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Molecular Methods (molecular + methods)
Selected AbstractsNatural Killer Cell Protocols: Cellular and Molecular Methods.HEMATOLOGICAL ONCOLOGY, Issue 1 20012000., Kerry S. Campbell, Marco Colonna (Eds.). Humana Press Inc No abstract is available for this article. [source] DNA barcoding: a new module in New Zealand's plant biosecurity diagnostic toolboxEPPO BULLETIN, Issue 1 2010K. Armstrong Molecular methods for identification of high risk pests and pathogens have been employed for more than a decade to supplement standard diagnostic protocols. However, as the volume of traded goods continues to increase so does the breadth of taxa that diagnosticians need to deal with. Keeping pace by introducing more molecular tests that are typically species-group specific is not an efficient way to progress. Since 2005 classical DNA barcoding using cytochrome oxidase I sequence has been employed routinely in New Zealand for the highest risk insect species (fruit flies and lymantriid moths). Subsequently a broader range of pests have been considered. Case studies are presented here for three important lepidopteran pests, Lymantria mathura (pink gypsy moth), Conogethes punctiferalis (yellow peach moth) and Hyphantria cunea (fall web worm), as well as a trial to identify miscellaneous border interceptions. While the data support the effectiveness of DNA barcoding for border diagnostics, they also raise issues around cryptic species identification and potential species discovery that could impact on operational biosecurity systems. [source] PERSPECTIVE: GENE DIVERGENCE, POPULATION DIVERGENCE, AND THE VARIANCE IN COALESCENCE TIME IN PHYLOGEOGRAPHIC STUDIESEVOLUTION, Issue 6 2000ScottV. Abstract Molecular methods as applied to the biogeography of single species (phylogeography) or multiple codistributed species (comparative phylogeography) have been productively and extensively used to elucidate common historical features in the diversification of the Earth's biota. However, only recently have methods for estimating population divergence times or their confidence limits while taking into account the critical effects of genetic polymorphism in ancestral species become available, and earlier methods for doing so are underutilized. We review models that address the crucial distinction between the gene divergence, the parameter that is typically recovered in molecular phylogeographic studies, and the population divergence, which is in most cases the parameter of interest and will almost always postdate the gene divergence. Assuming that population sizes of ancestral species are distributed similarly to those of extant species, we show that phylogeographic studies in vertebrates suggest that divergence of alleles in ancestral species can comprise from less than 10% to over 50% of the total divergence between sister species, suggesting that the problem of ancestral polymorphism in dating population divergence can be substantial. The variance in the number of substitutions (among loci for a given species or among species for a given gene) resulting from the stochastic nature of DNA change is generally smaller than the variance due to substitutions along allelic lines whose coalescence times vary due to genetic drift in the ancestral population. Whereas the former variance can be reduced by further DNA sequencing at a single locus, the latter cannot. Contrary to phylogeographic intuition, dating population divergence times when allelic lines have achieved reciprocal monophyly is in some ways more challenging than when allelic lines have not achieved monophyly, because in the former case critical data on ancestral population size provided by residual ancestral polymorphism is lost. In the former case differences in coalescence time between species pairs can in principle be explained entirely by differences in ancestral population size without resorting to explanations involving differences in divergence time. Furthermore, the confidence limits on population divergence times are severely underestimated when those for number of substitutions per site in the DNA sequences examined are used as a proxy. This uncertainty highlights the importance of multilocus data in estimating population divergence times; multilocus data can in principle distinguish differences in coalescence time (T) resulting from differences in population divergence time and differences in T due to differences in ancestral population sizes and will reduce the confidence limits on the estimates. We analyze the contribution of ancestral population size (,) to T and the effect of uncertainty in , on estimates of population divergence (,) for single loci under reciprocal monophyly using a simple Bayesian extension of Takahata and Satta's and Yang's recent coalescent methods. The confidence limits on , decrease when the range over which ancestral population size , is assumed to be distributed decreases and when increases; they generally exclude zero when /(4Ne) > 1. We also apply a maximum-likelihood method to several single and multilocus data sets. With multilocus data, the criterion for excluding = 0 is roughly that l/(4Ne)> 1, where l is the number of loci. Our analyses