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Evaluation of DNA polymorphisms amplified by arbitrary primers (RAPD) as genetically associated elements to differentiate virulent and non-virulent Paracoccidioides brasiliensis isolates

Motta, Teresa R.; Moreira-Filho, Carlos A.; Mendes, Rinaldo P.; Souza, Lenice R.; Sugizak, Maria F.; Baueb, Sonia; Calich, Vera L.G.; Vaz, Celideia A.C.
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 151-157
Português
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46.23%
Randomly amplified polymorphic DNA (RAPD) analysis of 35 Paracoccidioides brasiliensis isolates was carried out to evaluate the correlation of RAPD profiles with the virulence degree or the type of the clinical manifestations of human paracoccidioidomycosis. The dendrogram presented two main groups sharing 64% genetic similarity. Group A included two isolates from patients with chronic paracoccidioidomycosis; group B comprised the following isolates showing 65% similarity: two non-virulent, six attenuated, five virulent, eight from patients with chronic paracoccidioidomycosis and two from patients with acute paracoccidioidomycosis. The virulent Pb18 isolate and six attenuated or non-virulent samples derived from it were genetically indistinguishable (100% of similarity). Thus, in our study, RAPD patterns could not discriminate among 35 P. brasiliensis isolates according to their differences either in the degree of virulence or in the type of the clinical manifestation of this fungal infection. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Polymerase Chain Reaction Screening for Fungemia and/or Invasive Fungal Infections in Patients with Hematologic Malignancies

Ribeiro, P; Costa, F; Monteiro, A; Caldas, J; Silva, M; Ferreira, G; Veiga, J; Sousa, MO; Viegas, MP; Santos, E; Gonçalves, AJ; Sousa, AB
Fonte: Springer-Verlag Publicador: Springer-Verlag
Tipo: Artigo de Revista Científica
Publicado em //2006 Português
Relevância na Pesquisa
46.19%
INTRODUCTION: Invasive fungal infections (IFIs) are a life-threatening complication in patients with hematologic malignancies, mainly in acute leukemia patients, following chemotherapy. IFI incidence is increasing, and associated mortality remains high due to unreliable diagnosis. Antifungal drugs are often limited by inadequate antimicrobial spectrum and side effects. Thus, the detection of circulating fungal DNA has been advocated as a rapid, more sensitive diagnostic tool. PATIENTS AND METHODS: Between June 01 and January 03, weekly blood samples (1,311) were screened from 193 patients undergoing intensive myelosuppressive or immunosuppressive therapy. IFI cases were classified according to European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Fungal DNA was extracted from whole blood and amplified using polymerase chain reaction (PCR) published primers that bind to the conserved regions of the fungal 18S rRNA gene sequence. In our study, two or more consecutive positive samples were always associated with fungal disease. RESULTS: PCR screening predicted the development of IFI to be 17 days (median). This test had a specificity of 91.1% and a sensitivity of 75%. IFI incidence was 7.8%. DISCUSSION: Therefore...

Polymerase Chain Reaction Screening for Fungemia and/or Invasive Fungal Infections in Patients with Hematologic Malignancies

Ribeiro, P; Costa, F; Monteiro, A; Caldas, J; Silva, M; Ferreira, G; Veiga, J; Sousa, MO; Viegas, MP; Santos, E; Gonçalves, AJ; Sousa, AB
Fonte: Springer-Verlag Publicador: Springer-Verlag
Tipo: Artigo de Revista Científica
Publicado em //2006 Português
Relevância na Pesquisa
46.19%
INTRODUCTION: Invasive fungal infections (IFIs) are a life-threatening complication in patients with hematologic malignancies, mainly in acute leukemia patients, following chemotherapy. IFI incidence is increasing, and associated mortality remains high due to unreliable diagnosis. Antifungal drugs are often limited by inadequate antimicrobial spectrum and side effects. Thus, the detection of circulating fungal DNA has been advocated as a rapid, more sensitive diagnostic tool. PATIENTS AND METHODS: Between June 01 and January 03, weekly blood samples (1,311) were screened from 193 patients undergoing intensive myelosuppressive or immunosuppressive therapy. IFI cases were classified according to European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. Fungal DNA was extracted from whole blood and amplified using polymerase chain reaction (PCR) published primers that bind to the conserved regions of the fungal 18S rRNA gene sequence. In our study, two or more consecutive positive samples were always associated with fungal disease. RESULTS: PCR screening predicted the development of IFI to be 17 days (median). This test had a specificity of 91.1% and a sensitivity of 75%. IFI incidence was 7.8%. DISCUSSION: Therefore...

