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Kontaminace pitných vod původci virové gastroenteritidy (př. noroviry, rotaviry, adenoviry) představuje ve vodním hospodářství výzvu, zejména při ochraně zdrojů surové vody a dezinfekci pitné vody. V této práci mapujeme výskyt a možnosti odstraňování virových původců gastroenteritidy v oběhovém vodním hospodářství s cílem omezit riziko ohrožení veřejného zdraví v důsledku virové kontaminace. Enterické viry se do odpadních vod dostávají stolicí infikovaných jedinců. Při konvenčním mechanicko‐biologickém čištění odpadních vod jsou odstraňovány jen částečně a společně s viry v odlehčených odpadních vodách mohou pronikat až do zdrojů surové vody pro úpravny vod. Během procesů úpravy vody dochází ke snižování jejich množství, zejména díky pokročilým oxidačním procesům a dezinfekci. Zatímco v rozvojových zemích je přítomnost těchto virů v distribučních sítích běžná a dlouhodobá, v České republice je jejich výskyt zaznamenáván pouze výjimečně, a to při haváriích, například v Praze v roce 2015 nebo v Libereckém kraji v roce 2022. Z důvodu snížení potenciálních rizik při úpravě vody je vhodné sledovat míru znečištění viry v celém antropogenním vodním cyklu a zamezit únikům nevyčištěné odpadní vody do prostředí.
Contamination of drinking water by viral agents of gastroenteritis (e.g., norovirus, rotavirus, adenovirus) poses a challenge for water management, especially in protecting the freshwater resources and water disinfection. Mapping the occurrence of these viral pathogens within the anthropogenic water cycle can help reduce the risk to public health resulting from viral contamination. Enteric viruses are excreted in the feces of infected individuals and enter the wastewater. During conventional mechanical‐biological wastewater treatment, the excreted viruses are removed only partially and, together with viruses in overflow discharges, may reach raw water sources for drinking water production. During water treatment processes, their concentrations are reduced, especially by advanced oxidation processes and disinfection. The presence of these viruses in distribution systems has been documented even in the Czech Republic during incidents (e.g., Prague 2015, Liberec region 2022) and is a persistent issue in developing countries. Therefore, it is essential to monitor viral contamination throughout the entire anthropogenic water cycle and to prevent the release of untreated wastewater into the environment.
Autentizace rybích výrobků s využitím analýzy DNA vyžaduje zisk kvalitní DNA bez přítomnosti inhibitorů. V současné době jsou dostupné různé metody pro izolaci nukleových kyselin; pro svou rychlost a nenáročnost extrakčního postupu se staly velmi oblíbenými zejména silikátové centrifugační kolonky. Jejich nevýhodou však může být princip využívající záporný náboj DNA, který může být ovlivněn složením potravin, nebo jejich ucpání v důsledku špatné předúpravy vzorků. Cílem této práce bylo porovnat tři metody izolace DNA využívající různé principy (silikátové centrifugační kolonky, modifikované magnetické kuličky, cetyltrimethylamonium-bromid (CTAB) a chloroformová extrakce) a zhodnotit jejich vhodnost pro izolaci DNA z rybí svaloviny. Posuzovanými kritérii byla výtěžnost, čistota a amplifikovatelnost izolované DNA. Analyzována byla tkáň makrely obecné bez a s přídavkem přídatných látek běžně používaných při výrobě rybích produktů, konkrétně difosforečnanů (E 450) a barviv (E 110 a E 124), a následně byla vybraná metoda aplikována i na komerčně nabízené výrobky z ryb. Jako nejvhodnější se ukázala upravená metoda využívající detergent CTAB.
Authentication of fish products by DNA analysis requires the extraction of high quality DNA without the presence of inhibitors. Many nucleic acid isolation methods are currently available; silicate centrifugal columns have become very popular due to their speed and ease of extraction. However, their disadvantage may be the principle based on DNA charge, which may be affected by food composition, or clogging due to a poor sample pretreatment. The aim of this work was to compare three DNA isolation methods using different principles (silicate centrifugal columns, modified magnetic beads, Cetrimonium bromide and chloroform extraction) and to evaluate their suitability for DNA isolation from fish muscle. The criteria assessed were the recovery, purity and amplifiability of the isolated DNA. Mackerel tissue was analysed without and with the addition of additives commonly used in the manufacture of fish products, namely diphosphates (E 450) and colorants (E 110 and E 124), and the selected method was subsequently applied to commercial fish products. The modified method using the detergent CTAB proved to be the most suitable.