corroborate recent suggestions that increasing the number of loci is critical to decreasing the uncertainty in estimates of population divergence time. [source] Diagnosis of Helicobacter pylori InfectionHELICOBACTER, Issue 2009Lurdes Monteiro Abstract The articles published this last year in the field of Helicobacter pylori diagnosis reported the development of in vivo histology, small improvements in some invasive methods (urease test, culture, and histology) and new kits for the stool antigen tests. They also contributed to increasing our knowledge, by further exploration into specific conditions for the urea breath test and into the significance of cagA antibodies. The role of serum markers of atrophy was also confirmed. Molecular methods are still being developed for direct genotyping, detection of H. pylori and its clarithromycin resistance, either by polymerase chain reaction or fluorescent in-situ hybridization. For the first time, there was a report on a possible interest of magnetic resonance spectroscopy. [source] Diagnosis of Helicobacter pyloriHELICOBACTER, Issue 2008Marta Granstrom Abstract The different invasive and noninvasive diagnostic tests for Helicobacter pylori have been applied mainly in emerging countries. Molecular methods have been developed, especially a test for detection of H. pylori and its clarithromycin resistance directly from stools. The long-term effects of eradication on histologic lesions have been studied in a meta-analysis and the prognostic value of post-treatment in gastric mucosa-associated lymphoid tissue lymphoma has been assessed. An operating link for gastritis assessment (the OLGA staging) has also been published. Attempts to simplify the urea breath test protocol have been made, and new stool antigen tests have been proposed and compared to those previously available. [source] Molecular epidemiological study of HEV-B enteroviruses involved in the increase in meningitis cases occurred in Spain during 2006JOURNAL OF MEDICAL VIROLOGY, Issue 6 2008María Cabrerizo Abstract Human enteroviruses are one of the main etiological agents of aseptic meningitis and other central nervous system infections, particularly the serotypes included in the enterovirus B species. Molecular methods have proved useful to identify serotypes in clinical samples, facilitating the epidemiological study of these viruses. In the spring of 2006, there was a significant increase in meningitis cases caused by enteroviruses in Spain. In the present study, 138 enteroviruses directly detected in clinical samples of patients with aseptic meningitis (n,=,116) and other neurological pathologies (n,=,22) received by the National Center for Microbiology during the year, were genotyped by amplification and sequencing part of the VP1 region and phylogenetic analysis. Echovirus 30 was the most frequent serotype, followed in decreasing order by echovirus 6, 9, 13, 18, enterovirus 75, coxsackievirus A9, echovirus 11, 14, 29, 4, and coxsackievirus B4 and B5. Phylogenetic analysis with all Spanish echovirus 30 strains detected in 2006 and other reported echovirus 30 sequences, demonstrated that Spanish strains formed a new lineage, different from others previously described. In conclusion, echovirus 30 is the most commonly reported enterovirus serotype associated with aseptic meningitis in Spain. Direct molecular typing of clinical samples also allows rapid identification of the serotypes involved in an epidemic alert and phylogenetic analysis in the 3,-VP1 region is useful to study viral epidemiology. J. Med. Virol. 80:1018,1024, 2008. © 2008 Wiley-Liss, Inc. [source] Molecular detection and characterization of human enteroviruses directly from clinical samples using RT-PCR and DNA sequencingJOURNAL OF MEDICAL VIROLOGY, Issue 2 2006Miren Iturriza-Gómara Abstract Enteroviruses are common human pathogens associated with a wide spectrum of symptoms ranging from asymptomatic infection to acute flaccid paralysis and neonatal multi-organ failure. Molecular methods that provide rapid diagnosis and increased sensitivity have been developed for the diagnosis of enterovirus infection using oligonucleotide primers complementary to conserved sequences located in the 5, untranslated region (UTR), but data generated from these regions are not sufficiently discriminatory for typing due to the lack of correlation between their nucleic acid sequence and serotype specificity. Sequences derived from the gene encoding the capsid VP1 correlate with serotype, and therefore provide the opportunity for the development of molecular typing methods consistent with present serogical methods. In this study, oligonucleotide primers that amplify a region of the 5,UTR to detect enterovirus RNA, and the region encoding the enterovirus VP1 N-terminus to characterize virus strains were used in nested and semi-nested RT-PCRs, respectively. The ability of the VP1 RT-PCR to amplify diverse viruses within genotypes and genogroups was confirmed by the correct identification of both prototype strains, and strains circulating currently of the same genotypes. The mole-cular methods proved their utility through the detection of enteroviruses that failed to grow in cell culture, their subsequent characterization and the characterization of strains that failed to serotype in neutralization assays. Molecular methods increased significantly the sensitivity of detection (P,<,0.001) and of characterization (P,<,0.01) of enteroviruses when compared to classical methods. J. Med. Virol. 