RESUMO
Staphylococcus aureus plasmids are the main factor in the spreading of antibacterial resistance among bacterial strains that has emerged on a worldwide scale. Plasmids recovered from 12 clinical and food isolates of S. aureus were treated with 10 mM free lanthanide Nd(3+) ions (non-enzymatic cleavage agent) in Hepes buffer (pH 7.5) at 70 °C. Topological forms of plasmids-closed circular (ccc), open circular (oc), and linear (lin)-produced by cleavage at different times were separated using pulsed-field agarose gel electrophoresis. The method is proposed to detect and differentiate several plasmids in the same bacterial strain according to their size.
Assuntos
DNA Bacteriano/efeitos dos fármacos , DNA Bacteriano/metabolismo , Neodímio/farmacologia , Plasmídeos/metabolismo , Staphylococcus aureus/genética , DNA Bacteriano/genética , Íons/química , Íons/farmacologia , Neodímio/química , Plasmídeos/genéticaRESUMO
Superparamagnetic particles have been attractive for molecular diagnostics and analytical chemistry applications due to their unique magnetic properties and their ability to interact with various biomolecules of interest. This paper presents a critical overview of magnetic nano- and microparticles used as a solid phase for extraction and purification of DNAs. The mechanisms of DNA binding to the surface of functionalised magnetic particles are described. The most widely used materials including silica supports, organic polymers and other materials, mostly containing magnetite or paramagnetic metallic elements are reviewed. The main application areas of magnetic particles for DNA separation are briefly described.
Assuntos
DNA/isolamento & purificação , Nanopartículas de Magnetita/química , Extração em Fase Sólida , Extração em Fase Sólida/instrumentaçãoRESUMO
Magnetic La(0.75)Sr(0.25)MnO(3) nanoparticles possessing an approximately 20-nm-thick silica shell (LSMO(0.25)@SiO(2) ) were characterised and tested for the isolation of PCR-ready bacterial DNA. The results presented here show that the nanoparticles do not interfere in PCR. DNA was apparently reversibly adsorbed on their silica shell from the aqueous phase system (16% PEG 6000-2 M NaCl). The method proposed was used for DNA isolation from complex food samples (dairy products and probiotic food supplements). The isolated DNA was compatible with PCR. The main advantages of the nanoparticles tested for routine use were their high colloidal stability allowing a more precise dosage and therefore high reproducibility of DNA isolation.
Assuntos
DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Lantânio/química , Fenômenos Magnéticos , Compostos de Manganês/química , Nanopartículas/química , Óxidos/química , Dióxido de Silício/química , Estrôncio/química , Lactobacillus/citologia , Lactobacillus/genética , Magnetismo , Reação em Cadeia da PolimeraseRESUMO
Innovative nanotechnology aims to develop particles that are small, monodisperse, smart, and do not cause unintentional side effects. Uniform magnetic Fe3O4 nanoparticles (12 nm in size) were prepared by thermal decomposition of iron(III) oleate. To make them colloidally stable and dispersible in water and cell culture medium, they were modified with phosphonic acid- (PA) and hydroxamic acid (HA)-terminated poly(ethylene glycol) yielding PA-PEG@Fe3O4 and HA-PEG@Fe3O4 nanoparticles; conventional γ-Fe2O3 particles were prepared as a control. Advanced techniques were used to evaluate the properties and safety of the particles. Completeness of the nanoparticle coating was tested by real-time polymerase chain reaction. Interaction of the particles with primary human peripheral blood cells, cellular uptake, cytotoxicity, and immunotoxicity were also investigated. Amount of internalized iron in peripheral blood mononuclear cells was 72, 38, and 25 pg Fe/cell for HA-PEG@Fe3O4, γ-Fe2O3, and PA-PEG@Fe3O4, respectively. Nanoparticles were localized within the cytoplasm and in the extracellular space. No cytotoxic effect of both PEGylated nanoparticles was observed (0.12-75 µg/cm2) after 24 and 72-h incubation. Moreover, no suppressive effect was found on the proliferative activity of T-lymphocytes and T-dependent B-cell response, phagocytic activity of monocytes and granulocytes, and respiratory burst of phagocytes. Similarly, no cytotoxic effect of γ-Fe2O3 particles was observed. However, they suppressed the proliferative activity of T-lymphocytes (75 µg/cm2, 72 h) and also decreased the phagocytic activity of monocytes (15 µg/cm2, 24 h; 3-75 µg/cm2, 72 h). We thus show that newly developed particles have great potential especially in cancer diagnostics and therapy.
