PCR methods
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Two DNA-based techniques were used for species identification of enterococci. PvuII digestion of the genus-specific PCR product yielded four different restriction profiles among 20 enterococcal species; one of them was species-specific for E. faecium. In the second case, 32 reference strains belonging to 20 enterococcal species were divided to 12 groups by amplification of internal transcribed spacer of rRNA operon. Interspecies and some intraspecies profile variability was determined. Both methods gave similar results.
- MeSH
- druhová specificita MeSH
- Enterococcus klasifikace genetika metabolismus MeSH
- intergenová DNA chemie genetika MeSH
- polymerázová řetězová reakce metody MeSH
- protozoální DNA chemie genetika MeSH
- restrikční endonukleasy typu II metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- CAGCTG-specific type II deoxyribonucleases MeSH Prohlížeč
- intergenová DNA MeSH
- protozoální DNA MeSH
- restrikční endonukleasy typu II MeSH
PCR detection of fungal pathogens in clinical samples has been discussed in journals for more than two decades. However, its use for diagnosing invasive aspergillosis is still controversial, despite the fact that molecular methods are routinely used in various fields of modern microbiology. These are e. g. genotyping of bacterial strains resistant to antibiotics, molecular epidemiology or routine detection of viral infections in clinical material. PCR methods have made the diagnostic applications faster, simpler and more accurate. This review deals with issues related to molecular methods for diagnosing invasive fungal infections and the main factors limiting their use in everyday clinical practice.
- MeSH
- Aspergillus izolace a purifikace MeSH
- aspergilóza diagnóza MeSH
- DNA fungální analýza MeSH
- lidé MeSH
- polymerázová řetězová reakce * MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- anglický abstrakt MeSH
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- DNA fungální MeSH
The practices based on microbe cultivation are the corner stone of laboratory diagnostics of infectious diseases etiology. However, cultivation-independent molecular methods for microorganism detection are increasingly being used in a routine clinical setting as an important complement to the conventional procedures and, in some cases, they have already become the golden diagnostic standard. The PCR-based methods are used the most frequently.Key words: molecular genetic detection of microorganisms - polymerase chain reaction (PCR).
- MeSH
- Cardiobacterium genetika MeSH
- dospělí MeSH
- gramnegativní bakteriální infekce diagnóza MeSH
- infekce bakteriemi řádu Actinomycetales diagnóza MeSH
- lidé středního věku MeSH
- lidé MeSH
- polymerázová řetězová reakce * MeSH
- Tropheryma genetika MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
The authors give an account of hybridization methods, in particular DNA probes and the polymerase chain reaction (PCR) and their application in microbiological diagnosis. They deal with the principle of the two methods and their applications in the diagnosis not only of genera and species but also different factors of pathogenicity and virulence. The authors mention the advantages and disadvantages of radioactively and non-radioactively labelled probes such as PCR. They draw attention to the perspective application of these methods in research and diagnostic laboratories.
Since its discovery, PCR has become a conventional method of molecular biology research laboratories and an indispensable tool in diagnostic medicine. Multiple variants of the PCR technique were developed, which enable the analysis of different bio-logical materials at different amounts and reaction conditions. This article briefly summarizes the PCR approaches and points out their applications in oncological research and practice.
- MeSH
- biomedicínský výzkum MeSH
- lidé MeSH
- nádory genetika MeSH
- polymerázová řetězová reakce metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- anglický abstrakt MeSH
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
UNLABELLED: Epidemiological data indicate that raw vegetables are associated with outbreaks of Listeria monocytogenes. Therefore, there is a demand for the availability of rapid and sensitive methods, such as PCR assays, for the detection and accurate discrimination of L. monocytogenes. However, the efficiency of PCR methods can be negatively affected by inhibitory compounds commonly found in vegetable matrices that may cause false-negative results. Therefore, the sample processing and DNA isolation steps must be carefully evaluated prior to the introduction of such methods into routine practice. In this study, we compared the ability of three column-based and four magnetic bead-based commercial DNA isolation kits to extract DNA of the model micro-organism L. monocytogenes from raw vegetables. The DNA isolation efficiency of all isolation kits was determined using a triplex real-time qPCR assay designed to specifically detect L. monocytogenes. The kit with best performance, the PowerSoil(™) Microbial DNA Isolation Kit, is suitable for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. Coupled with the triplex real-time qPCR assay, this DNA isolation kit is applicable to the samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Several recent outbreaks of Listeria monocytogenes have been associated with the consumption of fruits and vegetables. Real-time PCR assays allow fast detection and accurate quantification of microbes. However, the success of real-time PCR is dependent on the success with which template DNA can be extracted. The results of this study suggest that the PowerSoil(™) Microbial DNA Isolation Kit can be used for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. This method is applicable to samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes.