78:243,253, 2006. © 2005 Wiley-Liss, Inc. [source] Molecular methods for arthropod bloodmeal identification and applications to ecological and vector-borne disease studiesMOLECULAR ECOLOGY RESOURCES, Issue 1 2009REBEKAH J. KENT Abstract DNA-based methods have greatly enhanced the sensitivity and specificity of hematophagous arthropod bloodmeal identification. A variety of methods have been applied to study the blood-feeding behaviour of mosquitoes, ticks, black flies and other blood-feeding arthropods as it relates to host,parasite interactions and pathogen transmission. Overviews of the molecular techniques used for bloodmeal identification, their advantages, disadvantages and applications are presented for DNA sequencing, group-specific polymerase chain reaction primers, restriction fragment length polymorphism, real-time polymerase chain reaction, heteroduplex analysis, reverse line-blot hybridization and DNA profiling. Technical challenges to bloodmeal identification including digestion and analysis of mixed bloodmeals are discussed. Analysis of bloodmeal identification results remains a challenge to the field, particularly with regard to incorporation of vertebrate census and ecology data. Future research directions for molecular analysis of arthropod bloodmeals are proposed. [source] Detection of the Dinozoans Pfiesteria piscicida and P. shumwayae: A Review of Detection Methods and Geographic Distribution,THE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2 2005PARKE A. RUBLEE Abstract. Molecular methods, including conventional PCR, real-time PCR, denaturing gradient gel electrophoresis, fluorescent fragment detection PCR, and fluorescent in situ hybridization, have all been developed for use in identifying and studying the distribution of the toxic dinoflagellates Pfiesteria piscicida and P. shumwayae. Application of the methods has demonstrated a worldwide distribution of both species and provided insight into their environmental tolerance range and temporal changes in distribution. Genetic variability among geographic locations generally appears low in rDNA genes, and detection of the organisms in ballast water is consistent with rapid dispersal or high gene flow among populations, but additional sequence data are needed to verify this hypothesis. The rapid development and application of these tools serves as a model for study of other microbial taxa and provides a basis for future development of tools that can simultaneously detect multiple targets. [source] Comparison of methods of DNA extraction for real-time PCR in a model of pleural tuberculosisAPMIS, Issue 1 2010ANA SANTOS Santos A, Cremades R, Rodríguez JC, García-Pachón E, Ruiz M, Royo G. Comparison of methods of DNA extraction for real-time PCR in a model of pleural tuberculosis. APMIS 2010; 118: 60,5. Molecular methods have been reported to have different sensitivities in the diagnosis of pleural tuberculosis and this may in part be caused by the use of different methods of DNA extraction. Our study compares nine DNA extraction systems in an experimental model of pleural tuberculosis. An inoculum of Mycobacterium tuberculosis was added to 23 pleural liquid samples with different characteristics. DNA was subsequently extracted using nine different methods (seven manual and two automatic) for analysis with real-time PCR. Only two methods were able to detect the presence of M. tuberculosis DNA in all the samples: extraction using columns (Qiagen) and automated extraction with the TNAI system (Roche). The automatic method is more expensive, but requires less time. Almost all the false negatives were because of the difficulty involved in extracting M. tuberculosis DNA, as in general, all the methods studied are capable of eliminating inhibitory substances that block the amplification reaction. The method of M. tuberculosis DNA extraction used affects the results of the diagnosis of pleural tuberculosis by molecular methods. DNA extraction systems that have been shown to be effective in pleural liquid should be used. [source] Speciation and susceptibility of Nocardia isolated from ocular infectionsCLINICAL MICROBIOLOGY AND INFECTION, Issue 8 2010A.K. Reddy Clin Microbiol Infect 2010; 16: 1168,1171 Abstract Twenty Nocardia spp. isolated from ocular infections were identified by 16S rRNA gene sequencing and susceptibility was determined using the E-test (AB Biodisk, Sweden). Species distribution among the 20 isolates was as follows: Nocardia levis (n = 7), Nocardia farcinica (n = 3), Nocardia abscessus (n = 2), Nocardia brasiliensis (n = 2), Nocardia amamiensis (n = 2), Nocardia puris (n = 1), Nocardia beijingensis (n = 1), Nocardia otitidiscaviarum (n = 1) and Nocardia thailandica (n = 1). All isolates were sensitive to amikacin. Eighteen (90%) isolates were sensitive to tobramycin, 11 (55%) to ciprofloxacin and gatifloxacin, and seven (35%) to azithromycin and clarithromycin. Molecular methods are useful for