Several methods of molecular analysis of microbial diversity, including terminal restriction fragment length polymorphism (T-RFLP) analysis are based on measurement of the DNA fragment length. Significant variation between sequence-determined and measured length of restriction fragments (drift) has been observed, which can affect the efficiency of the identification of microorganisms in the analyzed communities. In the past, this variation has been attributed to varying fragment length and purine content. In this study, principal component analysis and multiple regression analysis were applied to find the contributions of those and several other fragment characteristics. We conclude that secondary structure melting point and G+C nucleotide content, besides the fragment length, contribute to the variation observed, whereas the contribution of purine content is less important. Incomplete denaturation of the sample at the start of electrophoretic separation of fragments has been excluded as a major cause of the variation observed. Our regression model explains the observed drift variation by approximately 56%, with standard deviation of the prediction equal to approximately 1.3 bp.

• MeSH
• analýza polymorfismu délky amplifikovaných restrikčních fragmentů metody   normy   MeSH
• DNA fungální chemie   genetika   MeSH
• houby chemie   genetika   izolace a purifikace   MeSH
• konformace nukleové kyseliny MeSH
• Phalaris mikrobiologie   MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• tranzitní teplota MeSH
• zastoupení bazí MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

Streptococcus suis is an important pathogen of pigs but is also transmissible to humans, with potentially fatal consequences. Among 29 serotypes currently recognized, some are clinically and epidemiologically more important than others. This is particularly true for serotypes 2 and 14, which have a large impact on pig production and also on human health. Conventional PCR-based serotyping cannot distinguish between serotype 1/2 and serotype 2 or between serotype 1 and serotype 14. Although serotype 1/2 and serotype 2 have a very similar cps locus, they differ in a single-nucleotide substitution at nucleotide position 483 of the cpsK gene. Similarly, serotypes 1 and 14 have a very similar cps locus but also differ in the same nucleotide substitution of the cpsK gene. Fortunately, this cpsK 483G→C/T substitution can be detected by BstNI restriction endonuclease. A PCR-restriction fragment length polymorphism (RFLP) detection method amplifying a fragment of the cpsK gene digested by BstNI restriction endonuclease was developed and tested in reference strains of these serotypes and also in field isolates.

• MeSH
• polymerázová řetězová reakce MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• prasata MeSH
• séroskupina MeSH
• sérotypizace MeSH
• Streptococcus suis * genetika   MeSH
• streptokokové infekce * diagnóza   veterinární   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

During a survey on grapevine yellows disease complex in vineyards of Lombardy region (northern Italy), phytoplasmas associated with Flavescence dorée disease were identified in symptomatic grapevines. Polymerase chain reaction and restriction fragment length polymorphism (RFLP) analyses of 16S rDNA revealed the prevalence of phytoplasmal subgroup 16SrV-D. Bioinformatic analyses of nucleotide sequences of rplV and rpsC genes, amplified from 16SrV-D phytoplasma infected grapevines and cloned, underscored the presence of five confirmed rpsC single nucleotide polymorphism (SNP) lineages, determined by different combination of SNPs at nucleotide positions 29, 365, 680, and 720 of rpsC gene. Virtual and actual RFLP analyses with the enzyme TaqI validated the presence of these SNPs. Co-infections by up to four distinct rpsC SNP lineages of 16SrV-D phytoplasma were found in grapevines. These results could open new perspectives for the study of the ecology and the epidemiology of Flavescence dorée.

• MeSH
• bakteriální proteiny genetika   MeSH
• DNA bakterií genetika   chemie   MeSH
• DNA fingerprinting MeSH
• financování organizované MeSH
• fylogeneze MeSH
• jednonukleotidový polymorfismus MeSH
• molekulární sekvence - údaje MeSH
• nemoci rostlin mikrobiologie   MeSH
• Phytoplasma genetika   izolace a purifikace   klasifikace   MeSH
• polymerázová řetězová reakce MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• ribozomální DNA genetika   MeSH
• ribozomální proteiny genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA MeSH
• sekvenční homologie MeSH
• shluková analýza MeSH
• Vitis mikrobiologie   MeSH
• Geografické názvy
• Itálie MeSH

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.

