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PCR Technology (pcr + technology)
Selected AbstractsCFTR Rearrangements in Spanish Cystic Fibrosis Patients: First New Duplication (35kb) Characterised in the Mediterranean CountriesANNALS OF HUMAN GENETICS, Issue 5 2010Marķa D. Ramos Summary Developments in quantitative PCR technologies have greatly improved our ability to detect large genome rearrangements. In particular oligonucleotide-based array comparative genomic hybridisation has become a useful tool for appropriate and rapid detection of breakpoints. In this work, we have analysed 80 samples (42 unknown CF alleles) applying three quantitative technologies (MLPA, qPCR and array-CGH) to detect recurrent as well as novel large rearrangements in the Spanish CF population. Three deletions and one duplication have been identified in five alleles (12%). Interestingly, we provide the comprehensive characterisation of the first duplication in our CF cohort. The new CFTRdupProm-3 mutation spans 35.7 kb involving the 5,-end of the CFTR gene. Additionally, the RNA analysis has revealed a cryptic sequence with a premature termination codon leading to a disrupted protein. This duplication has been identified in five unrelated families from Spain, France and Italy with all patients showing the same associated haplotype, which is further evidence for its likely common Mediterranean origin. Overall, considering this and other previous studies, CFTR rearrangements account for 1.3% of the Spanish CF alleles. [source] Methods of detection of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis in periodontal microbiology, with special emphasis on advanced molecular techniques: a reviewJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 12 2004Mariano Sanz Abstract Background: Certain specific bacterial species from the subgingival biofilm have demonstrated aetiological relevance in the initiation and progression of periodontitis. Among all the bacteria studied, three have shown the highest association with destructive periodontal diseases: Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Tannerella forsythensis (Tf). Therefore, the relevance of having accurate microbiological diagnostic techniques for their identification and quantification is clearly justified. Aim: To evaluate critically all scientific information on the currently available microbial diagnostic techniques aimed for the identification and quantification of Aa, Pg and Tf. Summary: Bacterial culturing has been the reference diagnostic technique for many years and, in fact, most of our current knowledge on periodontal microbiology derives from cultural data. However, the advent of new microbial diagnostics, mostly based on immune and molecular technologies, has not only highlighted some of the shortcomings of cultural techniques but has also allowed their introduction as easy and available adjunct diagnostic tools to be used in clinical research and practice. These technologies, mostly polymerase chain reaction (PCR), represent a field of continuous development; however, we still lack the ideal diagnostic to study the subgingival microflora. Qualitative PCR is still hampered by the limited information provided. Quantitative PCR is still in development; however, the promising early results reported are still hampered by the high cost and the equipment necessary for the processing. Conclusion: Quantitative PCR technology may have a major role in the near future as an adjunctive diagnostic tool in both epidemiological and clinical studies in periodontology. However, culture techniques still hold some inherent capabilities, which makes this diagnostic tool the current reference standard in periodontal microbiology. [source] The use of biomagnetic separation to recover DNA suitable for PCR from Claviceps species ,LETTERS IN APPLIED MICROBIOLOGY, Issue 2 2000D.L. Scott Jr DNA analysis of agriculturally important fungi using polymerase chain reaction (PCR)-based methods is becoming routine in research and for diagnostic purposes. Rapid, small-scale DNA isolation methods that take advantage of the sensitivity, speed and automation potential of PCR technology are needed for timely analysis of important plant pathogens. DNA isolated from Claviceps africana (causal agent of ergot of sorghum) using several standard DNA extraction protocols was found to be unamplifiable using PCR. The standard methods apparently failed to separate DNA from substances inhibitory to the Taq polymerase enzyme. We obtained DNA amenable to PCR analysis using a novel method involving magnetic beads and high salt extraction buffer. The biomagnetic purification method allowed us to obtain reliable PCR amplification of the internal transcribed spacer (ITS) regions of rDNA of Claviceps africana, making genetic comparisons possible. [source] Nuclear DNA analyses in genetic studies of populations: practice, problems and prospectsMOLECULAR ECOLOGY, Issue 3 2003De-Xing Zhang Abstract Population-genetic studies have been