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Search results for: PCR RFLP
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PCR-RFLP assays for species-specific identification of fungi belonging to Scopulariopsis and related genera
PublicationFungi of the Scopulariopsis genus, commonly found in the environment, are opportunistic pathogens that can cause various types of human infections. So far, no efficient molecular method has been developed for species differentiation among Scopulariopsis and related genera. In order to advance this field, we have evaluated performance of polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays, based...
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Różnicowanie C. albicans i C. dubliniensis w oparciu o polimorfizm genu syntazy homocytrynianowej z wykorzystaniem metody PCR RFLP
PublicationNajczęstszym czynnikiem etiologicznym zakażeń wywołanych przez drożdżaki z rodzaju Candida jest C. albicans. Gatunek ten posiada bardzo zbliżone cechy fenotypowe do C. dubliniensis co często wiąże się z błędną identyfikacją. Skutkuje to wtórnym zakażeniem wywołanym przez współwystepujący gatunek C. dubliniensis, który nabywa oporność na flikonazol stosowany w terapii C. albicans. Celem badań była konstrukcja układu diagnostycznego...
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Amplification of a single-locus variable-number direct-repeats with restriction fragment length polymorphism (DR-PCR/RFLP) for genetic typing of Acinetobacter baumannii strains
PublicationW celu poszukiwania nowych metod typowania genetycznego szczepów Acinetobacter baumannii przydatnych w szpitalnych badaniach epidemiologicznych przeprowadzono analizę dostępnych w banku genów sekwencji szczepów referencyjnych na obecność nowych motywów repetytywnych. Do badania polimorfizmu sekwencji repetytywnych w szczepach Acinetobacter sp. wykorzystano proste powtórzenia typu "direct repeat" (DR) zlokalizowane w jednym miejscu...
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PCR-ELISA: inexpensive alternative to quantitative PCR
PublicationPCR-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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Specific detection of Alternaria alternata by PCR and real-time PCR
PublicationFungi 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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PCR and real-time PCR assays to detect fungi of Alternaria alternata species
PublicationFungi of the Alternaria genus are mostly associated with allergic diseases. However, with a growing number of immunocompromised patients, these fungi, with A. alternata being the most prevalent one, are increasingly recognized as etiological agents of infections (phaeohyphomycoses) in humans. Nowadays, identification of Alternaria spp. requires their pure culture and is solely based on morphological criteria. Clinically, Alternaria...
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PCR melting profile (PCR MP) - a new tool for differentiation of Candida albicans strains
PublicationPo 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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Technika PCR MP (PCR Melting Profile) - możliwości zastosowania w badaniach epidemiologicznych
PublicationUżyteczność 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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PCR detection of Scopulariopsis brevicaulis
PublicationScopulariopsis brevicaulis is known as a most common etiological factor of the mould toenail infections. There are also reports indicating that S. brevicaulis could cause organ and disseminated infections. Nowadays microscopic observations from the direct sample and culture are crucial for the appropriate recognition of the infection. In this paper is presented a PCR-based method for S. brevicaulis detection. The specificity of...
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A comparative evaluation of PCR ribotyping and ERIC PCR for determining the diversity of clinical Pseudomonas aeruginosa isolates
PublicationW pracy porównano dwie metody stosowane do badania różnorodności genetycznej drobnoustrojów. Modelem do badań była grupa 62 klinicznych izolatów bakterii P. aeruginosa. Zdecydowanie wyższy potencjał różnicujący stwierdzono w przypadku metody ERIC PCR, której zastosowanie pozwoliło na wyodrębnienie 36 różnych genotypów. W przypadku rybotypowania wyróżniono tylko 9 genotypów.
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Optimised five-hour multiplex PCR test for the detection of Tinea ungium: performance in a routine PCR laboratory
PublicationWe recently published the development of a 5-hour multiplex PCR test for the detection of dermatophyte nail infection. We have optimized this test by inclusion of an inhibition control and evaluated the test in a routine laboratory when compared to the conventional microscopy and culture. A total of 109 clinical samples received at the mycology reference lab at Statens Serum Institute were included. The samples were divided equally...
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DDLMS PCR double digestion Ligation Mediated Suppression PCR - a new technique for bacterial specific differentiation
PublicationA 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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Optimized 5-hour multiplex PCR test for the detection of tinea unguium: performance in a routine PCR laboratory
PublicationWe 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%)...