• MeSH
• Bifidobacterium genetika   izolace a purifikace   klasifikace   MeSH
• DNA bakterií genetika   chemie   MeSH
• druhová specificita MeSH
• financování organizované MeSH
• fylogeneze MeSH
• lidé MeSH
• mezerníky ribozomální DNA genetika   chemie   MeSH
• polymerázová řetězová reakce metody   MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• RNA ribozomální 16S genetika   chemie   MeSH
• RNA ribozomální 23S genetika   chemie   MeSH
• sekvence nukleotidů MeSH
• sekvenční seřazení MeSH
• Check Tag
• lidé MeSH

AIMS: Brewing yeasts are classified into two species-Saccharomyces pastorianus and Saccharomyces cerevisiae. Most of the brewing yeast strains are natural interspecies hybrids typically polyploids and their identification is thus often difficult giving heterogenous results according to the method used. We performed genetic characterization of a set of the brewing yeast strains coming from several yeast culture collections by combination of various DNA-based techniques. The aim of this study was to select a method for species-specific identification of yeast and discrimination of yeast strains according to their technological classification. METHODS AND RESULTS: A group of 40 yeast strains were characterized using PCR-RFLP analysis of ITS-5·8S, NTS, HIS4 and COX2 genes, multiplex PCR, RAPD-PCR of genomic DNA, mtDNA-RFLP and electrophoretic karyotyping. Reliable differentiation of yeast to the species level was achieved by PCR-RFLP of HIS4 gene. Numerical analysis of the obtained RAPD-fingerprints and karyotype revealed species-specific clustering corresponding with the technological classification of the strains. Taxonomic position and partial hybrid nature of strains were verified by multiplex PCR. Differentiation among species using the PCR-RFLP of ITS-5·8S and NTS region was shown to be unreliable. Karyotyping and RFLP of mitochondrial DNA evinced small inaccuracies in strain categorization. CONCLUSIONS: PCR-RFLP of HIS4 gene and RAPD-PCR of genomic DNA are reliable and suitable methods for fast identification of yeast strains. RAPD-PCR with primer 21 is a fast and reliable method applicable for differentiation of brewing yeasts with only 35% similarity of fingerprint profile between the two main technological groups (ale and lager) of brewing strains. SIGNIFICANCE AND IMPACT OF THE STUDY: It was proved that PCR-RFLP method of HIS4 gene enables precise discrimination among three technologically important Saccharomyces species. Differentiation of brewing yeast to the strain level can be achieved using the RAPD-PCR technique.

• MeSH
• DNA fungální genetika   MeSH
• druhová specificita MeSH
• mitochondriální DNA genetika   MeSH
• pivo analýza   mikrobiologie   MeSH
• polymerázová řetězová reakce metody   MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• Saccharomyces genetika   izolace a purifikace   metabolismus   MeSH
• technika náhodné amplifikace polymorfní DNA metody   MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

• MeSH
• alkaloidy biosyntéza   MeSH
• Claviceps genetika   izolace a purifikace   metabolismus   MeSH
• DNA primery genetika   MeSH
• fungální RNA genetika   MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• sekvence nukleotidů MeSH
• technika náhodné amplifikace polymorfní DNA MeSH

In Europe the Borrelia burgdorferi sensu lato complex is represented by five distinct genospecies: Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, and Borrelia lusitaniae. These taxonomic entities are known to differ in their specific associations with vertebrate hosts and to provoke distinct clinical manifestations in human patients. However, exceptions to these rules have often been observed, indicating that strains belonging to a single genospecies may be more heterogeneous than expected. It is, therefore, important to develop alternative identification tools which are able to distinguish Borrelia strains not only at the specific level but also at the intraspecific level. DNA from a sample of 370 Ixodes ricinus ticks collected in the Czech Republic was analyzed by PCR for the presence of a approximately 230-bp fragment of the rrfA-rrlB intergenic spacer of Borrelia spp. A total of 20.5% of the ticks were found to be positive. The infecting genospecies were identified by analyzing the amplified products by the restriction fragment length polymorphism (RFLP) method with restriction enzyme MseI and by single-strand conformation polymorphism (SSCP) analysis. The two methods were compared, and PCR-SSCP analysis appeared to be a valuable tool for rapid identification of spirochetes at the intraspecific level, particularly when large samples are examined. Furthermore, by using PCR-SSCP analysis we identified a previously unknown Borrelia genotype, genotype I-77, which would have gone unnoticed if RFLP analysis alone had been used.