- Klíčová slova
- DNA isolation, Listeria monocytogenes, inhibition, real-time PCR, vegetable,
- MeSH
- DNA bakterií genetika izolace a purifikace MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- Listeria monocytogenes genetika izolace a purifikace MeSH
- listeriové infekce prevence a kontrola MeSH
- nemoci přenášené potravou prevence a kontrola MeSH
- potravinářská mikrobiologie metody MeSH
- zelenina mikrobiologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA bakterií MeSH
BACKGROUND: Quantification of fluorescence labelled PCR products on capillary electrophoresis (QF PCR) has limits primarily in the possibility of more sensitive analyses to detect minority cell lines and small time related variations. PCR efficiency and human factor affect measuring error and reproducibility of results in these cases. The aim of this work was to assess and optimise of innovated (I)QF PCR in quantification of Y sequences in gonosomal mosaics. METHODS AND RESULTS: Artificially prepared Y/X mosaics were tested and quantified by IQF PCR, which replaces real-time PCR. Comparison of relative fluorescence to PCR cycles in different Y/X dilutions was plotted on the graphs. Calibration curve for Y sequences quantification was set by the analyses of ratio of Y/X fluorescent signals. An empirical formula was created for the rare mosaic calculation. CONCLUSIONS: QF PCR refined by manual real-time PCR eliminates limits of QF PCR and specifies quantitative analyses based on PCR. The outstanding feature of IQF PCR is its high sensitivity and accuracy in quantification of Y/X gonosomal mosaics.
The polymerase chain reaction (PCR) and biological methods for detection of enterotoxigenic strains of Escherichia coli were compared. The tests for LT, STa and STb were done in the cell line Y1, suckling mice, and ligated piglet intestinal loops, respectively. The production of STb was tested only in strains negative for LT and STa. The polymerase chain reaction has proved to be specific and reliable method than the biological methods. Unlike the latter, PCR allows the detection of strains producing in vitro only low quantities of the toxin. On the other hand, PCR fails to identify strains producing toxic substances with a different structure but the same or similar biological properties.
- MeSH
- bakteriologické techniky MeSH
- enterotoxiny biosyntéza MeSH
- Escherichia coli klasifikace genetika metabolismus MeSH
- myši MeSH
- polymerázová řetězová reakce MeSH
- prasata MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- Názvy látek
- enterotoxiny MeSH
The author compared nine methods for isolation of specific DNA of the etiological agent of human granulocytic ehrlichiosis from human blood for examination of the PCR. To full blood of healthy donors a laboratory culture of the agent was added and the effectiveness of isolation an stability of the template was tested under conditions when the blood was fresh or frozen. For universal use QIAamp (QIAGEN) was most suitable, frozen blood was extracted best using NucleoSpin Tissue (Macherey-Nagel).
- MeSH
- Anaplasma phagocytophilum genetika izolace a purifikace MeSH
- DNA bakterií krev izolace a purifikace MeSH
- ehrlichióza diagnóza MeSH
- lidé MeSH
- polymerázová řetězová reakce * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- anglický abstrakt MeSH
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- DNA bakterií MeSH
Enteroviruses belonging to the family Picornaviridae are important human pathogens. Although most cases of infection caused by these viruses are asymptomatic, a wide range of clinical syndromes is observed in manifest cases. Conventional laboratory diagnostic methods based on virus isolation and identification, or on the detection of specific antiviral antibodies, are costly and time consuming. Therefore, they are of little benefit to treatment. We have implemented a commercially available PCR-based test for the detection of enteroviral infections. In 2008, we analyzed biological specimens from 125 patients with suspected enteroviral disease, most often involving the nervous system. The presence of enterovirus was detected in 39 patients. The results were compared with those obtained by the conventional methods. PCR appeared to be a valuable method for rapid, accurate and reliable diagnosis of enteroviral infections which is of major benefit to patient management.
- MeSH
- enterovirové infekce diagnóza MeSH
- Enterovirus genetika MeSH
- lidé MeSH
- myokarditida diagnóza virologie MeSH
- polymerázová řetězová reakce * MeSH
- RNA virová analýza MeSH
- sérologické testy MeSH
- virové nemoci CNS diagnóza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- anglický abstrakt MeSH
- časopisecké články MeSH
- Názvy látek
- RNA virová MeSH
