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

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

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

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

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 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

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

In order to develop a rapid and reliable method for B. cereus genotyping, factors influencing PFGE results, including preparation of bacterial cells embedded in agarose, lysis of embedded cells, enzymatic digestion of intact genomic DNA, and electrophoresis parameters allowing for reproducible and meaningful DNA fragment separation, were controlled. Optimal cellular growth (Luria-Bertani agar plates for 12-18 h) and lysis conditions (4 h incubation with 500 µg/mL lysozyme) produced sharp bands on the gel. Restriction enzyme NotI was chosen as the most suitable. Twenty-two isolates were analyzed by NotI digestion, using three electrophoretic parameters (EPs). The EP-a was optimal for distinguishing between isolates. The optimized protocol could be completed within 40 h which is a significant improvement over the previous methods.
Bacillus cereus ; genetics ; isolation & purification ; Bacteriological Techniques ; DNA, Bacterial ; chemistry ; genetics ; Electrophoresis, Gel, Pulsed-Field ; 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 study the resistance mechanism and homology of carbapenems-resistant Pseudomonas aeruginosa (PA).

METHODSA total of 812 strains of PA (identified) were isolated from sputum, urine, blood, pus, and drainage of patients with burn, severe pneumonia, diabetes, chronic obstructive pneumonia, myocarditis, liver transplantation, or brainstem hemorrhage hospitalized from January to September 2012. Drug resistance of the 812 strains of PA to 15 antibiotics commonly used in clinic, including piperacillin, imipenem, etc., was tested using the automatic microorganism identifying and drug sensitivity analyzer. Among the carbapenems-resistant PA isolates, synergism test with imipenem-ethylene diamine tetraacetic acid (EDTA) and enhancement test with imipenem-EDTA and ceftazidime-EDTA were used to screen metallo-β-lactamase (MBL)-producing strains; modified Hodge test was used to screen strains producing Klebsiella pneumoniae carbapenemases (KPC); the carbapenemase gene, plasmid mediated quinolone resistant (PMQR) gene, and mobile genetic elements (MGE) were detected by polymerase chain reaction (PCR). In addition, a comparative analysis of the PMQR gene carrying level between the carbapenemase gene positive strains and carbapenemase gene negative strains was carried out. The repetitive consensus sequence of Enterobacteriaceae genome PCR (ERIC-PCR) was carried out for gene typing. Moreover, the source and resistance genes of strains with the same genotype were analyzed. Data were processed with Fisher's exact probability test.

RESULTSThe sensitive rates of the 812 strains of PA to ceftriaxone and trimethoprim-sulfamethoxazole were high, respectively 83.07% and 88.19%, and those of the other antibiotics ranged from 17.30% to 55.18%. Twenty-four carbapenems-resistant PA strains were screened, including 11 MBL-producing strains and 2 KPC-producing strains. Eleven carbapenems-resistant PA strains were found to harbor the blaVIM-2 gene, accounting for 45.83%; 2 carbapenems-resistant PA strains carried the blaKPC-2 gene, accounting for 8.33%. Fourteen carbapenems-resistant PA strains only harbored the PMQR gene acc (6')-Ib-cr, accounting for 58.33%; 3 carbapenems-resistant PA strains (12.50%) harbored the PMQR genes acc (6')-Ib-cr and qnr, including 1 strain with qnr A1 and 2 strains with qnr B4. Ten carbapenems-resistant PA strains carried the MGE gene ISCR1, accounting for 41.67%; 6 carbapenems-resistant PA strains carried the MGE gene ISEcp1, accounting for 25.00%. In addition, 3 carbapenems-resistant PA strains co-harbored the MGE genes ISCR1 and ISEcp1 (accounting for 12.50%), while only 1 carbapenems-resistant PA strain co-harbored the MGE genes class 1 integron and ISEcp1, accounting for 4.17%. Twelve out of the 13 carbapenemase gene positive strains carried one or two PMQR gene (s), which was significantly higher than that of the carbapenemase gene negative strains (with only five strains harboring one PMQR gene, P = 0.023). The 24 carbapenems-resistant PA strains were classified into 6 genotypes by the ERIC-PCR. Thirteen strains (accounting for 54.17%), mainly isolated from pus and blood samples, which were collected from burn department, were in genotype A. Eight out of the 13 strains harbored genes blaVIM-2, acc (6')-Ib-cr, and ISCR1. Five strains (accounting for 20.83%), mainly isolated from sputum samples which were collected from ICU, were in genotype B. Only 2 out of the 5 strains co-harbored the carbapenemase gene, PMQR gene, and MGE gene. There were respectively 2 strains in genotypes C and D, both accounting for 8.33%; the strains in different pattern were isolated from different wards, and they harbored diverse resistance genes. There were respectively 1 strain in genotypes E and F, both accounting for 4.17%.

CONCLUSIONSThe resistance mechanism of PA to carbapenems is mainly mediated by the VIM-2 type MBL in our hospital during 2012, followed by KPC-2 type carbapenemase, and the prevalent genotype is type A. The carbapenemase genes and PMQR genes co-carrying phenomenon exists among these strains of PA, which disseminated by clones.

Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; Carbapenems ; pharmacology ; DNA, Bacterial ; Drug Resistance, Bacterial ; Humans ; Microbial Sensitivity Tests ; Pseudomonas aeruginosa ; drug effects ; genetics ; isolation & purification ; beta-Lactamases ; genetics