Assuntos
Proliferação de Células/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Nanopartículas de Magnetita/toxicidade , Nanomedicina/métodos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Células Cultivadas , Citocinas/metabolismo , Humanos , Ácidos Hidroxâmicos/química , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Nanopartículas de Magnetita/química , Tamanho da Partícula , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Ácidos Fosforosos/química , Polietilenoglicóis/química , Explosão Respiratória/efeitos dos fármacos , Explosão Respiratória/imunologia , Propriedades de SuperfícieRESUMO
Molecular biological methods based on genus-specific PCR, species-specific PCR, and amplified ribosomal DNA restriction analysis (ARDRA) of two PCR amplicons (523 and 914bp) using six restriction enzymes were used to differentiate among species of Bifidobacterium. The techniques were established using DNA from 16 type and reference strains of bifidobacteria of 11 species. The discrimination power of 914bp amplicon digestion was higher than that of 523bp amplicon digestion. The 914bp amplicon digestion by six restrictases provided unique patterns for nine species; B. catenulatum and B. pseudocatenulatum were not differentiated yet. The NciI digestion of the 914bp PCR product enabled to discriminate between each of B. animalis, B. lactis, and B. gallicum. The reference strain B. adolescentis CCM 3761 was reclassified as a member of the B. catenulatum/B. pseudocatenulatum group. The above-mentioned methods were applied for the identification of seven strains of Bifidobacterium spp. collected in the Culture Collection of Dairy Microorganisms (CCDM). The strains collected in CCDM were differentiated to the species level. Six strains were identified as B. lactis, one strain as B. adolescentis.
Assuntos
Bifidobacterium/classificação , Polimorfismo de Fragmento de Restrição , Sequência de Bases , Bifidobacterium/genética , Bifidobacterium/isolamento & purificação , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Humanos , Filogenia , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/química , RNA Ribossômico 23S/genética , Alinhamento de Sequência , Especificidade da EspécieRESUMO
DNA amplification by real-time polymerase chain reaction (RT-PCR) was used for the evaluation of efficiency of polymer coating of magnetic hydrophilic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) (P(HEMA-co-GMA)) and poly(glycidyl methacrylate) (PGMA) microspheres with/without carboxyl groups. The inhibition effect of magnetic microspheres on real-time polymerase chain reaction (RT-PCR) course was evaluated by regression analysis after the addition of different concentrations of tested microspheres to PCR mixtures. Microspheres mostly did not interfere in RT-PCR till the concentration 50 µg/25 µl PCR mixture. No relationship between Fe content (and microsphere diameter) and inhibition effect was found. Microspheres containing carboxyl groups extinguished the fluorescence at lower concentrations (10-20 µg/25 µl PCR mixture) without inhibition of DNA amplification as PCR products were detected using agarose gel electrophoresis. Negative effect of maghemite on PCR course was partially reduced by coating of magnetic core by silica or polymers. Two inhibition mechanisms of DNA amplification were discussed in this work.
Assuntos
Nanopartículas de Magnetita/química , Microesferas , Patologia Molecular/métodos , Ácidos Polimetacrílicos/química , Reação em Cadeia da Polimerase em Tempo Real , DNA Bacteriano/análise , DNA Bacteriano/genética , Eletroforese em Gel de ÁgarRESUMO
Carboxyl groups containing magnetic and non-magnetic microspheres were used in solid-phase reversible immobilization (SPRI) of genomic DNA. Magnetic non-porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate)--P(HEMA-co-EDMA), poly(glycidyl methacrylate)--PGMA and P(HEMA-co-GMA) microspheres with hydrophilic properties were prepared by dispersion copolymerization of the respective monomers in the presence of colloidal iron oxides. DNA from chicken erythrocytes and DNA isolated from bacterial cells of Bifidobacterium longum was used for testing of adsorption/desorption properties of magnetic microspheres. The occurrence of false negative results in polymerase chain reaction (PCR) caused by the presence of extracellular inhibitors in DNA samples has been solved using SPRI. The P(HEMA-co-EDMA) and P(HEMA-co-GMA) microspheres were used for isolation of DNA from different dairy products followed by PCR identification of Bifidobacterium strains.
