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Celem pracy było zaprojektowanie i optymalizacja warunków reakcji w metodzie real-time PCR z wykorzystaniem sondy typu TaqMan do wykrywania enterowirusów człowieka. Użyto starterów oraz sondy komplementarnych dla konserwatywnych sekwencji w obrębie regionu niekodującego genomu enterowirusów (5'UTR). Sprawdzono swoistość metody wobec 19 taksonów enterowirusów z grup Coxsackie A, Coxsackie B, echowirusów i enterowirusów 68/71, a także innych wirusów zakażających człowieka. Określono zakres czułości metody względem szeregu dziesiętnych rozcieńczeń zawiesiny wzorcowych szczepów enterowirusów.
Badano przydatność zaprojektowanych starterów FopA F/R, Tul4 F/R i sond hybrydyzujących FopA S1/S2, Tul4 S1/S2 dla genów fopA i tul4 do wykrywania F. tularensis. W badaniach, w których użyto 50 szczepów F. tularensis uzyskano wyniki dodatnie. Swoistość reakcji wykazano badając szczepy bakterii non-Francisella tularensis. Przy użyciu starterów FopA F/R i sond hybrydyzujących FopA S1/S2 zaprojektowanych dla genów fopA dodatnie wyniki amplifikacji uzyskano ze wszystkimi badanymi szczepami F. tularensis. Identyczne wyniki otrzymano w reakcji real - time PCR z zastosowaniem starterów Tul4 F/R i sond hybrydyzujących Tul4 S1/ S2 zaprojektowanych dla genu tul4. Swoiste produkty reakcji amplifikacji pojawiły się między 16 a 18 cyklem reakcji. Badania z użyciem starterów i sond hybrydyzujących zaprojektowanych dla genu fopA wykazały, że charakterystyczna temperatura topnienia produktów wynosiła 61°C, a dla genu tul4 60°C. Przy użyciu starterów Tul4 F/R i sond hybrydyzujących Tul4 S1/ S2 czułość oznaczeń wynosiła 10 fg/µl, a przy użyciu starterów FopA F/R i sond hybrydyzujących FopA S1/S2 1 fg/µl.
Traditional detection methods such as baiting or direct isolation take a long time and are incapable to handling large volume of material to be tested. The real−time PCR−based techniques are faster, more sensitive, more easily automated, and do not require post−amplification procedures. Species−specific primers for Phytophthora were designed based on the internal transcribed spacer regions (ITS) of rDNA collected from the NCBI DNA database. Primers and probes were designed using the Allele ID 7 at default search criteria. Specific probes were labeled with the reporter dyes JOE (6−carboxy−4,5−dichloro−2,7−dimethoxyfluorescein) at the 5' end and HBQ1 quencher at the 3' end (Sigma−Aldrich). The specificity of primers and fluorogenic probes was tested against genomic DNA of P. alni subsp. multiformis, P. lacustris and P. taxon hungarica. The real−time PCR reactions with the specific probes and primers yielded positive results with five concentrations of standards obtained by standard PCR reaction for corresponding Phytophthora species. The negative control (lack of DNA pathogens) yielded no amplification products. Standard curves showed a linear correlation between input DNA and cycle threshold (Ct) values with R² from 0.994 (P. alni) to 0.998 (P. taxon hungarica). The amplification efficiency of target DNA varied from 94.6% (P. alni) to 100% (P. taxon hungarica). The validation of the primers and probes designed for analysed Phytophthora species was performed on pure cultures, on soil samples from the forest nursery and declining oak stands. The designed probes displayed the high specificity of the detection of investigated species in pure cultures. The presented new molecular TaqMan probes can fully assist the integrated pest management as a powerful tool for a quick detection of above pathogenic organisms in forest nurseries. The molecular detection of harmful phytophthoras and in consequences diminishing of fungicides use for their control in forestry fully support European Union directives as well as the ‘Good plant protection practice measures' elaborated by European and Mediterranean Organisation of Plant Protection.
Despite the permanent development of laboratory techniques, various types of adulterations are still a problem in the food industry. An important group among different frauds is adulterations connected with meat species authenticity. Uncovering of adulterated meat products is important inter alia for allergic individuals, for those who can’t intake certain species because of religious beliefs, and for maintaining fair-trade. More subtle techniques used for the mentioned adulterations generate a need for the elaboration of better analytical methods to provide effective control of meat and meat products. The aim of this review is to present currently used laboratory methods applied for meat species identification and detection of adulterations in the declared composition of meat products. The first group of the described methods enables identification and analysis of proteins and the second presented group contains techniques of DNA analysis. Apart from their short characteristics, some disadvantages and potential problems found during work with certain methods are described.
Dietetic and nutritional value of fish and shellfish products have made them very attractive food products for consumers. This results in both the increase of seafood consumption per capita and more diverse offers of finfish/shellfish species on the market. Nevertheless, while pursuing profits some of the producers/importers may replace valuable, more costly raw materials with cheaper substitutes. In order to efficiently monitor what is in the product and not to allow such practices to take place it is necessary to apply reliable, fast methods as to verify their contents. Until recently the methods used to test the products’ components were based mostly on electrophoretic, chromatographic or the immunological tests. At presents to detect food adulteration the PCR technique and its variety based on species polymorphy (multiplex PCR, PCR-RFLP, PCR-RAPD, PCR-AFLP, PCR-FIN or PCR-SSCP) are used. The latest achievements in identification and differentiation between the species are real-time PCR and microarray DNA. Discussions on how to improve the methods of identification/differentiation and on the possible application of such methods in routine quality control of seafood by the appropriate authorities are in progress.
Listeria monocytogenes is an important foodborne pathogen that causes a disease known as listeriosis, which is especially dangerous for pregnant women. Infection with L. monocytogenes may also result in stillbirths, abortions and premature deliveries, as well as meningitis, septicaemia, encephalitis, and meningoencephalitis. Conventional detection methods of these bacteria are time-consuming; therefore, rapid alternative methods, including those based on molecular tests are needed. PCR is sensitive and specific; however, it requires the use of an agarose gel, which increases the time of analysis. A technique that allows the elimination of this step is real-time PCR, which enables the quantitative determination of L. monocytogenes in foods. A modification of PCR is multiplex PCR that allows detection of several genes at the same time and distinguishes between different species of microorganisms. Techniques such as RT-PCR or NASBA, where the target molecule is RNA, are used to detect viable cells and also allow quantitative analyses to be performed. Another rapid and specific method is LAMP, which can be performed in one hour in a water bath or heating block, without the use of a thermocycler. Biosensors and microarrays are examples of new technologies that due to the possibility to use anywhere and immediate interpretation of the results can be routinely used in the future for identification of L. monocytogenes in food.
Celem pracy była optymalizacja metody real-time PCR do wykrywania oraz różnicowania zakażeń ludzkimi wirusami opryszczki typu 1 i 2, zwłaszcza w przypadkach infekcji przebiegających z niewielkim nasileniem replikacji patogenu.
Diagnostics of Mycoplasma synoviae (MS) is generally based on serological, culturing and molecular methods. Rapid diagnosis and identification of infections is very important in the poultry industry. The use of PCR or real-time PCR makes it possible to shorten the time for obtaining results from research and the effective detection of genetic material of MS. There are many variations of the PCR method, one of them is the LAMP method, which is rapid, very sensitive and does not require specialist equipment. This article reviews the molecular methods used for the diagnosis of Mycoplasma synoviae infection in poultry.
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