Identification of Contaminating Fungal DNA Sequences in Zymolyase

Rimek, Dagmar; Garg, Amar P.; Haas, Walter H.; Kappe, Reinhard
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /03/1999 Português
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45.94%
When different preparations of Zymolyase were included in the pretreatment protocol of a panfungal PCR assay using a primer system for the 18S rRNA gene, an amplification product occurred in negative controls. The amplified fragment showed 100.0% sequence identity to the Saccharomyces sensu stricto complex and Kluyveromyces lodderae. Lyticase, lysing enzymes, and proteinase K appeared to be free from fungal DNA.

Detection and Identification of Fungal Pathogens by PCR and by ITS2 and 5.8S Ribosomal DNA Typing in Ocular Infections

Ferrer, Consuelo; Colom, Francisca; Frasés, Susana; Mulet, Emilia; Abad, José L.; Alió, Jorge L.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /08/2001 Português
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36.28%
The goal of this study was to determine whether sequence analysis of internal transcribed spacer/5.8S ribosomal DNA (rDNA) can be used to detect fungal pathogens in patients with ocular infections (endophthalmitis and keratitis). Internal transcribed spacer 1 (ITS1) and ITS2 and 5.8S rDNA were amplified by PCR and seminested PCR to detect fungal DNA. Fifty strains of 12 fungal species (yeasts and molds) were used to test the selected primers and conditions of the PCR. PCR and seminested PCR of this region were carried out to evaluate the sensitivity and specificity of the method. It proved possible to amplify the ITS2/5.8S region of all the fungal strains by this PCR method. All negative controls (human and bacterial DNA) were PCR negative. The sensitivity of the seminested PCR amplification reaction by DNA dilutions was 1 organism per PCR, and the sensitivity by cell dilutions was fewer than 10 organisms per PCR. Intraocular sampling or corneal scraping was undertaken for all patients with suspected infectious endophthalmitis or keratitis (nonherpetic), respectively, between November 1999 and February 2001. PCRs were subsequently performed with 11 ocular samples. The amplified DNA was sequenced, and aligned against sequences in GenBank at the National Institutes of Health. The results were PCR positive for fungal primers for three corneal scrapings...

Comparison of Six DNA Extraction Methods for Recovery of Fungal DNA as Assessed by Quantitative PCR

Fredricks, David N.; Smith, Caitlin; Meier, Amalia
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/2005 Português
Relevância na Pesquisa
46.37%
The detection of fungal pathogens in clinical samples by PCR requires the use of extraction methods that efficiently lyse fungal cells and recover DNA suitable for amplification. We used quantitative PCR assays to measure the recovery of DNA from two important fungal pathogens subjected to six DNA extraction methods. Aspergillus fumigatus conidia or Candida albicans yeast cells were added to bronchoalveolar lavage fluid and subjected to DNA extraction in order to assess the recovery of DNA from a defined number of fungal propagules. In order to simulate hyphal growth in tissue, Aspergillus fumigatus conidia were allowed to form mycelia in tissue culture media and then harvested for DNA extraction. Differences among the DNA yields from the six extraction methods were highly significant (P < 0.0001) in each of the three experimental systems. An extraction method based on enzymatic lysis of fungal cell walls (yeast cell lysis plus the use of GNOME kits) produced high levels of fungal DNA with Candida albicans but low levels of fungal DNA with Aspergillus fumigatus conidia or hyphae. Extraction methods employing mechanical agitation with beads produced the highest yields with Aspergillus hyphae. The MasterPure yeast method produced high levels of DNA from C. albicans but only moderate yields from A. fumigatus. A reagent from one extraction method was contaminated with fungal DNA...