the identification and for the detection of Nocardia species that have not so far been reported in human infections. [source] College of American pathologists practical guide to gynecologic cytopathology: Morphology, management, and molecular methodsDIAGNOSTIC CYTOPATHOLOGY, Issue 9 2010Martha Bishop Pitman M.D. No abstract is available for this article. [source] Life as an endodontic pathogenENDODONTIC TOPICS, Issue 1 2003Ecological differences between the untreated, root-filled root canals This review describes the type of microbial flora in the untreated root canal and the root-filled canal with persistent infection. Recent contributions of molecular methods of microbial identification are outlined along with a discussion of advantages and limitations of these methods. Ecological and environmental factors are the prime reasons for differences in the microbial flora in these distinct habitats. Shared phenotypic traits and an ability to respond to the modified environment select for the species that establish a persistent root canal infection. [source] Development of Live Cell Chips to Monitor Cell Differentiation ProcessesENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 1 2008C. Maercker Abstract A big demand exists for high-throughput functional in vitro assays which can measure cellular phenotypes by molecular methods and therefore improve the resources of primary cells for cell therapy, tissue engineering and high-content screenings in drug development. This approach focuses on cellular adhesion which is an important differentiation process during homing of stem cells. Moreover, it is a promising method especially for adherent cells which are not accessible by classical cell sorting methods. The chip design includes a housing with electrodes to measure electric field densities and impedance, respectively. Moreover, specific coatings of the wells permit a perfect growth of the selected cell types. In parallel, protein biomarkers can be followed by light microscopy. So far, experiments have been started to discriminate between different cell densities and cell types. In addition, after stimulating human cardiac fibroblasts and human umbilical vein endothelial cells, concentrations of proteins involved in adhesion had been increased, and proteins were translocated within the cells. In ongoing experiments, different human cell lines and fibroblastoid mesenchymal stem cells isolated from fat tissue, umbilical cord, or bone marrow are tested in the chip. To optimize the adhesion conditions, the surfaces within the vials of the chip were specifically activated. Microscopy was adjusted to be able to measure cellular morphology in parallel. This concept allows to identify the behavior of mesenchymal stem cells, which cannot be described so far by standard biomarkers. In addition, simulation of the homing process of the cells within its stem cell niche in an in vitro assay is a promising setup for large-scale gain-of-function or loss-of-function screenings in functional genomics as well as for generating precursor cells relevant for the therapy of various diseases. [source] Microbial communities in a porphyry copper tailings impoundment and their impact on the geochemical dynamics of the mine wasteENVIRONMENTAL MICROBIOLOGY, Issue 2 2007Nouhou Diaby Summary The distribution and diversity of acidophilic bacteria of a tailings impoundment at the La Andina copper mine, Chile, was examined. The tailings have low sulfide (1.7% pyrite equivalent) and carbonate (1.4% calcite equivalent) contents and are stratified into three distinct zones: a surface (0-70-80 cm) ,oxidation zone' characterized by low-pH (2.5,4), a ,neutralization zone' (70,80 to 300,400 cm) and an unaltered ,primary zone' below 400 cm. A combined cultivation-dependent and biomolecular approach (terminal restriction enzyme fragment length polymorphism and 16S rRNA clone library analysis) was used to characterize the indigenous prokaryotic communities in the mine tailings. Total cell counts showed that the microbial biomass was greatest in the top 125 cm of the tailings. The largest numbers of bacteria (109 g,1 dry weight of tailings) were found at the oxidation front (the junction between the oxidation and neutralization zones), where sulfide minerals and oxygen were both present. The dominant iron-/sulfur-oxidizing bacteria identified at the oxidation front included bacteria of the genus Leptospirillum (detected by molecular methods), and Gram-positive iron-oxidizing acidophiles related to Sulfobacillus (identified both by molecular and cultivation methods). Acidithiobacillus ferrooxidans was also detected, albeit in relatively small numbers. Heterotrophic acidophiles related to Acidobacterium capsulatum were found by molecular methods, while another Acidobacterium -like bacterium and an Acidiphilium sp. were isolated from oxidation zone samples. A conceptual model was developed, based on microbiological and geochemical data derived from the tailings, to account for the biogeochemical evolution of the Piuquenes tailings impoundment. [source] Molecular investigation on strain genetic relatedness and population structure of Beauveria bassianaENVIRONMENTAL MICROBIOLOGY, Issue 10 2003Chengshu Wang Summary Triplicate molecular methods, i.e. polymerase chain reaction-restriction fragment length polymorphism of the pr1 gene, microsatellite markers and 28S rDNA haplotyping by detecting the presence or absence of group I introns, were used for population study of the entomopathogenic fungus, Beauveria bassiana. The findings showed that the average genetic diversity index of geographical populations was significantly smaller than that of populations derived from insect host orders, indicating that the genetic relatedness of B. bassiana strains was highly associated with geographical locality rather than insect host species. The reproductive style of all the B. bassiana populations was found to be non-clonal. Population structure analysis revealed that the average divergent coefficient among populations of B. bassiana was far below 1 (0.1112), which indicated that there was no significant genetic differentiation between populations, and that the overall genetic diversity mainly resulted from the genetic variations within geographical populations. Statistically, genetic distances between populations were positively correlated with geographical distances, suggesting that geographical separation poses an obstacle to the possibility and frequency of genetic exchanges between populations. On the other hand, gene flow was indirectly established to occur between B. bassiana populations. [source] Analysis of Genetically Complex EpilepsiesEPILEPSIA, Issue 2005Ruth Ottman Summary:, During the last decade, great progress has been made in the discovery of genes that influence risk for epilepsy. However, these gene discoveries have been in epilepsies with Mendelian modes of inheritance, which comprise only a tiny fraction of all epilepsy. Most people with epilepsy have no affected relatives, suggesting that the great majority of all epilepsies are genetically complex: multiple genes contribute to their etiology, none of which has a major effect on disease risk. Gene discovery in the genetically complex epilepsies is a formidable task. It is unclear which epilepsy phenotypes are most advantageous to study, and chromosomal localization and mutation detection are much more difficult than in Mendelian epilepsies. Association studies are very promising for the identification of complex epilepsy genes, but we are still in the earliest stages of their application in the epilepsies. Future studies should employ very large sample sizes to ensure adequate statistical power, clinical phenotyping methods of the highest quality, designs and analytic techniques that control for population stratification, and state-of-the-art molecular methods. Collaborative studies are essential to achieve these goals. [source] A comparison of molecular methods for the routine detection of viroids,EPPO BULLETIN, Issue 3-4 2000R. A. Mumford Viroids, such as Chrysanthemum stunt viroid (CSVd) and Potato spindle tuber viroid (PSTVd), are important plant pathogens. However, because of their unique biological properties, viroids have proved, in the past, difficult to diagnose. The use of molecular methods has now changed this and this paper reports the comparison of three such methods (dot-blot hybridization using DIG-labelled cRNA probes, reverse transcription-polymerase chain reaction (RT-PCR) and TaqMan), which have been developed for routine detection of CSVd. Sensitivity comparisons show that the TaqMan assay is more sensitive than either RT-PCR (100 times) and hybridization (1000 times). RT-PCR and TaqMan assays have also been developed to detect PSTVd. In addition to the development of sensitive detection methods, considerable emphasis has been placed on making these assays amenable to mass-scale detection through the use of internal controls and the development of a rapid, reliable probe capture extraction system. [source] A REAPPRAISAL OF BATEMAN'S CLASSIC STUDY OF INTRASEXUAL SELECTIONEVOLUTION, Issue 11 2007Brian F. Snyder Bateman's (1948) study showing greater variances in number of mates and reproductive success in male than female Drosophila melanogaster is a foundational paper in sexual selection. Here we show for the first time that his methods had flaws, including the elimination of genetic variance, sampling biases, miscalculations of fitness variances, statistical pseudo-replication, and selective presentation of data. We conclude that Bateman's results are unreliable, his conclusions are questionable, and his observed variances are similar to those expected under random mating. Despite our analysis, we do not intend this article as a criticism of Bateman; he accomplished his work without modern computational tools, and his approach was groundbreaking emphasizing the significance of fitness variance for sexual selection. However, this reanalysis has implications for what counts as evidence for sexual selection and we believe that our concerns should be of interest to contemporary students of sexual selection. We call for repetitions of Bateman's study using modern statistical and molecular methods. [source] Genomic and phenotypic heterogeneity of Acidithiobacillus spp. strains isolated from diverse habitats in ChinaFEMS MICROBIOLOGY ECOLOGY, Issue 2 2008Yong-Qing Ni Abstract The genetic variability among 32 Chinese Acidithiobacillus spp. environmental isolates and four reference strains representing three recognized species of the genus Acidithiobacillus was characterized by using a combination of molecular methods, namely restriction fragment length polymorphisms of PCR-amplified 16S rRNA genes and 16S,23S rRNA gene intergenic spacers, repetitive element PCR, arbitrarily primed PCR and 16S rRNA gene sequence analyses. 