• MeSH
• Borrelia burgdorferi komplex klasifikace   genetika   MeSH
• DNA bakterií genetika   izolace a purifikace   MeSH
• fylogeneze MeSH
• genetická variace * MeSH
• genotyp MeSH
• klíště mikrobiologie   MeSH
• lidé MeSH
• mezerníky ribozomální DNA genetika   MeSH
• molekulární sekvence - údaje MeSH
• polymerázová řetězová reakce metody   MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• polymorfismus konformace jednovláknové DNA * MeSH
• sekvenční analýza DNA MeSH
• techniky typizace bakterií MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

OBJECTIVES: Asians (including Chinese, Japanese and Koreans), who generally have a relatively smaller body size and a lower mean body mass index (BMI), have a relatively higher risk of developing android-type obesity than westerners. Substitution of alanine for threonine (Ala54Thr) on the FABP2 gene (rs 1799883) is related to insulin resistance and obesity. However, few studies have examined this substitution in Koreans, and the number of Korean subjects in those studies is limited. For this reason, we investigated the differences between the FABP2 Ala54Thr polymorphism and obesity, hemodynamic variables, blood lipid profile results, and insulin resistance among middle-aged Korean women with abdominal obesity. METHODS: We studied 243 middle-aged community-dwelling Korean women with abdominal obesity from Gyeonggi Province, Republic of Korea, who had no history of taking chronic medications. We examined each subject (n = 243) for the presence of FABP2 Ala54Thr polymorphism using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Subjects were also examined for obesity hemodynamic variables (n = 243), lipid profiles (n = 142), and insulin resistance (n = 142). RESULTS: Of the 243 subjects, 117 had AA ("normal") homozygotic genotype, 100 had AT heterozygotic genotype, and 26 had TT homozygotic genotype for the FABP2 Ala54Thr polymorphism. The AT heterozygotic individuals had a significantly higher mean waist-to-hip ratio, abdominal fat area, and visceral fat area than individuals with other genotypes. TT homozygotic individuals had higher mean triglyceride and fasting glucose levels than individuals with other genotypes. CONCLUSIONS: The results of this study show that the FABP2 Ala54Thr polymorphism was associated with central obesity and obesity-related metabolic syndrome among middle-aged Korean women.

• MeSH
• abdominální obezita genetika   patofyziologie   MeSH
• analýza polymorfismu délky amplifikovaných restrikčních fragmentů MeSH
• genotyp MeSH
• lidé středního věku MeSH
• lidé MeSH
• metabolický syndrom genetika   patofyziologie   MeSH
• polymerázová řetězová reakce metody   MeSH
• polymorfismus genetický MeSH
• poměr pasu a boků MeSH
• proteiny vázající mastné kyseliny genetika   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Korejská republika MeSH

Impulsivní a hyperaktivní chování souvisí s dysfunkcí centrální serotoninové aktivity a výsledky nedávných studií uvádějí možný vztah mezi polymorfismy genu pro serotoninový přenašeč (SLC6A4) a hyperkinetickou poruchou/ADHD. Soubor: Do souboru bylo zařazeno 90 chlapců s hyperkinetickou poruchou v průměrném věku 9,97 roku (SD = 1,66) a kontrolní skupinu tvořilo 82 chlapců ze základních škol. Metodika: Diagnóza byla stanovena dle kritérií ICD 10 a pomocí škál Connersové (Dotazníku pro rodiče CPQ Children Parent Questionnaire a Dotazníku pro učitele CTQ - Conners Teacher Rating Scale). K hodnocení pozornosti byl použit Test diskriminace tvarů, pro impulsivitu test TENAZO a počítačová sestava Neurobehavioral evaluation systém (NES 2). Detekce délkového polymorfismu VNTR genu SLC6A4 byla provedena pomocí fragmentační analýzy na přístroji Genetic Analyzer 3130. Výsledky byly vyhodnoceny v softwaru GeneMapper (Applied Biosystems). Výsledky: Kratší alela S a genotyp SS se signifikantně častěji (P < 0,01) vyskytovaly u chlapců s hyperkinetickou poruchou ve srovnání s kontrolní skupinou (Risk Ratio = 1,2673, 95% CI of RR = 1,0684 to 1,5032, Odds Ratio = 1, 843; 95% CI of OR =1,1863 to 2,8631). Závěr: Lze předpokládat, že dysregulace 5-HT systému může ovlivňovat vývoj emoční a behaviorální regulace u dětí s hyperkinetickou poruchou a více odráží symptomatologii pro hyperkinetické poruchy dle kritérií MKN-10 než užší spektrum symptomů pro ADHD dle DSM-IV. V souladu s údaji v literatuře výskyt alely S a genotypu SS hypoteticky může disponovat tyto jedince k výskytu vysoce frekventních komorbidních úzkostných poruch.