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The construction and use of a PCR internal control
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Modified DNA polymerases for PCR troubleshooting
PublicationPCR 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 test for Microsporum canis identification
PublicationMicrosporum 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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Effect of bio-polyol molecular weight on the structure and properties of polyurethane-polyisocyanurate (PUR-PIR) foams
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Effect of bio-polyol molecular weight on the structure and properties of polyurethane-polyisocyanurate (PUR-PIR) foams
PublicationThe increasing interest in polyurethane materials has raised the question of the environmental impact of these materials. For this reason, the scientists aim to find an extremely difficult balance between new material technologies and sustainable development. This work attempts to validate the possibility of replacing petrochemical polyols with previously synthesized bio-polyols and their impact on the structure and properties...
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PCR and real-time PCR approaches to the identification of Arthroderma otae species Microsporum canis and Microsporum audouinii/Microsporum ferrugineum
PublicationObjectives The identification of species in the Arthroderma otae complex is essential to determine the origin of infection and to eliminate the risk of transmission. Microsporum canis is a zoophilic species, whereas Microsporum audouinii and Microsporum ferrugineum are anthropophilic species. In this paper, we propose alternative methods that permit species-specific identification of both anthropophilic and zoophilic members of...
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Diagnostic PCR assay for Microsporum and Trichophyton infections
PublicationWe have previously described a highly sensitive 5-hour PCR test for the rapid diagnosis of onychomycosis which detects any dermatophyte and species-identify T. rubrum from patient specimens. We have subsequently developed and evaluated new PCR tests for detection of Trichophyton and Microsporum canis/audouinii infections.58 dermatophyte isolates (21) Microsporum spp. (4 different species), 35 Trichophyton spp (10 different species)...
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Ligation Mediated PCR methods, their possibilities and limitations
PublicationThe application of molecular diagnostics for bacterial strain typing or identifying bacteria at the strain level is of particular importance at present. However, among the many genotyping methods that are currently available, no single one is universally ideal. In this article, we thus review the Ligation Mediated PCR group methods as prospects from which to choose. We discuss different strategies for selecting the amplified PCR...
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The Influence of Substitution of a Phosphorus-Containing Polyol with the Bio-polyol on the Properties of Bio-based PUR/PIR Foams
PublicationIn this work, effects of incorporating of a phosphorus-containing polyol into rigid polyurethane/polyisocyanurate foams’ formulations developed with use of two different bio-based polyols, derived from crude glycerol or liquefied cellulose were examined. The bio-polyol derived from crude glycerol was synthesized via two-step process from crude glycerol and castor oil, whereas the bio-polyol derived from liquefied cellulose was...
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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
PublicationThis 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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Study on the Structure-Property Dependences of Rigid PUR-PIR Foams Obtained from Marine Biomass-Based Biopolyol
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Study on the Structure-Property Dependences of Rigid PUR-PIR Foams Obtained from Marine Biomass-Based Biopolyol
PublicationThe paper describes the preparation and characterization of rigid polyurethane-polyisocyanurate (PUR-PIR) foams obtained with biopolyol synthesized in the process of liquefaction of biomass from the Baltic Sea. The obtained foams differed in the content of biopolyol in polyol mixture (0–30 wt%) and the isocyanate index (IISO = 200, 250, and 300). The prepared foams were characterized in terms of processing parameters (processing...
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Physico‐Mechanical Properties and Flammability of PUR/PIR Foams Containing Expandable Graphite Core‐Shell Composite Particles
PublicationIn this work, polyurethane/polyisocyanurate (PUR/PIR) foams were modified by two types of expandable graphite (EG) core-shell composite particles. The pulverized EG core-shell composite particles were prepared during emulsion polymerization using methyl methacrylate or glycidyl methacrylate for the synthesis of the polymeric shells (poly(methyl methacrylate) (PMMA) and poly (glycidyl methacrylate) (PGMA)), and then the obtained EG_PMMA...
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PRZYDATNOŚĆ DIAGNOSTYCZNA REAKCJI PCR W ROZPOZNAWANIU BORELIOZY
PublicationBorelioza jest wieloukładową chorobą wywoływaną przez bakterie zaliczane do kompleksu Borrelia burgdorferi sensu lato. Wektorem przenoszącym infekcję są kleszcze z rodzaju Ixodes, które zakażają kolejnych żywicieli krętka podczas spożywania ich krwi. Zróżnicowany przebieg boreliozy uniemożliwia jej rozpoznanie na podstawie objawów klinicznych. W związku z tym diagnostyka choroby z Lyme opiera się głównie na metodach laboratoryjnych,...