Assuntos
DNA/isolamento & purificação , Genoma , Magnetismo , Metacrilatos/química , DNA/genética , Eletroforese em Gel de Ágar , Microscopia Eletrônica de Varredura , Microesferas , Reação em Cadeia da PolimeraseRESUMO
The influence of cobalt ferrite particles, with non-modified or modified surface, on the course of polymerase chain reaction (PCR) was investigated. DNA isolated from bacterial cells of Bifidobacterium bifidum was used in PCR evaluation of magnetic microspheres. The presence of cobalt ferrite particles inhibits PCR amplification. The effect is not dependent on the functional groups of the modifying reagents used (none, amino, carboxyl). Amplification was improved after the magnetic separation of magnetic particles. Proposed indirect method enabled verification of the suitability of designed particles for their application in PCR assays. Magnetic particles coated with alginic acid under high PEG and sodium chloride concentration were used for the isolation of PCR-ready bacterial DNA from various dairy products. DNA was isolated from crude bacterial cell lysates without phenol extraction of samples. Bifidobacterium and Lactobacillus DNAs were identified in dairy products using PCR.
Assuntos
DNA Bacteriano/isolamento & purificação , Compostos Férricos/química , Reação em Cadeia da Polimerase/métodos , Bifidobacterium/genética , Lactobacillus/genéticaRESUMO
BACKGROUND: Reduced microbial diversity has been associated with inflammatory bowel disease (IBD) and probiotic bacteria have been proposed for its prevention and/or treatment. Nevertheless, comparative studies of strains of the same subspecies for specific health benefits are scarce. Here we compared two Bifidobacterium longum ssp. longum strains for their capacity to prevent experimental colitis. METHODS: Immunomodulatory properties of nine probiotic bifidobacteria were assessed by stimulation of murine splenocytes. The immune responses to B. longum ssp. longum CCM 7952 (Bl 7952) and CCDM 372 (Bl 372) were further characterized by stimulation of bone marrow-derived dendritic cell, HEK293/TLR2 or HEK293/NOD2 cells. A mouse model of dextran sulphate sodium (DSS)-induced colitis was used to compare their beneficial effects in vivo. RESULTS: The nine bifidobacteria exhibited strain-specific abilities to induce cytokine production. Bl 372 induced higher levels of both pro- and anti-inflammatory cytokines in spleen and dendritic cell cultures compared to Bl 7952. Both strains engaged TLR2 and contain ligands for NOD2. In a mouse model of DSS-induced colitis, Bl 7952, but not Bl 372, reduced clinical symptoms and preserved expression of tight junction proteins. Importantly, Bl 7952 improved intestinal barrier function as demonstrated by reduced FITC-dextran levels in serum. CONCLUSIONS: We have shown that Bl 7952, but not Bl 372, protected mice from the development of experimental colitis. Our data suggest that although some immunomodulatory properties might be widespread among the genus Bifidobacterium, others may be rare and characteristic only for a specific strain. Therefore, careful selection might be crucial in providing beneficial outcome in clinical trials with probiotics in IBD.
Assuntos
Bifidobacterium/classificação , Bifidobacterium/fisiologia , Colite/prevenção & controle , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Intestinos/fisiopatologia , Probióticos/farmacologia , Animais , Colite/induzido quimicamente , Células Dendríticas/microbiologia , Células Dendríticas/patologia , Feminino , Células HEK293 , Humanos , Técnicas Imunoenzimáticas , Intestinos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismoRESUMO
The HEMA-BIO 1000 support, which is based on a copolymer of 2-hydroxyethyl methacrylate and ethylene dimethacrylate, was used for separation of lambda DNA and its fragments and plasmid pBR322 DNA. The separation of fragments greater than 6.6 kbp was demonstrated according to the slalom chromatography mechanism on column for size-exclusion chromatography in the case of linear lambda DNA fragments. The influence of particle size of column packing, mobile phase rate, and KCl concentration in mobile phase is discussed. The purification of plasmid DNA pBR322 using size-exclusion chromatography was more rapid compared to gel electrophoresis. The presence of salts in the eluate is not disadvantageous. DNA can be recovered from the eluate by ethanol precipitation. Plasmid DNA pBR322 isolated in this way was suitable for different biological applications (cleavage with restrictases, electrotransformation into bacterial cells).
