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Usefulness of PCR Melting Profile Method for Genotyping Analysis of Klebsiella oxytoca Isolates from Patients of a Single Hospital Unit
PublikacjaOpracowanie szybkich i prostych metod typowania jest wymagane w celu identyfikacji potencjalnych źródeł zakażenia ludzi drobnoustrojami oportunistycznymi. Klebsiella spp. należą do grupy bakterii oportunistycznych odpowiedzialnych za wzrost liczby wieloopornych zakażeń szpitalnych. Ostatnio pokazaliśmy wysoką różnorodność genetyczną K. oxytoca używając dużej kolekcji szczepów izolowanych przez 50 lat od pacjentów z kilku szpitali...
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Identification and cloning of C. albicans SC5314 genes encoding L-methionine biosynthetic pathway enzymes.
Dane BadawczeEnzymes of fungal L-methionine biosynthetic pathway: homoserine O-acetyltransferase (Met2p), O-acetylhomoserine sulfhydrylase (Met15p) and cystathionine-γ-synthase (Str2p) could be exploited as molecular targets for antifungal chemotherapy. The goal of the study was to identify and clone genes encoding mentioned above enzymes. MET2, MET15 and STR2 genes...
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Retrospective analysis of genetic diversity of Klebsiella oxytoca isolated in Poland over a 50-year period.
PublikacjaAnaliza genetyki populacji i określenie powiązań filogenetycznych pomiędzy szczepami może być niezwykle użytecznym podejściem w określaniu rozprzestrzeniania się wzorów genetycznych różnych gatunków bakterii na potrzeby epidemiologiczne. Nie ma danych, które opisywałyby długoterminowe sytuacje epidemiologiczne powodowane przez wielolekooporne, oportunistyczne szczepy Klebsiella oxytoca metodami molekularnymi. Celem przedstawionych...
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Antiviral activity of bee bread derived from polish apiaries.
Dane BadawczeBee bread is a product of fermentation of bee-collected pollen and revealed a high nutritional value. Other bee products, such as honey and propolis, are known for their antiviral activity, but bee bread is still under investigation, thus its antiviral potential is still unspecified. For investigation antiviral activity of bee bread samples, cytotoxicity...
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PCR test for Microsporum canis identification
PublikacjaMicrosporum canis, for which the natural hosts are cats and dogs, is the most prevalent zoophilic agent causing tinea capitis and tinea corporis in humans. We present here a diagnostic PCR test for M. canis, since its detection and species identification is relevant to the choice of treatment and to the understanding of a probable source of infection. An M. canis-specific PCR was evaluated using 130 clinical isolates of dermatophytes...
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Phylogenetic trees of genus Oncidium Sw. based on analysis of DNA sequences
Dane BadawczeGenus Oncidium Sw. is widely regarded as a polyphiletic, and the taxonomic boundaries between him and such genera as Odontoglossum Kunth. or Miltonia Lindley remain blurred. The goal of the study was to determine the phylogenetic relationships within the genus Oncidium s.lato based on the DNA sequences analysis. The correlation between molecular data...
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A New Double Digestion Ligation Mediated Suppression PCR Method for Simultaneous Bacteria DNA-Typing and Confirmation of Species: An Acinetobacter sp. Model
PublikacjaWe have designed a new ddLMS PCR (double digestion Ligation Mediated Suppression PCR) method based on restriction site polymorphism upstream from the specific target sequence for the simultaneous identification and differentiation of bacterial strains. The ddLMS PCR combines a simple PCR used for species or genus identification and the LM PCR strategy for strain differentiation. The bacterial identification is confirmed in the...
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Fuzyjne polimerazy DNA – otrzymywanie, charakterystyka i zastosowanie
PublikacjaObecnie reakcje PCR (ang. Polymerase Chain Reaction) wykazują bardzo szerokie zastosowanie w diagnostyce medycznej, biologii molekularnej czy inżynierii genetycznej. Efektywność tych reakcji rozumiana jako wydajność i wierność przeprowadzonej amplifikacji jest nieodłącznie związana ze stosowaną polimerazą DNA i warunkami prowadzenia reakcji PCR. Aby sprostać wymaganiom stawianym przez nowoczesne metody diagnostyczne oraz współczesną...
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Principles and applications of Ligation Mediated PCR methods for DNA-based typing of microbial organisms
PublikacjaA significant number of DNA-based techniques has been introduced into the field of microorganisms’ characterization and taxonomy. These genomic fingerprinting methods were developed to detect DNA sequence polymorphisms by using general principles, such as restriction endonuclease analysis, molecular hybridization, and PCR amplification. In recent years, some alternative techniques based on ligation of oligonucleotide adapters before...
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The New LM-PCR/Shifter Method for the Genotyping of Microorganisms Based on the Use of a Class IIS Restriction Enzyme and Ligation Mediated PCR
PublikacjaThis study details and examines a novel Ligation-Mediated - Polymerase Chain Reaction (LM-PCR) method. Named the LM-PCR/Shifter, it relies on the use of a Class IIS restriction enzyme giving restriction fragments with different 4 base, 5' overhangs, this being the Shifter, and the ligation of appropriate oligonucleotide adapters. A sequence of 4-base, 5' overhangs of the adapter and a 4-base sequence of the 3' end of the primer(s)...