Development and Clinical Application of a Panfungal PCR Assay To Detect and Identify Fungal DNA in Tissue Specimens▿

Lau, Anna; Chen, Sharon; Sorrell, Tania; Carter, Dee; Malik, Richard; Martin, Patricia; Halliday, Catriona
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Português
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46.21%
Given the rise in the incidence of invasive fungal infections (IFIs) and the expanding spectrum of fungal pathogens, early and accurate identification of the causative pathogen is essential. We developed a panfungal PCR assay that targets the internal transcribed spacer 1 (ITS1) region of the ribosomal DNA gene cluster to detect fungal DNA in fresh and formalin-fixed, paraffin-embedded (PE) tissue specimens from patients with culture-proven (n = 38) or solely histologically proven (n = 24) IFIs. PCR products were sequenced and compared with sequences in the GenBank database to identify the causal pathogen. The molecular identification was correlated with results from histological examination and culture. The assay successfully detected and identified the fungal pathogen in 93.6% and 64.3% of culture-proven and solely histologically proven cases of IFI, respectively. A diverse range of fungal genera were identified, including species of Candida, Cryptococcus, Trichosporon, Aspergillus, Fusarium, Scedosporium, Exophiala, Exserohilum, Apophysomyces, Actinomucor, and Rhizopus. For five specimens, molecular analysis identified a pathogen closely related to that identified by culture. All PCR-negative specimens (n = 10) were PE tissues in which fungal hyphae were visualized. The results support the use of the panfungal PCR assay in combination with conventional laboratory tests for accurate identification of fungi in tissue specimens.

Toll-Like Receptor 9-Dependent Immune Activation by Unmethylated CpG Motifs in Aspergillus fumigatus DNA▿ †

Ramirez-Ortiz, Zaida G.; Specht, Charles A.; Wang, Jennifer P.; Lee, Chrono K.; Bartholomeu, Daniella C.; Gazzinelli, Ricardo T.; Levitz, Stuart M.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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46.13%
Phagocytic defenses are critical for effective host defenses against the opportunistic fungal pathogen Aspergillus fumigatus. Previous studies found that following challenge with A. fumigatus, Toll-like receptor 9 (TLR9) knockout mice survived longer than wild-type mice. However, the mechanism responsible was not defined. Here we demonstrate that A. fumigatus contains unmethylated CpG sequences, the natural ligands for TLR9. A. fumigatus DNA and synthetic CpG-rich oligodeoxynucleotides (ODNs) containing sequences found in the A. fumigatus genome potently stimulated the production of proinflammatory cytokines in mouse bone marrow-derived dendritic cells (BMDCs) and human plasmacytoid dendritic cells. The response was decreased when the fungal DNA was treated with a CpG methylase or with CpG-specific endonucleases. A role for TLR9 was demonstrated as cytokine production was abolished in BMDCs from TLR9-deficient mice. Moreover, transfection of HEK293 cells with human TLR9 conferred responsiveness to synthetic CpG-rich ODNs containing sequences found in A. fumigatus DNA. Taken together, these data demonstrate that TLR9 detects A. fumigatus DNA, resulting in the secretion of proinflammatory cytokines, which may contribute to the immune response to the pathogen.