Assuntos
Bacteriófago lambda/genética , Cromatografia em Gel/métodos , DNA Viral/isolamento & purificação , DNA/isolamento & purificação , Metacrilatos/química , Plasmídeos , DNA/química , DNA Viral/química , Eletroforese em Gel de ÁgarRESUMO
Adsorption separation techniques as an alternative to laborious traditional methods (e.g., based on phenol extraction procedure) have been applied for DNA purification. In this work we used two types of particles: silica and cobalt ferrite (unmodified or modified with a reagent containing weakly basic aminoethyl groups, aminophenyl groups, or alginic acid). DNA from chicken erythrocytes and DNA isolated from bacteria Lactococcus lactis were used for testing of adsorption/desorption properties of particles. The cobalt ferrite particles modified with different reagents were used for isolation of PCR-ready bacterial DNA from different dairy products.
Assuntos
Cobalto/química , DNA/isolamento & purificação , Compostos Férricos/química , Dióxido de Silício/química , Adsorção , Animais , Galinhas , DNA/química , Eletroforese em Gel de Ágar , Magnetismo , Microscopia Eletrônica de Varredura , Reação em Cadeia da PolimeraseRESUMO
Magnetic nonporous poly(HEMA-co-EDMA) and poly(HEMA-co-GMA) microspheres were prepared by dispersion copolymerisation of 2-hydroxyethyl methacrylate (HEMA) and ethylene dimethacrylate (EDMA) or glycidyl methacrylate (GMA) in the presence of magnetite. They were functionalized by polyclonal Salmonella antibodies via the trichlorotriazine method. Salmonella cells were then successfully identified using cultural and polymerase chain reaction (PCR) methods after their immunomagnetic separation. The PCR sensitivity of target cell detection was negatively influenced by the presence of some compounds used in the process of particle preparation. In some cases, magnetic poly(HEMA-co-EDMA) microspheres with immobilized proteinase K were used for degradation of intracellular inhibitors present in Salmonella cells.
Assuntos
Salmonella/citologia , Eletroforese em Gel de Ágar , Separação Imunomagnética , Metacrilatos , Microscopia Eletrônica de Varredura , Reação em Cadeia da PolimeraseRESUMO
Magnetic hydrophilic non-porous P(HEMA-co-EDMA), P(HEMA-co-GMA) and PGMA microspheres were prepared by dispersion (co)polymerization of 2-hydroxyethyl methacrylate (HEMA) and ethylene dimethacrylate (EDMA) or glycidyl methacrylate (GMA) in the presence of several kinds of magnetite. It was found that some components used in the preparation of magnetic carriers interfered with polymerase chain reaction (PCR). Influence of non-magnetic and magnetic microspheres, including magnetite nanoparticles and various components used in their synthesis, on the PCR course was thus investigated. DNA isolated from bacterial cells of Bifidobacterium longum was used in PCR evaluation of non-interfering magnetic microspheres. The method enabled verification of the incorporation of magnetite nanoparticles in the particular methacrylate-based polymer microspheres and evaluation of suitability of their application in PCR. Preferably, electrostatically stabilized colloidal magnetite (ferrofluid) should be used in the design of new magnetic methacrylate-based microspheres by dispersion polymerization.
Assuntos
Metacrilatos/química , Microesferas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Bifidobacterium/química , DNA Bacteriano/isolamento & purificação , Eletroforese em Gel de Ágar , Compostos de Epóxi , Magnetismo , Microscopia Eletrônica de VarreduraRESUMO
The use of free lanthanide ions and their complexes for plasmid DNA pBR322 and chromosomal DNA cleavage was studied. Plasmid pBR322 DNA was treated by lanthanide chlorides (Eu(3+), La(3+), Nd(3+), Pr(3+), Gd(3+)) in HEPES buffer (pH 7.0, 7.5 and 8.0) at 24, 37, 50, 63, and 76 degrees C. The formation of linear and nicked plasmid forms was investigated depending on the reaction conditions. Heterogeneous lanthanide complexes of ethylenediamine tetraacetic acid (EDTA) immobilized on insoluble methacrylate support and iminodiacetic acid (IDA) immobilized on styrene support were used as catalysts plasmid for DNA pBR322 cleavage, too. The temperature of reaction mixture had substantial influence on cleavage rate. The precipitation of DNA occurred during the measurement of interactions between chromosomal DNA and La(3+) ions.