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The New LM-PCR/Shifter Method for the Genotyping of Microorganism
PublicationTechniques 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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Real-Time PCR: molecular technique of many applications
PublicationReal-Time PCR is a sensitive DNA amplification technique initially applied in genetics and molecular biology. It enables in vivo copying of the selected DNA fragment (flanked by two primers) by the thermostable polymerase (in the presence of magnesium ions and deoxynucleotide triphosphates) and simultaneous measurement of the fluorescence. For one or more specific sequences in a DNA sample, real-time PCR enables both detection...
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Novel application of terminal restriction fragments length polymorphism method (t-RFLP) for identification of six clinically important Candida sp.
PublicationTerminal restriction fragments length polymorphism method (t-RFLP) is a modified classical RFLP method. The main difference is based on carrying out PCR using a fluorescently labeled primer or primers. The result of such modification is that after digestion of amplicons only terminal fragments of PCR product (containing fluorescent dye) are visualized. T-RFLP method has found application in environmental microbiology and epidemiology....
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Wykorzystanie reakcji PCR do badania charakterystycznych obszarów genomu
PublicationW pracy zostały opisane metody wykorzystujące technię PCR do długo- i krótkoterminowych badań epidemiologicznych. Typowanie szczepów oparte jest na analizie fragmentów genów, bądź całego genomu w zależności od potrzeby posiadania potencjału różnicującego metody. Przez ostatnie 30 lat rozwijały się nie tylko metody badawcze, doszło również do rozwoju międzynarodowych baz danych i przepływu informacji o obecnych sytuacjach epidemiologicznych...
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Ewaluation of a PCR Melting Profile technique for bacterial strain differentiation
PublicationBadano przydatność techniki PCR MP w efektywnym typowaniu szczepów w badaniach epidemiologicznych zakażeń szpitalnych. Technika oparta jest na użyciu niskiej temperatury denaturacji podczas LM PCR. Badaniu zostały poddane izolaty Escherichia coli pochodzące od pacjentów Szpitala Klinicznego w Gdańsku. Wykazano, że technika jest szybka, o wysokim potencjale różnicującym i bardzo dobrej powtarzalności i może być zastosowana w badaniach...
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Detection of sulfonamide resistance genes via in situ PCR-FISH
PublicationDue to the rising use of antibiotics and as a consequence of their concentration in the environment an increasing number of antibiotic resistant bacteria is observed. The phenomenon has a hazardous impact on human and animal life. Sulfamethoxazole is one of the sulfonamides commonly detected in surface waters and soil. The aim of the study was to detect sulfamethoxazole resistance genes in activated sludge biocenosis by use of...
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Effect of Some Sulphur Additives on The Degradation of 9Cr-1Mo Steel After Its 10 Years Service in The CCR Platforming Unit
PublicationThe work shows the results of the tests of 9Cr-1Mo steel, which was for 10 years operated in the CCR platforming unit in Group Lotos SA in Gdańsk, and then in the laboratory was sulphidised during 166 h at a temperature of 600 °C. Sulphidation was performed in a mixture of H2-H2S gases at the vapour pressure of sulphur 4.1·10-14 atm, so the order of magnitude of vapour pressure was less than that of the dissociation pressure of...
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Diagnostic PCR tests for Microsporum audouinii, M. canis and Trichophyton infections
PublicationSince traditional diagnosis of dermatophyte infections is slow, we present a rapid new pcr test for detection of trichophyton spp., microsporum canis and m. audouinii infections. the performance of the test was evaluated with: 58 dermatophyte isolates; 10 yeast, mould and human dna control samples; 25 routine specimens from patients suspected of having dermatophytosis; 10 hair specimens from guinea pigs experimentally infected...
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Modyfikacje polimeraz DNA jako rozwiązania problemów w reakcjach PCR
PublicationReakcja PCR już od wielu lat jest podstawowym narzędziem stosowanym w biotechnologii molekularnej. Nadal napotykamy jednak liczne problemy podczas jej przeprowadzania związane z trudnymi matrycami, obecnością inhibitorów itp. Optymalizacja reakcji PCR jest więc niezbędna w celu poprawy wyników. Jednym z rozwiązań tych problemów może być modyfikowanie polimeraz DNA. W powyższym artykule opisano problemy i rozwiązania zależne polimerazy...
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A simple modification of PCR thermal profile applied to evade persisting contamination
PublicationThe 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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Real-time PCR approach in dermatophyte detection and Trichophyton rubrum identification
PublicationDermatophytes are keratinophilic molds that infect human hair, nails and skin. Diagnosis of dermatophytosis is based on morphological, serological and biochemical features. However, identification is difficult and laborious due to similarities between microorganisms. Thus, there is considerable interest to develop mycological diagnostic procedures based on molecular biology methods. In this study, fast, two-step DNA extraction...