16S rRNA gene sequences revealed that all Acidithiobacillus spp. strains could be assigned to seven groups, three of which encompassed the Acidithiobacillus ferrooxidans strains from various parts of the world. A comparative analysis of the phylogenetic Group 1 and 2 was undertaken. Restriction fragment length polymorphism results allowed us to separate the 35 Acidithiobacillus strains into 15 different genotypes. An integrated phenotypic and genotypic analysis indicated that the distribution of A. ferrooxidans strains among the physiological groups were in agreement with their distribution among the genomic groups, and that no clear correlation was found between the genetic polymorphism of the Acidithiobacillus spp. strains and either the geographic location or type of habitats from which the strains were isolated. In addition, five unidentified sulfur-oxidizing isolates may represent one or two novel species of the genus Acidithiobacillus. The results showed that the Chinese Acidithiobacillus spp. isolates exhibited a high degree of genomic and phenotypic heterogeneity. [source] Distribution and ecophysiology of the nitrifying bacteria emphasizing cultured speciesFEMS MICROBIOLOGY ECOLOGY, Issue 1 2001Hans-Peter Koops Abstract Nitrification is an important factor in the global nitrogen cycle. Therefore, an increasing number of publications deal with in situ studies of natural bacterial populations participating in this process. However, some crucial points complicate suchlike investigations. At the time being, a total of 25 species of ammonia-oxidizers and eight species of nitrite-oxidizers are cultured but the existence of many more species has been indicated by molecular in situ investigations. With that, only a part of the existing nitrifiers has been defined via isolation and subsequent physiological and molecular characterization. Furthermore, the distribution patterns of the distinct species of nitrifiers depend on various environmental parameters. Hence the composition of nitrifying bacterial communities is complex and divers in heterogeneous habitats. In consequence of the above-mentioned problems, the representation of nitrifying community structures obtained from in situ investigations often has been incomplete and unbalanced in many respects. Polyphasic approaches, applying a combination of classical as well as molecular methods in parallel, could help to find the way for overcoming these problems in the future. Isolation and characterization of as many as possible new species seems to be one of the most important missing steps to advance at this way. [source] Methods for the isolation and identification of Listeria spp. and Listeria monocytogenes: a reviewFEMS MICROBIOLOGY REVIEWS, Issue 5 2005Uta Gasanov Abstract Listeria monocytogenes is an important food-borne pathogen and is widely tested for in food, environmental and clinical samples. Identification traditionally involved culture methods based on selective enrichment and plating followed by the characterization of Listeria spp. based on colony morphology, sugar fermentation and haemolytic properties. These methods are the gold standard; but they are lengthy and may not be suitable for testing of foods with short shelf lives. As a result more rapid tests were developed based on antibodies (ELISA) or molecular techniques (PCR or DNA hybridization). While these tests possess equal sensitivity, they are rapid and allow testing to be completed within 48 h. More recently, molecular methods were developed that target RNA rather than DNA, such as RT-PCR, real time PCR or nucleic acid based sequence amplification (NASBA). These tests not only provide a measure of cell viability but they can also be used for quantitative analysis. In addition, a variety of tests are available for sub-species characterization, which are particularly useful in epidemiological investigations. Early typing methods differentiated isolates based on phenotypic markers, such as multilocus enzyme electrophoresis, phage typing and serotyping. These phenotypic typing methods are being replaced by molecular tests, which reflect genetic relationships between isolates and are more accurate. These new methods are currently mainly used in research but their considerable potential for routine testing in the future cannot be overlooked. [source] The use of killer sensitivity patterns for biotyping yeast strains: the state of the art, potentialities and limitationsFEMS YEAST RESEARCH, Issue 6 2007Pietro Buzzini Abstract In recent years molecular techniques have been the most useful tools for the unequivocal identification of undetermined strains at the species level. In many instances, however, a further discrimination at the strain level (biotyping) is required, such as during epidemiological