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Modified DNA polymerases for PCR troubleshooting
PublikacjaPCR has become an essential tool in biological science. However, researchers often encounter problems with difficult targets, inhibitors accompanying the samples, or PCR trouble related to DNA polymerase. Therefore, PCR optimization is necessary to obtain better results. One solution is using modified DNA polymerases with desirable properties for the experiments. In this article, PCR troubleshooting, depending on the DNA polymerase...
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PCR melting profile (PCR MP) - a new tool for differentiation of Candida albicans strains
PublikacjaPo raz pierwszy przedstawiliśmy użyteczność techniki PCR MP opartej na metodzie ligacji adaptorów (LM PCR) i obniżonej temperaturze denaturacji w cyklach PCR do wewnątrzgatunkowego różnicowania szczepów Candida albicans. Przebadaliśmy 123 szczepy Candida albicans z zastosowaniem trzech metod genotypowania: PCR MP, REA-PFGE i RAPD i uzyskaliśmy odpowiednio 27, 26 i 25 unikalnych genotypów. Zaprezentowane dane pokazały, że technika...
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A simple modification of PCR thermal profile applied to evade persisting contamination
PublikacjaThe polymerase chain reaction (PCR), one of the most commonly applied methods of diagnostics and molecular biology has a frustrating downside known as the false positive signal or contamination. Several solutions to avoid and to eliminate PCR contaminations have been worked out to date but the implementation of these solutions to laboratory practice may be laborious and time consuming. A simple approach to circumvent the problem...
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The New LM-PCR/Shifter Method for the Genotyping of Microorganism
PublikacjaTechniques relies on the ligation of appropriates adapters (LM-PCR) as AFLP, PCR MP and ADSRRS are successfully used for epidemiological studies for prokaryotic and eukaryotic microorganisms. In this study we propose a new method, called the LM-PCR/Shifter, based on the use of a Class IIS restriction enzyme giving restriction fragments with different 4 base 5' overhangs (Shifter) and the ligation of appropriate oligonucleotide...
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PCR-ELISA: inexpensive alternative to quantitative PCR
PublikacjaPCR-ELISA (polymerase chain reaction-enzyme linked immunosorbent assay), a combination of PCR and ELISA methods, has been used since late 1980s. The technique is based on specially labelled DNA fragments which are captured by specific DNA sequences and detected by antibodies. The whole procedure of PCR-ELISA is divided into three steps: DNA extraction, PCR reaction and detection by ELISA. The method has been found as very specific...
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A universal method for the identification of genes encoding amatoxins and phallotoxins in poisonous mushrooms
PublikacjaABSTRACT Background. As the currently known diagnostic DNA targets amplified in the PCR assays for detection of poisonous mushrooms have their counterparts in edible species, there is a need to design PCR primers specific to the genes encoding amanitins and phallotoxins, which occur only in poisonous mushrooms. Objective. The aim of the study was testing of PCR-based method for detection of all genes encoding hepatotoxic cyclic...
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DDLMS PCR double digestion Ligation Mediated Suppression PCR - a new technique for bacterial specific differentiation
PublikacjaA new diagnostic kit for K. oxytoca specific differentiation based on ddLMS PCR (ang. double digest ligation Mediated PCR) technique is shown. As a species-specific DNA fragment pehX gene, encoding the enzyme polygalactouronase, was chosen. The genome sequence of K. oxytoca is digested with two endonucleases: AclI and BclI which cut DNA before and after pehX gene. The polymorphic DNA fragments are ligated with AclI-end-specific...
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Specific detection of Alternaria alternata by PCR and real-time PCR
PublikacjaFungi of Alternaria genus are cosmopolitan organisms, which spores can be found in the air, soil, water, clothing and food. They commonly occur as saprotrophs on the plant remains, contributing to the decomposition of organic matter. Additionally, they are components of the normal human and animal skin flora. Alternaria spp. are also known human allergens, causing hay fever and allergic reactions that can lead to the development...
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Technika PCR MP (PCR Melting Profile) - możliwości zastosowania w badaniach epidemiologicznych
PublikacjaUżyteczność PCR MP (ang. PCR Melting Profile), stosunkowo nowej techniki różnicowania genetycznego szczepów mikroorganizmów, pokazano na przykładach badań epidemiologicznych, dotyczących wykrywania zakażeń szpitalnych, analizy sytuacji epidemiologicznej i identyfikacji transmisji bakteryjnej u poszczególnych pacjentów. Technikę PCR MP charakteryzuje prostota, niskie koszty wykonania oraz potencjał różnicujący porównywalny do metody...
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Optimized 5-hour multiplex PCR test for the detection of tinea unguium: performance in a routine PCR laboratory
PublikacjaWe recently reported the development of a 5-hour multiplex PCR test for the detection of tinea unguium and the optimization of this test by the inclusion of an inhibition control. Here we report the performance of this procedure as used in a routine clinical laboratory as compared to conventional microscopy and culture-based techniques performed in a mycology reference laboratory. We found in processing 109 samples that 22 (20.2%)...