Impulsive and hyperactive behavior is related to a central serotonin dysfunction and the results of recent studies indicate the possible relationship between polymorphisms of the serotonin transporter gene (SLC6A4) and hyperkinetic disorder/ADHD. Sample: The sample included 90 boys with ADHD and the control group consisted of 82 boys. Method: The diagnosis was based on the ICD-10 criteria and the Conners' Parent Rating Scales. Attention was evaluated using the Shape Discrimination Test; TE-NA-ZO (Familiar Figures Finding Test) was used for impulsivity measures and a computerized set of Neurobehavioral Evaluation System (NES-2) was used for evaluation of both. The detection of the VNTR length polymorphism of the SLC6A4 gene was done using the fragment analysis on capillary sequencing instrument. The GeneMapper software was used for results analysis. Results: The short S-allele and the SS-genotype was significantly more frequently (P < 0.01) found in boys with hyperkinetic disorder compared to the control group. The results show a relationship between hyperkinetic disorder and the short alleles in contrast to studies published so far. Conclusion: It may be hypothesized that dysregulation in the 5-HT system could mainly influence the development of emotional and behavioral regulation in children with hyperkinetic disorder and reflects the symptomatology of hyperkinetic disorders as defined in ICD-10 rather than the narrower spectrum of symptoms of ADHD defined in DSM-IV. In compliance with previously published data the presence of the S-allele and the S/S-genotype may hypothetically make these individuals prone to a high prevalence of co-morbid anxiety disorders.

• Klíčová slova
• děti, anxieta, serotoninový transportér, SLC6A4 gen,
• MeSH
• analýza polymorfismu délky amplifikovaných restrikčních fragmentů MeSH
• dítě MeSH
• genetická predispozice k nemoci genetika   MeSH
• genotyp * MeSH
• hyperkinetická porucha * epidemiologie   etiologie   genetika   MeSH
• lidé MeSH
• polymorfismus délky restrikčních fragmentů * genetika   MeSH
• poruchy se sníženou pozorností a vyrušováním * diagnóza   genetika   MeSH
• receptory serotoninové * genetika   MeSH
• úzkostné poruchy * genetika   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• práce podpořená grantem MeSH

Projekt předpokládá vyšetření desítek až set kmenů a izolátů poddruhu T. pallidum ssp. pallidum v hypervariabilních oblastech genomu. Rozdíly v těchto sekvencích budou použity pro identifikaci individuálních kmenů tohoto poddruhu. Rozdíly v genomech kmenů a izolátů poddruhu T. pallidum ssp. pallidum budou zjišťovány po PCR amplifikaci sekvenací PCR produktů. Bude navržen zjednodušený protokol na identifikaci nejčastějších kmenů na základě PCR amplifikace a RFLP.; The main aim of this project is to collect over one hundred strains and clinical isolates of T. pallidum ssp. pallidum, the causative agent of syphilis. Variable parts of the genomes will be amplified and sequenced. Molecular identification of individualTreponema pallidum ssp. pallidum strains and isolates is important for epidemiologic consequences and for clinical discrimination between reinfection and reactivation of the syphylitic process.

• MeSH
• genomika MeSH
• jednonukleotidový polymorfismus MeSH
• molekulární typizace MeSH
• polymerázová řetězová reakce MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• Treponema pallidum klasifikace   MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR