We isolated and amplified by PCR 16S rDNA from bacteria attached to the bovine rumen wall and analyzed it by denaturing gradient gel electrophoresis (DGGE) with subsequent sequence analysis. The attached bacterial community differed from the bacteria of rumen content; however, no differences were observed among the five epithelial sampling sites taken from each animal. The DGGE profile of the bacterial population attached to the rumen wall represented a high inter-animal variation.

• MeSH
• Rumen microbiology   MeSH
• Bacteria genetics   classification   MeSH
• Biodiversity MeSH
• Nucleic Acid Denaturation MeSH
• DNA, Bacterial genetics   MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Financing, Organized MeSH
• Polymerase Chain Reaction MeSH
• DNA, Ribosomal genetics   MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Cattle MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Cattle MeSH
• Female MeSH
• Animals MeSH

• Publication type
• Meeting Abstract MeSH

A feeding study was performed to monitor the effect of chitosan intake on the fecal microbiota of ten healthy human subjects. Diversity of microflora was monitored during 8 weeks including 4 weeks of chitosan supplementations. Using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene amplicons and quantitative PCR method we revealed possible changes originating in the overall bacterial composition and also in the subpopulation of Bifidobacterium group. DGGE profiles displayed high complexity and individuality for each subject. Considerable variations in the composition of band patterns were observed among different persons. A raised level of fecal Bacteroides in response to chitosan intake was found in all samples. Bifidobacterium levels following chitosan intake increased or remain unchanged. Non-significant increase was, surprisingly, found in the numbers of butyrate-producing bacteria.

• MeSH
• Bacteroides genetics   isolation & purification   MeSH
• Bifidobacterium genetics   isolation & purification   MeSH
• Biodiversity MeSH
• Chitosan metabolism   MeSH
• Nucleic Acid Denaturation MeSH
• DNA, Bacterial genetics   MeSH
• Adult MeSH
• Electrophoresis, Polyacrylamide Gel MeSH
• Human Experimentation MeSH
• Feces microbiology   MeSH
• Financing, Organized MeSH
• Middle Aged MeSH
• Humans MeSH
• Polymerase Chain Reaction MeSH
• DNA, Ribosomal genetics   MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Female MeSH

Katétrizace patří k běžným postupům při řadě zákroků, s čímž souvisí fakt že přes 40 % nozokomiálních infekcí jsou právě inf. močového traktu, zejména inf. katétrizovaných pacientů. U polymikrobiálních infekcí ran je známo, že kultivačně lze zachytit pouze část mikrobů a část lze prokázat pouze metodami molekulárními. Mikrobiální diverzita biofilmových společenstev močových stentů a katétrů nebyla dosud uspokojivě prozkoumána. Vhodnou metodou molekulární analýzy se zdá denaturační gradientová gelová elektroforéza (DGGE). Většinou, když byla PCR-DGGE použita, byly zachyceny i bakterie nekultivovatelné, navíc byly prokázány bakterie u daných infekcí dosud neznámé. Cílem projektu je studium druhového složení biofilmových společenstev kultivačně a molekulárními technikami a zhodnocení rozdílů v polymikrobiálních společenstvech ve vztahu k selhání terapie, délce zavedení katétru, jeho druhu a dalším faktorům včetně predominujících mikrobiálních druhů.; Catheterization of patient is common during many interventions. This is one of reasons, why over 40% of nosocomial infections are infections of urinary tract, esp. of catheterized patients. Poly-microbial wound infection are known to be only partially cultivable, part of microbes can be proved only by molecular techniques. The microbial diversity of biofilm communities of urethral stents and catheters was not satisfactory examined. Suitable method for poly-microbial diagnostic is Denaturing Gradient Gel Electrophoresis (DGGE). In most studies the PCR-DGGE proved also non-cultivable bacteria and even bacteria unknown with those infections. The aim of this project is to study of species composition of biofilm communities by cultivation and molecular methods and assessment of the differences in poly-microbial communities in relation to therapy failure, length of insertion of catheter, its type and other factors including predominating microbial species.

• MeSH
• Antibiotic Prophylaxis MeSH
• Biofilms MeSH
• Denaturing Gradient Gel Electrophoresis MeSH
• Catheter-Related Infections MeSH
• Urinary Catheters microbiology   MeSH
• Stents microbiology   MeSH

• Conspectus
• Mikrobiologie
• NML Fields
• mikrobiologie, lékařská mikrobiologie
• infekční lékařství
• urologie
• NML Publication type
• závěrečné zprávy o řešení grantu IGA MZ ČR

• MeSH
• Electrophoresis, Agar Gel methods   utilization   MeSH
• Mutation genetics   MeSH
• Publication type
• Congress MeSH

Complex samples are a challenge for sequencing-based broad-range diagnostics. We analysed 19 urinary catheter, ureteral Double-J catheter, and urine samples using 3 methodological approaches. Out of the total 84 operational taxonomic units, 37, 61, and 88% were identified by culture, PCR-DGGE-SS (PCR denaturing gradient gel electrophoresis followed by Sanger sequencing), and PCR-DGGE-RM (PCR- DGGE combined with software chromatogram separation by RipSeq Mixed tool), respectively. The latter approach was shown to be an efficient tool to complement culture in complex sample assessment.

• MeSH
• Bacteria classification   genetics   isolation & purification   MeSH
• Denaturing Gradient Gel Electrophoresis methods   MeSH
• Molecular Diagnostic Techniques methods   MeSH
• Diagnostic Techniques, Urological MeSH
• DNA, Bacterial MeSH
• Humans MeSH
• Urine chemistry   microbiology   MeSH
• Urinary Catheters microbiology   MeSH
• Polymerase Chain Reaction methods   MeSH
• Software * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Cílem je mutační analýza genů ATM a p53 u pacientek s karcinomem prsu z rizikových rodin; v neselektovaných rodinách budou studována mutace genu BRCA1 5382insC specifická pro naši populaci. Používané techniky zahrnují: extrakci genetického materiálu, amplifikací genových fragmentů pomocí PCR a RT-PCR, prescreening PCR produktů pomocí PTT nebo DGGE, charakterizaci mutací sekvenováním, analýzy LOH.; The aim of study is mutation analysis of ATM and p53 genes in breast cancer patients from risk families. The recurrent mutation in BRCA1 gene, 5382insC, will be detected in patients not selected for a family history of cancer. Methods used in this studyinvolte: DNA and RNA izolation, PCR and RT-PCR amplification, PTT and DGGE analyses, direct suquencing, LOH analysis.

• MeSH
• Anticipation, Genetic MeSH
• Genetic Predisposition to Disease genetics   MeSH
• Genes, p53 MeSH
• Breast Neoplasms genetics   prevention & control   MeSH
• Polymerase Chain Reaction MeSH
• BRCA1 Protein MeSH
• BRCA2 Protein MeSH
• Telangiectasis MeSH
• Loss of Heterozygosity MeSH

• Conspectus
• Biochemie. Molekulární biologie. Biofyzika
• NML Fields
• onkologie
• gynekologie a porodnictví
• histologie
• biologie
• genetika, lékařská genetika
• NML Publication type
• závěrečné zprávy o řešení grantu IGA MZ ČR

Objasnění genetických příčin FAP, AFAP a mnohočetných adenomů ve vybraném souboru rodin bez prokázané mutace v genu APC. Analýza skrytých zárodečných mutací v genu APC a zárodečných mutací v genu MYH. Využití metod kvantitativní PCR v reálném čase (RQ-PCR), analýza jednonukleotidových polymorfismů, PCR amplifikace, screeningové metody (HA, DGGE, SSCP), sekvenační analýza aberantního fragmentu. Korelace genotyp - fenotyp, presymptomatická DNA diagnostika, frekvence a mutační spektrum v ČR, vztah genu MYHa kolorektální polypózy.; Genetic causes of FAP, AFAP and multiple adenomas in the selected set of families with no demonstrable germline APC mutation. Analysis of invisible germline APC mutations and of germline MYH mutations. Mutation analysis using real-time quantitative PCR,analysis of single nucleotide polymorphisms, PCR amplification, screening methods (HA, DGGE, SSCP), sequencing. Genotype-phenotype correlation, presymptomatic DNA diagnosis, frequence and type of germline mutations, MYH gene and colorectal polyposis.

• MeSH
• Adenomatous Polyps genetics   MeSH
• Diagnosis, Differential MeSH
• Adenomatous Polyposis Coli genetics   MeSH
• Phenotype MeSH
• Genotype MeSH
• DNA Mutational Analysis MeSH
• Polymerase Chain Reaction methods   utilization   MeSH
• Germ-Line Mutation MeSH

• Conspectus
• Biochemie. Molekulární biologie. Biofyzika
• NML Fields
• onkologie
• hematologie a transfuzní lékařství
• biologie
• genetika, lékařská genetika
• gastroenterologie
• NML Publication type
• závěrečné zprávy o řešení grantu IGA MZ ČR

Denaturant gradient gel electrophoresis (DGGE) enables insight into the diversity of the studied microbial communities on the basis of separation of PCR amplification products according to their nucleotide sequence composition. However, the success of the method is accompanied by the inherent appearance of various sequence artifacts that bias the impression of community structure by generating additional bands representing no virtual microbes. PCR-DGGE artifacts require optimization of the method when aiming at the phylogenetic identification of the selected DGGE bands. The aim of our study was to develop a procedure which will increase the reliability of the identification. Samples of rumen fluid were used for the optimization since they contain a complex microbial community that supports the generation of artifactual bands. An optimized procedure following band excision and elution of microbial DNA is proposed including nuclease treatment, selection of DNA polymerase with proofreading activity, and cloning prior to sequencing and identification analysis.

• MeSH
• Rumen microbiology   MeSH
• Bacteria classification   genetics   isolation & purification   MeSH
• Denaturing Gradient Gel Electrophoresis methods   MeSH
• DNA, Bacterial genetics   MeSH
• Phylogeny MeSH
• Bacterial Typing Techniques methods   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH

Mutační analýza duplikované oblasti genu PKD1 ve vybraném souboru rodin s ADPKD pomocí LR-PCR a screeningových metod s následnou sekvenační analýzou fragmentů s pozitivním nálezem. Studium vztahů mezi genotypem a klinickými projevy onemocnění.; Analysis of mutations in duplicated region of the PKD1 gene in selected families with ADPKD by LR-PCR , screening methods (SSCP,HA,DGGE) and following sequencing of positive fragments.

• MeSH
• Phenotype MeSH
• Genetic Predisposition to Disease MeSH
• Genetic Testing MeSH
• Genotype MeSH
• Molecular Sequence Data MeSH
• DNA Mutational Analysis MeSH
• Polycystic Kidney, Autosomal Dominant MeSH

• Conspectus
• Biochemie. Molekulární biologie. Biofyzika
• NML Fields
• nefrologie
• biologie
• NML Publication type
• závěrečné zprávy o řešení grantu IGA MZ ČR