Sequencing and Analysis of Fungal rRNA Operons for Development of Broad-Range Fungal PCR Assays▿ †

Khot, Prasanna D.; Ko, Daisy L.; Fredricks, David N.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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36.27%
rRNA genes are attractive targets for developing PCR assays targeting human fungal pathogens. Most studies have focused on the 18S rRNA gene, internal transcribed spacers, and the 5′ end of the 28S rRNA gene. An approximately 2,900-bp region of the 28S rRNA gene remains largely unexplored because sequences of many medically relevant fungi are either unavailable or undefined in genomic databases. The internal transcribed spacers and 28S rRNA gene of nine medically and phylogenetically important fungi were sequenced. In addition, 42 sequences from this region were acquired from public databases, resulting in an alignment of 51 fungal sequences spanning 30 fungal genera. For the nearly 3,950-bp region from the 3′ end of 18S rRNA gene to the 3′ end of the 28S rRNA gene, 27 broad-range PCR primers were designed such that their sequence homology with the human rRNA gene was minimal. All 62 possible amplicons in the size range from 75 to 400 bp from 27 primers were screened using fungal genomic DNA from 26 species spanning 14 genera. Eleven of the 62 amplicons did not cross-react with 1 μg/PCR human DNA but simultaneously amplified 10 fg of fungal DNA. Phylogenetic distance matrices were calculated for regions covered by these 11 amplicons based on 51 fungi. Two of these 11 amplicons successfully amplified 30 fg of fungal DNA from 25 of 26 fungi and provided the most phylogenetic information for species identification based on the distance matrices. These PCR assays hold promise for detection and identification of fungal pathogens in human tissues.

Improving Molecular Detection of Fungal DNA in Formalin-Fixed Paraffin-Embedded Tissues: Comparison of Five Tissue DNA Extraction Methods Using Panfungal PCR▿

Muñoz-Cadavid, C.; Rudd, S.; Zaki, S. R.; Patel, M.; Moser, S. A.; Brandt, M. E.; Gómez, B. L.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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46.18%
DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues is difficult and requires special protocols in order to extract small amounts of DNA suitable for amplification. Most described methods report an amplification success rate between 60 and 80%; therefore, there is a need to improve molecular detection and identification of fungi in FFPE tissue. Eighty-one archived FFPE tissues with a positive Gomori methenamine silver (GMS) stain were evaluated using five different commercial DNA extraction kits with some modifications. Three different panfungal PCR assays were used to detect fungal DNA, and two housekeeping genes were used to assess the presence of amplifiable DNA and to detect PCR inhibitors. The sensitivities of the five extraction protocols were compared, and the quality of DNA detection (calculated for each kit as the number of housekeeping gene PCR-positive samples divided by the total number of samples) was 60 to 91% among the five protocols. The efficiencies of the three different panfungals used (calculated as the number of panfungal-PCR-positive samples divided by the number of housekeeping gene PCR-positive samples) were 58 to 93%. The panfungal PCR using internal transcribed spacer 3 (ITS3) and ITS4 primers yielded a product in most FFPE tissues. Two of the five DNA extraction kits (from TaKaRa and Qiagen) showed similar and promising results. However...

Testing Potential Effects of Maize Expressing the Bacillus thuringiensis Cry1Ab Endotoxin (Bt Maize) on Mycorrhizal Fungal Communities via DNA- and RNA-Based Pyrosequencing and Molecular Fingerprinting

Verbruggen, Erik; Kuramae, Eiko E.; Hillekens, Remy; de Hollander, Mattias; Kiers, E. Toby; Röling, Wilfred F. M.; Kowalchuk, George A.; van der Heijden, Marcel G. A.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/2012 Português
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36.25%
The cultivation of genetically modified (GM) crops has increased significantly over the last decades. However, concerns have been raised that some GM traits may negatively affect beneficial soil biota, such as arbuscular mycorrhizal fungi (AMF), potentially leading to alterations in soil functioning. Here, we test two maize varieties expressing the Bacillus thuringiensis Cry1Ab endotoxin (Bt maize) for their effects on soil AM fungal communities. We target both fungal DNA and RNA, which is new for AM fungi, and we use two strategies as an inclusive and robust way of detecting community differences: (i) 454 pyrosequencing using general fungal rRNA gene-directed primers and (ii) terminal restriction fragment length polymorphism (T-RFLP) profiling using AM fungus-specific markers. Potential GM-induced effects were compared to the normal natural variation of AM fungal communities across 15 different agricultural fields. AM fungi were found to be abundant in the experiment, accounting for 8% and 21% of total recovered DNA- and RNA-derived fungal sequences, respectively, after 104 days of plant growth. RNA- and DNA-based sequence analyses yielded most of the same AM fungal lineages. Our research yielded three major conclusions. First, no consistent differences were detected between AM fungal communities associated with GM plants and non-GM plants. Second...