Assuntos
DNA/química , Elementos da Série dos Lantanídeos/química , Plasmídeos/química , Cromossomos/química , Desoxirribonuclease EcoRI/química , Ácido Edético , Eletroforese em Gel de Ágar , Indicadores e Reagentes , Metacrilatos , Piridinas/químicaRESUMO
Magnetic bead cellulose particles and magnetic poly(HEMA-co-EDMA) microspheres with immobilized DNase I were used for degradation of chromosomal and plasmid DNAs. Magnetic bead particles were prepared from viscose and magnetite powder. Magnetic poly(HEMA-co-EDMA) microspheres were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of magnetite. Divalent cations (Mg(2+), Ca(2+), Mn(2+) and Co(2+)) were used for the activation of DNase I. A comparison of free and immobilized enzyme (magnetic bead particles) activities was carried out in dependence on pH and activating cation. The maximum of the activity of immobilized DNase I was shifted to lower pH compared with free DNase I. DNase I immobilized on magnetic bead cellulose was used 20 times in the degradation of chromosomal DNA. Its residual activity was influenced by the nature of activating divalent cation. The immobilized enzyme with decreased activity was reactivated by Co(2+) ions.
Assuntos
Desoxirribonuclease I/metabolismo , Enzimas Imobilizadas/metabolismo , Polímeros/química , Cátions Bivalentes , Eletroforese em Gel de Ágar , Ativação Enzimática , Concentração de Íons de Hidrogênio , Magnetismo , Microesferas , PlasmídeosRESUMO
Eleven strains of Lactobacillus collected in the Culture Collection of Dairy Microorganisms (CCDM) were evaluated for selected probiotic properties such as survival in gastrointestinal fluids, antimicrobial activity, and competition with non-toxigenic Escherichia coli O157:H7 for adhesion on Caco-2 cells. The viable count of lactobacilli was reduced during 3-h incubation in gastric fluid followed by 3-h incubation in intestinal fluid. All strains showed antimicrobial activity and the three most effective strains inhibited the growth of at least 16 indicator strains. Antimicrobial metabolites of seven strains active against Lactobacillus and Clostridium indicator strains were found to be sensitive to proteinase K and trypsin, which indicates their proteinaceous nature. The degree of competitive inhibition of non-toxigenic E. coli O157:H7 adhesion on the surface of Caco-2 cells was strain-dependent. A significant decrease (P < 0.05) in the number of non-toxigenic E. coli O157:H7 adhering to Caco-2 cells was observed with all lactobacilli. Three strains were selected for additional studies of antimicrobial activity, i.e., Lactobacillus gasseri CCDM 215, Lactobacillus acidophilus CCDM 149, and Lactobacillus helveticus CCDM 82.
Assuntos
Anti-Infecciosos/metabolismo , Antibiose , Lactobacillus/fisiologia , Viabilidade Microbiana/efeitos dos fármacos , Probióticos/farmacologia , Aderência Bacteriana , Células CACO-2 , Células Epiteliais/microbiologia , Escherichia coli O157/fisiologia , Suco Gástrico/microbiologia , Humanos , Lactobacillus/crescimento & desenvolvimentoRESUMO
Wedelolactone is one of the active plant polyphenolic compounds. Anti-tumor effects of this drug have been demonstrated recently. We have described that wedelolactone acts as catalytic inhibitor of DNA topoisomerase IIα. The aim of this study was to further characterize the mechanism of its anti-tumor effects. We showed that wedelolactone inhibits binding of DNA topoisomerase IIα to plasmid DNA and antagonizes formation of etoposide-induced DNA cleavage complex. The inhibition of topoisomerase IIα by wedelolactone is reversible by excess of the enzyme but not DNA. The in vitro inhibitory effect of wedelolactone on the topoisomerase IIα activity is redox-dependent as it diminished in the presence of reducing agents. Cytotoxicity of wedelolactone was partially inhibited by N-acetylcysteine and glutathione ethyl ester in breast cancer MDA-MB-231 and MDA-MB-468 cells while the inhibitory effect of catalase was observed only in the former cell line. Finally, we found that wedelolactone can be oxidized in the presence of copper ions resulting in DNA strand break and abasic site formation in vitro. However, wedelolactone induced neither DNA damage in MDA-MB-231 cells nor mutations in bacterial cells detectable by Ames test suggesting that wedelolactone may not be an effective inducer of DNA damage. We conclude that the topoisomerase IIα inhibitory- and DNA damaging activities of wedelolactone in vitro depend on its redox state. Pro-oxidant activity could, however, explain only part of wedelolactone-induced cytotoxicity. Therefore, the major cellular target(s) of wedelolactone and the exact mechanism of wedelolactone-induced cytotoxicity still remain to be identified.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Cumarínicos/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Acetilcisteína , Antígenos de Neoplasias/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Catalase , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cumarínicos/química , Cumarínicos/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Glutationa/análogos & derivados , Humanos , Immunoblotting , Técnicas In Vitro , Estrutura Molecular , NAD/metabolismo , OxirreduçãoRESUMO