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Enhancement of PUR/PIR foam thermal stability after addition of Zostera marina biomass component investigated via thermal analysis and isoconversional kinetics
PublicationIn the present work, a thorough thermogravimetric (TG) analysis of bio-based polyurethane–polyisocyanurate (PUR–PIR) foams in both nitrogen and oxygen atmosphere is performed. A sustainable element of the foam is a biopolyol obtained via acid-catalyzed liquefaction of Zostera marina and Enteromorpha Algae biomass. Based on isoconversional analysis and apparent activation energies, several conclusions are obtained. In contradiction...
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BDNF Val66Met Polymorphism, the Allele-Specific Analysis by qRT-PCR - a Novel Protocol
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Molecular typing of clinical isolates of Escherichia coli by a new PCR MP method
PublicationTypowanie epidemiologiczne bakterii jest rutynową procedurą stosowaną w badaniach zakażeń szpitalnych i wymaga stosowania odpowiednich metod o wysokim potencjale różnicującym. Przedstawiliśmy możliwości nowej techniki PCR MP w różnicowaniu szczepów E. coli. Rezultaty zostały porównane z wynikami uzyskanymi metodą PFGE.
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Molecular typing of clinical isolates of Escherichia coli by a new PCR MP method
PublicationTypowanie epidemiologiczne bakterii jest rutynową procedurą stosowaną w badaniach zakażeń szpitalnych i wymaga stosowania odpowiednich metod o wysokim potencjale różnicującym. Przedstawiliśmy możliwości nowej techniki PCR MP w różnicowaniu szczepów E. coli. Rezultaty zostały porównane z wynikami uzyskanymi metodą PFGE.
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Diagnostyka zakażeń Candida spp. oparta o amplifikację DNA techniką PCR
PublicationZakażenia wywoływane przez oportunistyczne grzyby z rodzaju Candida są poważnym problemem klinicznym. Niezbędne jest poszukiwanie nowych szybkich, czułych i pewnych metod do identyfikacji gatunkowej tych drobnoustrojów. W artykule przedstawiono opisane w literaturze metody diagnostyki molekularnej drożdżaków z rodzaju Candida oparte o amplifikację DNA techniką PCR.
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Identification of anisakid nematodes from the Southern Baltic Sea usingPCR-based methods
PublicationZaproponowano metodę identyfikacji nicieni gatunków Anisakis, Hysterothyla-cium, Contracaecum przy użyciu techniki PCR/RFLP.
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Opracowanie zestawu diagnostycznego do genetycznego typowania szczepów bakteryjnych metodą PCR MP
PublicationOmówiono etapy tworzenia zestawu diagnostycznego przeznaczonego do genetycznego typowania szczepów bakteryjnych wykorzystującego procedurę PCR MP (PCR Melting Profiles). Dokonano optymalizacji warunków i walidacji metody w celu przystosowania jej do potrzeb epidemiologicznej oceny zakażeń szpitalnych. Opracowany zestaw stwarza możliwości zastosowania go w laboratoriach prowadzących rutynowo badania diagnostyczne.
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Identification of Leaf Rust Resistance Genes in Selected Wheat Cultivars and Development of Multiplex PCR
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ADSRRS-fingerprinting and PCR MP techniques for studies of intraspecies genetic relatedness in Staphylococcus aureus
PublicationW pracy pokazano użyteczność dwóch nowych metod do typowania molekularnego Staphylococcus aureus, ADSRRS-fingerprinting (Amplification of DNA fragments Surrounding Rare Restriction Sites) i PCR MP (PCR melting profile). Przebadano 37 szczepów Staphylococcus aureus izolowanych od pacjentów z czyracznością. Jako metodę referencyjną zastosowano REA-PFGE. Okazało się, że izolaty Staphylococcus aureus z nosa od niektórych pacjentów...
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Nested PCR for the detection of Aspergillus species in maxillary sinus samples of patients with chronic sinusitis
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Principles and applications of Ligation Mediated PCR methods for DNA-based typing of microbial organisms
PublicationA 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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Comparative studies of the Acinetobacter genus and the species identification method based on the recA sequences
PublicationZaproponowano metodę genotypowania bakterii rodzaju Acinetobacter, opartą o analizę sekwencji genów recA, amplifikację fragmentów genu recA i analizę profili fragmentów restrykcyjnych po cięciu produktów reakcji PCR enzymami restrykcyjnymi (recA PCR/RFLP). Profile RFLP otrzymane za pomocą enzymu Tsp509I są użyteczne w identyfikacji poszczególnych gatunków bakterii rodzaju Acinetobacter. Na podstawie analizy otrzymanych sekwencji...