Detection of Fungal DNA in Human Body Fluids and Tissues during a Multistate Outbreak of Fungal Meningitis and Other Infections

Gade, Lalitha; Scheel, Christina M.; Pham, Cau D.; Lindsley, Mark D.; Iqbal, Naureen; Cleveland, Angela Ahlquist; Whitney, Anne M.; Lockhart, Shawn R.; Brandt, Mary E.; Litvintseva, Anastasia P.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2013 Português
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46.28%
Exserohilum rostratum was the major cause of an outbreak of fungal infections linked to injections of contaminated methylprednisolone acetate. Because almost 14,000 persons were exposed to product that was possibly contaminated with multiple fungal pathogens, there was unprecedented need for a rapid throughput diagnostic test that could detect both E. rostratum and other unusual agents of fungal infection. Here we report development of a novel PCR test that allowed for rapid and specific detection of fungal DNA in cerebrospinal fluid (CSF), other body fluids and tissues of infected individuals. The test relied on direct purification of free-circulating fungal DNA from fluids and subsequent PCR amplification and sequencing. Using this method, we detected Exserohilum rostratum DNA in 123 samples from 114 case-patients (28% of 413 case-patients for whom 627 samples were available), and Cladosporium DNA in one sample from one case-patient. PCR with novel Exserohilum-specific ITS-2 region primers detected 25 case-patients with samples that were negative using broad-range ITS primers. Compared to fungal culture, this molecular test was more sensitive: of 139 case-patients with an identical specimen tested by culture and PCR, E. rostratum was recovered in culture from 19 (14%)...

Endotoxin, Ergosterol, Fungal DNA and Allergens in Dust from Schools in Johor Bahru, Malaysia- Associations with Asthma and Respiratory Infections in Pupils

Norbäck, Dan; Markowicz, Pawel; Cai, Gui-Hong; Hashim, Zailina; Ali, Faridah; Zheng, Yi-Wu; Lai, Xu-Xin; Spangfort, Michael Dho; Larsson, Lennart; Hashim, Jamal Hisham
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 11/02/2014 Português
Relevância na Pesquisa
46.22%
There are few studies on associations between respiratory health and allergens, fungal and bacterial compounds in schools in tropical countries. The aim was to study associations between respiratory symptoms in pupils and ethnicity, chemical microbial markers, allergens and fungal DNA in settled dust in schools in Malaysia. Totally 462 pupils (96%) from 8 randomly selected secondary schools in Johor Bahru, Malaysia, participated. Dust was vacuumed from 32 classrooms and analysed for levels of different types of endotoxin as 3-hydroxy fatty acids (3-OH), muramic acid, ergosterol, allergens and five fungal DNA sequences. Multiple logistic regression was applied. Totally 13.1% pupils reported doctor’s diagnosed asthma, 10.3% wheeze and 21.1% pollen or pet allergy. Indian and Chinese children had less atopy and asthma than Malay. Carbon dioxide levels were low (380–690 ppm). No cat (Fel d1), dog (Can f 1) or horse allergens (Ecu cx) were detected. The levels of Bloomia tropicalis (Blo t), house dust mite allergens (Der p 1, Der f 1, Der m 1) and cockroach allergens (Per a 1 and Bla g 1) were low. There were positive associations between levels of Aspergillus versicolor DNA and daytime breathlessness, between C14 3-OH and respiratory infections and between ergosterol and doctors diagnosed asthma. There were negative (protective) associations between levels of C10 3-OH and wheeze...