Bifidobacterium longum is considered to play an important role in health maintenance of the human gastrointestinal tract. Probiotic properties of bifidobacterial isolates are strictly strain-dependent and reliable methods for the identification and discrimination of this species at both subspecies and strain levels are thus required. Differentiation between B. longum ssp. longum and B. longum ssp. infantis is difficult due to high genomic similarities. In this study, four molecular-biological methods (species- and subspecies-specific PCRs, random amplified polymorphic DNA (RAPD) method using 5 primers, repetitive sequence-based (rep)-PCR with BOXA1R and (GTG)(5) primers and amplified ribosomal DNA restriction analysis (ARDRA)) and biochemical analysis, were compared for the classification of 30 B. longum strains (28 isolates and 2 collection strains) on subspecies level. Strains originally isolated from the faeces of breast-fed healthy infants (25) and healthy adults (3) showed a high degree of genetic homogeneity by PCR with subspecies-specific primers and rep-PCR. When analysed by RAPD, the strains formed many separate clusters without any potential for subspecies discrimination. These methods together with arabionose/melezitose fermentation analysis clearly differentiated only the collection strains into B. longum ssp. longum and B. longum ssp. infantis at the subspecies level. On the other hand, ARDRA analysis differentiated the strains into the B. longum/infantis subspecies using the cleavage analysis of genus-specific amplicon with just one enzyme, Sau3AI. According to our results the majority of the strains belong to the B. longum ssp. infantis (75%). Therefore we suggest ARDRA using Sau3AI restriction enzyme as the first method of choice for distinguishing between B. longum ssp. longum and B. longum ssp. infantis.
Assuntos
Bifidobacterium/classificação , Fezes/microbiologia , Tipagem Molecular/métodos , Técnica de Amplificação ao Acaso de DNA Polimórfico , Adulto , Bifidobacterium/química , Bifidobacterium/genética , Aleitamento Materno , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Humanos , Lactente , Filogenia , RNA Ribossômico 16S/genética , Mapeamento por RestriçãoRESUMO
Magnetic microspheres P(HEMA-co-EDMA) were used for PCR-ready phage DNA isolation from lysogenic strains of Staphylococcus aureus, including two new clinical isolates. The conditions of phage particle lysis were optimized. The quality of eluted phage DNA was evaluated by PCR. It was demonstrated that PCR-ready phage DNA can be isolated from small volumes of phage lysates (150 microl) by magnetic microspheres. The reported method is very expeditious without using toxic compounds such as phenol or chloroform. It can be used for phage identification and phage gene detection.
Assuntos
DNA Viral/isolamento & purificação , Técnicas Genéticas , Magnetismo , Microesferas , Extração em Fase Sólida/métodos , Fagos de Staphylococcus/genética , DNA Viral/genética , Reação em Cadeia da Polimerase , Fagos de Staphylococcus/isolamento & purificação , Staphylococcus aureus/virologiaRESUMO
Carboxyl group-containing magnetic nonporous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (P(HEMA-co-EDMA)) microspheres were used for the isolation of polymerase chain reaction (PCR)-ready DNA from samples of Baikal sediments. DNA was isolated using the phenol extraction method or the Soil DNA Isolation Kit. The occurrence of false-negative results in PCR caused by the presence of extracellular inhibitors in DNA samples was solved using solid phase reversible DNA immobilisation. PCR-ready DNA was reversibly adsorbed to the microspheres in the presence of 8.0% (w/v) poly(ethylene glycol) (PEG 6000) and 2.0M sodium chloride concentrations. The adsorbed DNA was released from the microspheres in a low ionic strength TE buffer. The quality of isolated DNA was checked by PCR amplification.