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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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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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PCR and real-time PCR assays to detect fungi of Alternaria alternata species
PublikacjaFungi 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
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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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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PCR detection of Scopulariopsis brevicaulis
PublikacjaScopulariopsis 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
PublikacjaW 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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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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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%)...
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Optimised five-hour multiplex PCR test for the detection of Tinea ungium: performance in a routine PCR laboratory
PublikacjaWe 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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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 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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The construction and use of a PCR internal control
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PCR and real-time PCR approaches to the identification of Arthroderma otae species Microsporum canis and Microsporum audouinii/Microsporum ferrugineum
PublikacjaObjectives 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
PublikacjaWe 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
PublikacjaThe 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 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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PRZYDATNOŚĆ DIAGNOSTYCZNA REAKCJI PCR W ROZPOZNAWANIU BORELIOZY
PublikacjaBorelioza 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
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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Real-Time PCR: molecular technique of many applications
PublikacjaReal-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...