Enhanced fungal DNA-Extraction from Formalin fixed, paraffin embedded tissue specimens by application of thermal energy

Rickerts, V.; Khot, P.D.; Ko, D.L.; Fredricks, D.N.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
46.31%
Determining the etiology of invasive fungal infections (IFI) is critical for patient management as fungi vary in their susceptibility to antifungals. However, the etiology remains obscure in many cases due to negative culture results. The identification of fungal DNA from pathology blocks by PCR and sequencing is an alternative approach to determine the etiology of IFI. Previous studies identified fungal DNA in only 50% of samples with positive histopathology, probably due to DNA damage by the tissue fixation. We used realtime PCR to quantify human and fungal DNA from Formalin-fixed, paraffin embedded tissue specimens in order to study the effect of thermal energy during extraction on the yield of amplifiable DNA and subsequent identification of fungal DNA. Tissue sections from eight patients with proven IFI were subjected to DNA extraction with varying exposure to thermal energy. Amplifiable DNA increased up to 76-fold by increasing incubation temperature from 65°C to 90°C. An additional increase was documented by incubation for up to 6 hours at 90°C. The augmented amplification of fungal DNA was associated with improved species identification by sequencing of the PCR amplicons. This may help illuminate the etiology of IFI and thereby improve patient management by guiding antifungal therapy.

Fungal DNA Detected in Blood Samples of Patients Who Received Contaminated Methylprednisolone Injections Reveals Increased Complexity of Causative Agents

Zhao, Yanan; Armeanu, Emilian; DiVerniero, Richard; Lewis, Terri A.; Dobson, Richard C.; Kontoyiannis, Dimitrios P.; Roilides, Emmanuel; Walsh, Thomas J.; Perlin, David S.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /06/2014 Português
Relevância na Pesquisa
46.03%
Using Exserohilum rostratum-specific and panfungal real-time PCR, we studied 24 blood samples and 2 synovial fluid specimens from 20 patients with persistent or worsening pain following injections of contaminated methylprednisolone. Seven blood specimens from 6 patients were significantly positive for fungal DNA by panfungal PCR, with multiple fungal species identified.

An assessment of the efficiency of fungal DNA extraction methods for maximizing the detection of medically important fungi using PCR

Karakousis, A.; Tan, L.W.; Ellis, D.; Alexiou, H.; Wormald, P.J.
Fonte: Elsevier Science BV Publicador: Elsevier Science BV
Tipo: Artigo de Revista Científica
Publicado em //2006 Português
Relevância na Pesquisa
46.22%
To date, no single reported DNA extraction method is suitable for the efficient extraction of DNA from all fungal species. The efficiency of extraction is of particular importance in PCR-based medical diagnostic applications where the quantity of fungus in a tissue biopsy may be limited. We subjected 16 medically relevant fungi to physical, chemical and enzymatic cell wall disruption methods which constitutes the first step in extracting DNA. Examination by light microscopy showed that grinding with mortar and pestle was the most efficient means of disrupting the rigid fungal cell walls of hyphae and conidia. We then trialled several published DNA isolation protocols to ascertain the most efficient method of extraction. Optimal extraction was achieved by incorporating a lyticase and proteinase K enzymatic digestion step and adapting a DNA extraction procedure from a commercial kit (MO BIO) to generate high yields of high quality DNA from all 16 species. DNA quality was confirmed by the successful PCR amplification of the conserved region of the fungal 18S small-subunit rRNA multicopy gene.; http://www.elsevier.com/wps/find/journaldescription.cws_home/506034/description#description; A. Karakousis, L. Tan, D. Ellis, H. Alexiou and P.J. Wormald

Optimierung eines vollautomatisierten, PCR-basierten Nachweisverfahrens von DNA aus humanpathogenen Pilzen; Optimisation of an fully automated, PCR – based essay fort the detection of DNA from human pathogenic fungi

