasive plasmid relative antigen existed both in plasmid and in genome. The primers of salmonella were designed from the 16SrRNA sequence. The primer of E. coli O157:H7 was taken from eaeA gene. Five random primers were selected for RAPD. The detection system included common PCR, semi-nested PCR and RAPD.

RESULTSThis method was more sensitive, specific and efficient and its processing was rapid and simple. For example, the method could be used to specifically detect and identify salmonella, shigella, and E. coli O157:H7, and its sensitivity ranged from 3 to 50 CFU, and its detection time was 4 hours.

CONCLUSIONThis PCR method, therefore, can serve as a routine and practical protocol for detecting and identifying pathogenic microorganisms from clinical samples.

DNA Primers ; DNA, Bacterial ; analysis ; Escherichia coli O157 ; isolation & purification ; Feces ; microbiology ; Humans ; Polymerase Chain Reaction ; Salmonella typhi ; isolation & purification ; Sensitivity and Specificity ; Shigella flexneri ; isolation & purification

Following Escherichia coli lysis with alkali, cetyltrimethylammonium bromide (CTAB) was directly titrated into the supernatant. An easy and feasible technology for plasmid purification was established with the optimized proportion between the quantity of CTAB and plasmid, combined with the specific solution for DNA release and TritonX-114 for endotoxin removal. Quality detection showed that the purified plasmid was free of contamination of host RNA. The host bacterial genomic DNA, endotoxin and bacterial protein were less than 10 microg, 50 EU and 10 microg per mg plasmids, respectively. The ratio of OD260/OD280 was between 1.75-1.85. Eighty percent of the prepared plasmids were presented in the supercoiled form. The plasmid purified with this technology can satisfy all criteria stipulated by FDA. The main advantages of the technology include the avoidance of animal-derived enzymes such as ribonucleases A, Proteinase K and toxic reagents like chloroform and phenol. In addition, the technology has low cost and no pollution.
Cetrimonium Compounds ; chemistry ; Chemical Precipitation ; DNA ; genetics ; isolation & purification ; DNA, Bacterial ; genetics ; isolation & purification ; Escherichia coli ; chemistry ; genetics ; Plasmids ; genetics ; isolation & purification

OBJECTIVETo identify the relationship between amino acid mutations in Neisseria gonorrhoeae isolates and their antibiotic resistance.

METHODSPI gene fragments of Neisseria gonorrhoeae from 17 clinical isolates were obtained with PCR amplification. They were cloned into the PCR cloning vector pBS-T to form pBS-T-PI and sequenced. The sequences of PI genes were analyzed. At the same time, minimum inhibitory concentration (MIC) of penicillin and tetracycline to these 17 isolates were measured and contrasted with the corresponding PI sequence.

RESULTSThe recombinants of PI gene from 17 clinical isolates of Neisseria gonorrhoeae were successfully constructed and sequenced. They were divided into PIA and PIB subtypes according to the results from blastn software by comparing the sequences with the GenBank. Mutations were found at the sites of 120 and 121. There were only some of the sequences having an aspartic acid (D) mutation on 120 and 121 sites, which was not the same as reported. On the other hand,there were two PI sequences,5-9 and 6-1, whose mutations on No. 120 were lysine, similar to those documented.

CONCLUSIONSome relationship between PI amino acids mutations at sites 120 and 121 in Neisseria gonorrhoeae isolates from Chengdu, China and their resistance to penicillin and tetracycline were found. However,further studies need to be done in the future to confirm this hypothesis.

Amino Acid Sequence ; Anti-Bacterial Agents ; pharmacology ; DNA Mutational Analysis ; DNA, Bacterial ; Drug Resistance, Bacterial ; Mutation ; Neisseria gonorrhoeae ; drug effects ; genetics ; isolation & purification ; Polymerase Chain Reaction

OBJECTIVEExpress a novel species of single-stranded DNA-binding protein (SSB) derived from Thermococcus kodakarensis KOD1, abbreviated kod-ssb. And evaluate the effect of kod-ssb on PCR-based DNA amplification and reverse transcription.

METHODSWe express kod-ssb with the Transrtta (DE3), and kod-ssb was purified by affinity chromatography on a Ni2+ Sepharose column, detected by SDS-PAGE. To evaluate the effect of kod-ssb on PCR-based DNA amplification, the human beta globin gene was used as template to amplify a 5-kb, 9-kb and 13-kb. And to detect the effect of kod-ssb on reverse transcription, we used RNA from flu cell culture supernatant extraction as templates to implement qRT-PCR reaction.

RESULTSThe plasmid pET11a-kod was transformed into Transetta (DE3) and the recombinant strain Transetta (pET11 a-kod) was obtained. The kod-ssb was highly expressed when the recombinant strain Transetta(pET11a-kod) was induced by IPTG. The specific protein was detected by SDS-PAGE. To confirm that kod-ssb can enhance target DNA synthesis and reduce PCR by-products, 5-, 9-, and 13-kb human beta globin gene fragments were used as templates for PCR. When PCR reactions did not include SSB proteins, the specific PCR product was contaminated with non-specific products. When kod -ssb was added, kod-ssb significantly enhanced amplification of the 5-, 9-and 13-kb target product and minimised the non-specific PCR products. To confirm that kod-ssb can enhance target cDNA synthesis, RNA from flu cell culture supernatant extraction was used as templates for qRT-PCR reaction. The results was that when kod-ssb was added, kod-ssb significantly enhanced the synthesis of cDNA, average Ct value is 19.42, and the average Ct value without kod-ssb is 22.15.

CONCLUSIONSkod-ssb may in future be used to enhance DNA and cDNA amplification.

Archaeal Proteins ; genetics ; isolation & purification ; metabolism ; Chromatography, Affinity ; DNA, Bacterial ; genetics ; metabolism ; DNA, Complementary ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; isolation & purification ; metabolism ; Gene Expression ; Thermococcus ; genetics ; metabolism