investigations, in which the distribution of pathogenic microorganisms is studied, and for patent protection purposes. Although molecular methods are routinely used also for yeast biotyping, several nonmolecular techniques have been proposed. One of these, the determination of the killer sensitivity pattern (KSP) towards a panel of selected killer toxins has proven to be a good auxiliary method. Despite the plethora of studies published, the potential and limitations of the determination of KSPs have never been critically evaluated. In this review the use of this nonmolecular technique as a biotyping tool is discussed and compared with some currently used DNA-based procedures. In addition, methodological, mechanistic and ecological implications are evaluated. [source] Microbial biodiversity in groundwater ecosystemsFRESHWATER BIOLOGY, Issue 4 2009C. GRIEBLER Summary 1. Groundwater ecosystems offer vast and complex habitats for diverse microbial communities. Here we review the current status of groundwater microbial biodiversity research with a focus on Bacteria and Archaea and on the prospects of modern techniques for enhancing our understanding of microbial biodiversity patterns and their relation to environmental conditions. 2. The enormous volume of the saturated terrestrial underground forms the largest habitat for microorganisms on earth. Up to 40% of prokaryotic biomass on earth is hidden within this terrestrial subsurface. Besides representing a globally important pool of carbon and nutrients in organisms, these communities harbour a degree of microbial diversity only marginally explored to date. 3. Although first observations of groundwater microbiota date back to Antonie van Leeuwenhoek in 1677, the systematic investigation of groundwater microbial biodiversity has gained momentum only within the last few decades. These investigations were initiated by an increasing awareness of the importance of aquifer microbiota for ecosystem services and functioning, including the provision of drinking water and the degradation of contaminants. 4. The development of sampling techniques suitable for microbiological investigations as well as the application of both cultivation-based and molecular methods has yielded substantial insights into microbial communities in contaminated aquifers, whereas knowledge of microbial biodiversity in pristine habitats is still poor at present. 5. Several novel phylogenetic lineages have been described from groundwater habitats, but to date no clearly ,endemic' subsurface microbial phyla have been identified. The future will show if the rather low diversity generally found in pristine oligotrophic aquifers is a fact or just a result of low abundances and insufficient resolution of today's methods. Refined approaches complemented by statistically rigorous applications of biodiversity estimates are urgently needed. 6. Factors identified to control microbial diversity in aquifers include spatial heterogeneity, temporal variability and disturbances such as pollution with chemical anthropogenic contaminants. Although first insights into the importance of individual biogeochemical processes may be obtained from surveys of microbial diversity within functional groups, direct links to groundwater ecosystem functioning have rarely been established so far. [source] Constraints on recovery: using molecular methods to study connectivity of aquatic biota in rivers and streamsFRESHWATER BIOLOGY, Issue 4 2007JANE M. HUGHESArticle first published online: 2 MAR 200 Summary 1. The ,Field of Dreams Hypothesis' states ,if we build it, they will come', referring to the assumption that if habitats are restored, species will recolonise them. However, the ability of a species to recolonise a restored site will depend not only on the appropriate habitat being present, but also on the ability to get there. This is likely to depend on both the species' dispersal behaviour and the position of a site in the landscape. 2. Animals with good potential for dispersal are more likely to be able to disperse to newly restored sites. Similarly, sites in lowland streams with limited altitudinal differences between sites may be easier to reach than upstream sites. This is because upstream sites are connected to one another via lowland streams that have different characteristics and therefore may be difficult for animals to traverse. 3. In this paper, genetic data from a range of freshwater species that have been analysed in my laboratory are used to assess the importance of life cycle and position in the landscape (i.e. upland versus lowland streams) on connectivity patterns (and thus recolonisation potential) among populations. 4. In general, contemporary dispersal across catchment boundaries is negligible, except for aquatic insects with an adult flight stage. Dispersal among streams within catchments appears to be more limited than was predicted from knowledge on life histories, except for fish in lowland rivers and streams. 5. As predicted, dispersal of fish, crustaceans and molluscs among streams within catchments is significantly greater in lowland rivers than in upland streams. 