Baumann, Andreas Florian Fürchtegott
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
Português
Relevância na Pesquisa
36.29%
Zielsetzung dieser Arbeit war die Optimierung neuer vollautomatisierter Testverfahren für die Extraktion, Amplifikation und Detektion von Nukleinsäuren aus humanpathogenen Pilzen. Insgesamt wurden 152 Vollblutproben von insgesamt 38 Patienten auf die Präsenz von Aspergillus DNA untersucht. Die Untersuchung beinhaltete die Extraktion fungaler DNA mit dem MagNA Pure LC Extraktionsautomaten und die anschließende Amplifikation und Detektion mit dem real-time PCR LightCycler Instrument. Parallel wurden die Nukleinsäuren manuell mit Qiagen extrahiert, mit der PCR - ELISA Methode amplifiziert und detektiert, und anschließend die Ergebnisse miteinander verglichen. 24 Proben von 11 Patienten konnten mit Qiagen und PCR - ELISA Aspergillus positiv getestet werden. Dem gegenüber konnten 3 Proben von 3 Patienten mit der Kombination MagNA Pure - LightCycler positiv getestet werden. Da das Erregerspektrum der invasiven Mykosen ständig zunimmt, wurde in diesem Zusammenhang der Extraktionskit MagNA Pure LC DNA Isolation Kit II für die Extraktion von DNA verschiedener Pilze evaluiert. Die Evaluierung erfolgte anhand verschiedener Sensitivitäts-, Reproduktions- und Kontaminationsversuche, wobei unter anderem auch die Notwendigkeit einer vorbereitenden Zellwanddisruption mit Glaspartikeln (glass beads) bewiesen werden konnte. In diesem Zusammenhang wurde eine Methode zur Homogenisierung und damit zur Extraktionsvorbereitung von murinen Gewebestücken (Herz...

Nachweis von Histoplasma capsulatum-DNA in Blutproben von Mäusen und humanen Gewebeproben mittels PCR-Verfahren; Detection of Histoplasma capsulatum DNA in murine models and human tissue by nested PCR assays

Feucht, Antje
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
Português
Relevância na Pesquisa
46.24%
IInvasive Mykosen sind lebensbedrohliche Erkrankungen vor allem bei Patienten mit Defekten des zellulären Immunsystems. Auch bei der Histoplasmose, verursacht durch den dimorphen Pilz Histoplasma capsulatum, ist ein disseminierter Befall nach Inhalation der Erreger bei Immungeschwächten möglich. Nur die frühzeitige Diagnostik einer Histoplasmose bietet die Möglichkeit einer rechtzeitigen antimykotischen Therapie. Diagnostische Methoden sollten sensitiv und spezifisch und möglichst innerhalb eines Arbeitstages durchführbar sein. Sowohl Verfahren zum Direktnachweis wie die kulturelle Anzucht der Pilzzellen oder die mikroskopische Identifizierung in Patientenmaterial als auch serologische Methoden mit Antikörper- und Antigen-Nachweis erfüllen diese Anforderungen jedoch nur teilweise. Bereits 2001 war von Bialek et al. eine 18 S DNA-PCR-Methode zum Nachweis von Histoplasma capsulatum-spezifischer DNA im Tiermodell aufgebaut worden. In der vorliegenden Arbeit zeigten ergänzende Untersuchungen, dass der Nachweis der Pilz-DNA auch im Blut infizierter Mäuse möglich ist. Dabei stellte sich jedoch heraus, dass es aufgrund der hoch konservierten Zielregion innerhalb der 18 S rDNA häufig zu einer Kreuzreaktion der Primer kommt. Wir entwickelten daher eine neue PCR mit Zielregion in einem Gen...