remarkably productive and successful in the last decade following the invention of PCR technology and the introduction of mitochondrial and microsatellite DNA markers. While mitochondrial DNA has proven powerful for genealogical and evolutionary studies of animal populations, and microsatellite sequences are the most revealing DNA markers available so far for inferring population structure and dynamics, they both have important and unavoidable limitations. To obtain a fuller picture of the history and evolutionary potential of populations, genealogical data from nuclear loci are essential, and the inclusion of other nuclear markers, i.e. single copy nuclear polymorphic (scnp) sequences, is clearly needed. Four major uncertainties for nuclear DNA analyses of populations have been facing us, i.e. the availability of scnp markers for carrying out such analysis, technical laboratory hurdles for resolving haplotypes, difficulty in data analysis because of recombination, low divergence levels and intraspecific multifurcation evolution, and the utility of scnp markers for addressing population-genetic questions. In this review, we discuss the availability of highly polymorphic single copy DNA in the nuclear genome, describe patterns and rate of evolution of nuclear sequences, summarize past empirical and theoretical efforts to recover and analyse data from scnp markers, and examine the difficulties, challenges and opportunities faced in such studies. We show that although challenges still exist, the above-mentioned obstacles are now being removed. Recent advances in technology and increases in statistical power provide the prospect of nuclear DNA analyses becoming routine practice, allowing allele-discriminating characterization of scnp loci and microsatellite loci. This certainly will increase our ability to address more complex questions, and thereby the sophistication of genetic analyses of populations. [source] PCR as a specific, sensitive and simple method suitable for diagnosticsBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 4 2000M. Gonzalo Claros PCR technology is a widespread method that has not reached students laboratory in anything else than a typical amplification reaction. We describe a simple application of PCR in pathogen diagnostics that enables students to identify which ampicillin-resistant organism is present in a cell culture. This experiment has been performed for one year in two "Experimental Biochemistry and Molecular Biology" courses with Biological and Chemical undergraduates. Using specific primers from the Escherichia coli ,-lactamase gene, they have been able to selectively amplify a ,-lactamase DNA fragment in E. coli but not in Staphylococcus aureus and, using different annealing temperatures, test the reaction specificity. By solving the "Study Questions", students understood the specificity and sensitivity of the method, as well as the rationale that should be applied when a molecular weight pattern is used for calculating unknown DNA sizes. © 2000 IUBMB. Published by Elsevier Science Ltd. All rights reserved. [source] Endocarditis due to Tropheryma whipplei: rapid detection, limited genetic diversity, and long-term clinical outcome in a local experienceCLINICAL MICROBIOLOGY AND INFECTION, Issue 8 2010R. Escher Clin Microbiol Infect 2010; 16: 1213,1222 Abstract The characteristic features of Whipple's disease include abdominal pain, diarrhoea, wasting, and arthralgias, with the causative agent, Tropheryma whipplei, being detected mainly in intestinal biopsies. PCR technology has led to the identification of T. whipplei in specimens from various other locations, including the central nervous system and the heart. T. whipplei is now recognized as one of the causes of culture-negative endocarditis, and endocarditis can be the only manifestation of the infection with T. whipplei. Although it is considered a rare disease, the true incidence of endocarditis due to T. whipplei is not clearly established. With the increasing use of molecular methods, it is likely that T. whipplei will be more frequently identified. Questions also remain about the genetic variability of T. whipplei strains, optimal diagnostic procedures and therapeutic options. In the present study, we provide clinical data on four new patients with documented endocarditis due to T. whipplei in the context of the available published literature. There was no clinical involvement of the gastrointestinal tract. Genetic analysis of the T. whipplei strains with DNA isolated from the excised heart valves revealed little to no genetic variability. In a selected case, we describe acridine orange staining for early detection of the disease, prompting early adaptation of the antibiotic therapy. We provide long-term follow-up data on the patients. In our hands, an initial 2-week course of intravenous antibiotics followed by cotrimoxazole for at least 1 year was a suitable treatment option for T. whipplei endocarditis. 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