6. Overall, these analyses suggest that, with the exception of most insects, and fishes in lowland rivers, natural recolonisation of restored sites is only likely from sites within the same stream. If a species has disappeared from the whole stream, then restoration of habitat alone may not be sufficient for its re-establishment. [source] High Level of Antimicrobial Resistance in French Helicobacter pylori IsolatesHELICOBACTER, Issue 1 2010Josette Raymond Abstract Background: Helicobacter pylori is a human pathogen responsible for serious diseases including peptic ulcer disease and gastric cancer. The recommended triple therapy included clarithromycin but increasing resistance has undermined its effectiveness. It is therefore important to be aware of the local prevalence of antimicrobial resistance to adjust treatment strategy. Materials and Methods: Overall, 530 biopsies were collected between 2004 and 2007. The antimicrobial susceptibility of H. pylori was determined by E-test and molecular methods. Results: Among these, 138/530 (26%) strains were resistant to clarithromycin, 324/530 (61%) to metronidazole and 70/530 (13.2%) to ciprofloxacin. Whereas no resistance against amoxicillin and tetracycline was observed, only one strain was resistant to rifampicin. Compared to the patients never treated for H. pylori infection, the prevalence of resistance was significantly higher in patients previously treated (19.1% vs 68% for clarithromycin; 13.2% vs 53.3% for both clarithromycin and metronidazole). The trend analysis revealed an increase of primary resistance to ciprofloxacin between 2004 and 2005 (7.3%) vs 2006,2007 (14.1%) (p = .04) and the secondary resistance reached 22.7% in 2007. Interestingly, 27 biopsies (19.6%) contained a double population of clarithromycin-susceptible and -resistant strains. Conclusions: The reported high prevalence of clarithromycin and multiple resistances of H. pylori suggest that the empiric therapy with clarithromycin should be abandoned as no longer pretreatment susceptibility testing has assessed the susceptibility of the strain. As culture and antibiogram are not routinely performable in most clinical laboratories, the use of molecular test should be developed to allow a wide availability of pretreatment susceptibility testing. [source] Diagnosis of Helicobacter pyloriHELICOBACTER, Issue 2007Meltem Yalinay Cirak Abstract Although there are attempts to perform Helicobacter pylori diagnosis directly in vivo using magnification endoscopy, most articles on diagnosis this year concerned non-invasive tests and molecular methods. For urea breath tests, there are attempts to have a quicker and cheaper test and to evaluate its role in cases of premalignant lesions. For stool antigens tests, evaluation of kits using monoclonal antibodies was carried out. Molecular tests have been applied for typing and detection of resistant mutants. [source] Diagnosis of Helicobacter pylori InfectionHELICOBACTER, Issue 2006Katarzyna Dzier, anowska-Fangrat Abstract A growing interest in non-invasive tests for the detection of Helicobacter pylori has been observed recently, reflecting a large number of studies published this year. New tests have been validated, and the old ones have been used in different clinical situations or for different purposes. Stool antigen tests have been extensively evaluated in pre- and post-treatment settings both in adults and children, and the urea breath test has been studied as a predictor of bacterial load, severity of gastric inflammation, and response to eradication treatment. Several studies have also explored the usefulness of some serologic markers as indicators of the gastric mucosa status. With regard to invasive tests, molecular methods are being used more and more, but the breakthrough this year was the direct in vivo observation of H. pylori during endoscopy. [source] Diagnosis of Helicobacter pylori InfectionHELICOBACTER, Issue 2005Karen A. Krogfelt ABSTRACT A large number of studies on diagnostic tests have been published this year. New tests were proposed for the detection of Helicobacter pylori antigens in stools and new molecular methods (real-time polymerase chain reaction) to look for antimicrobial susceptibility. The other standard tests have been applied in different situations to improve the diagnosis of the infection. [source] Diagnosis of Helicobacter pylori InfectionHELICOBACTER, Issue 2004Athanasios Makristathis ABSTRACT While there are some attempts to improve culture of Helicobacter pylori, molecular methods have been the main focus of this interest. Their main application concerns the development of rapid tests also allowing the determination of bacterial resistance, i.e. real-time polymerase chain reaction (PCR) or fluorescence in situ hybridization (FISH), or to genotype the strains. Attempts to improve, simplify or explain the discrepancies of urea breath test results have been made and new generation of stool antigen test with monoclonal antibodies either using the standard ELISA format or rapid immunoenzymatic detection have confirmed their value. With regard to serology, studies have mainly focused on the distinction of infections with more pathogenic strains and the ability to diagnose atrophic gastritis with the Gastropanel. [source] | |||||||||||||||||||||||||||||||||||||