Etablierung und Evaluierung einer vollautomatisierten Extraktionsmethode für DNA aus humanpathogenen Pilzen; Establishment and Evaluation of a fully automated extraction method for DNA from pathogenic fungi

Schmidt, Kathrin Dorothee
Fonte: Universität Tübingen Publicador: Universität Tübingen
Tipo: Dissertation; info:eu-repo/semantics/doctoralThesis
Português
Relevância na Pesquisa
46.37%
Die Anwendung von MagNA Pure LC (TM) und das entwickelte Protokoll zur Extraktion von Pilz-DNA erwies sich als schnelle, sensitive und reproduzierbare Methode zur molekularen Diagnostik invasiver Mykosen. Sie leistet damit einen Beitrag zur Standardisierung molekularer Nachweisverfahren von Pilzinfektionen. In dieser Arbeit wurde eine automatisierte Methode zur Extraktion von fungalen Nukleinsäuren entwickelt und an klinischen Proben evaluiert. Die Methode basiert auf dem Extraktionsautomaten MagNA Pure LC (TM) (Roche Diagnostics) und wurde zur Aufspaltung der rigiden Pilzzellwände durch die Anwendung von Glasperlen erweitert. Mit dieser Methode konnte die DNA von insgesamt 23 verschiedenen Hefe- und Schimmelpilzen erfolgreich isoliert werden. Serielle Verdünnungen von Aspergillus fumigatus Konidien und Candida albicans Zellen (jeweils von 106 bis 100 CFU / ml) wurden zur Evaluierung der Sensitivität des Verfahrens eingesetzt. Außerdem wurde die DNA aus 68 klinischen Proben von 31 hämatologischen Patienten (57 Blutproben und 11 BALs), extrahiert und die PCR-Ergebnisse mit denen der manuellen, etablierten Methode verglichen. Die mit der neu entwickelten Methode isolierte DNA war reproduzierbar bis zu einem Detektionslimit von 101 CFU nachweisbar. Für C. albicans und in 9 / 28 Extraktionsläufen auch für A. fumigatus waren 100 CFU detektierbar. Insgesamt ist die Sensitivität der entwickelten Methode mit der des Routineprotokolls vergleichbar und ausreichend...

Vergleich der Light-cycler-PCR mit anderen molekularbiologischen Nachweisverfahren zum Nachweis von Aspergillus- und Candida-DNA im Blut von Patienten nach allogener Knochenmarkstransplantation; Comparison between Light-cycler-PCR and other molecularbiological methods for detection of Aspergillus- and Canida-DNA in bloodsamples of bone marrow transplanted patients

Hunecken, Nicole Michaela
Fonte: Universität Tübingen Publicador: Universität Tübingen
Tipo: Dissertation; info:eu-repo/semantics/doctoralThesis
Português
Relevância na Pesquisa
46.18%
Patienten mit hämatologisch-onkologischen Erkrankungen oder Patienten nach Transplantationen sind gefährdet, sich eine lebensbedrohliche Pilzinfektion zuzuziehen. Mit einer möglichst früh eingeleiteten antimykotischen Therapie kann die Morbidität und die Mortalität dieser Patienten herabgesetzt werden. Dazu ist eine rasche, sensitive und speziesidentifizierende Diagnostik von großer Bedeutung. Das Ziel dieser Arbeit war es in erster Linie die LightCycler-Methode und die PCR-Elisa-Methode, aber auch andere Methoden, für die Routinediagnostik von Pilzen so anwendbar zu machen, dass der Verlust an Sensitivität und Spezifität möglichst gering gehalten wird, trotzdem aber am Tag der Blutentnahme ein Ergebnis vorliegen kann. Die Versuche zur Extraktion haben gezeigt, dass die Methode mit RCLB, WCLB und Lytikase, mit niedrigerer Zentrifugalkraft, dafür aber mit größeren Ausgangsvolumina die besten Ergebnisse lieferte. Diese Methode wurde zur Extraktion der Pilz-DNA für den PCR-Elisa als auch für das LightCycler-Verfahren angewendet. Im Hauptteil dieser Arbeit wurde die LightCycler- mit der PCR-Elisa-Methode anhand von 846 Blutproben von Patienten nach Knochenmarks- oder Stammzelltransplantation verglichen. Im Vergleich der Ergebnisse dieser Studie wird deutlich...