- MeSH
- analýza potravin metody MeSH
- DNA analýza izolace a purifikace MeSH
- lidé MeSH
- polymerázová řetězová reakce MeSH
- potrava z moře (živočišná) analýza MeSH
- potravinářské přísady analýza MeSH
- rybí výrobky * analýza MeSH
- ryby MeSH
- techniky amplifikace nukleových kyselin metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Many reports have documented that the presence of SARS-CoV-2 RNA in the influents of municipal wastewater treatment plants (WWTP) correlates with the actual epidemic situation in a given city. However, few data have been reported thus far on measurements upstream of WWTPs, i.e. throughout the sewer network. In this study, the monitoring of the presence of SARS-CoV-2 RNA in Prague wastewater was carried out at selected locations of the Prague sewer network from August 2020 through May 2021. Various locations such as residential areas of various sizes, hospitals, city center areas, student dormitories, transportation hubs (airport, bus terminal), and commercial areas were monitored together with four of the main Prague sewers. The presence of SARS-CoV-2 RNA was determined by reverse transcription - multiplex quantitative polymerase chain reaction (RT-mqPCR) after the precipitation of nucleic acids with PEG 8,000 and RNA isolation with TRIzolTM Reagent. The number of copies of the gene encoding SARS-CoV-2 nucleocapsid (N1) per liter of wastewater was compared with the number of officially registered COVID-19 cases in Prague. Although the data obtained by sampling wastewater from the major Prague sewers were more consistent than those obtained from the small sewers, the correlation between wastewater-based and clinical-testing data was also good for the residential areas with more than 7,000 registered inhabitants. It was shown that monitoring SARS-CoV-2 RNA in wastewater sampled from small sewers could identify isolated occurrences of COVID-19-positive cases in local neighborhoods. This can be very valuable while tracking COVID-19 hotspots within large cities.
- MeSH
- čištění vody * MeSH
- COVID-19 * epidemiologie MeSH
- lidé MeSH
- odpadní voda MeSH
- RNA virová MeSH
- SARS-CoV-2 MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Opium poppy (Papaver somniferum L.) is a versatile plant exploited by the pharmaceutical and food industries. Unfortunately, it is also infamously known as a source of highly addictive narcotics, primarily heroin. Drug abuse has devastating consequences for users and also has many direct or indirect negative impacts on human society as a whole. Therefore, developing a molecular genetic tool for the individualization of opium poppy, raw opium or heroin samples could help in the fight against the drug trade by retrieving more information about the source of narcotics and linking isolated criminal cases. Bioinformatic analysis provided insight into the distribution, density and other characteristics of roughly 150 thousand microsatellite loci within the poppy genome and indicated underrepresentation of microsatellites with the desired attributes. Despite this fact, 27 polymorphic STR markers, divided into three multiplexed assays, were developed in this work. Internal validation confirmed species-specific amplification, showed that the optimal amount of DNA is within the range of 0.625-1.25 ng per reaction, and indicate relatively well balanced assays according to the metrics used. Moreover, the stutter ratio (mean + 3 SD 2.28-15.59%) and allele-specific stutters were described. The analysis of 187 individual samples led to the identification of 158 alleles in total, with a mean of 5.85 alleles and a range of 3-14 alleles per locus. Most of the alleles (151) were sequenced by the Sanger method, which enabled us to propose standardized nomenclature and create three allelic ladders. The OpiumPlex system discriminates most of the varieties from each other and pharmaceutical varieties from the others (culinary, dual and ornamental).
This study is focused on the analysis of extracellular DNA (eDNA) from a biofilm matrix formed by Staphylococcus aureus, Listeria monocytogenes, and Salmonella enterica. The presence of eDNA in the biofilm of all the studied strains was confirmed by confocal laser scanning microscopy using fluorescent dyes with high affinity to nucleic acid. The protocol for eDNA isolation from the biofilm matrix was established, and subsequent characterization of the eDNA was performed. The purified eDNA obtained from the biofilm matrix of all three microorganisms was compared to the genomic DNA (gDNA) isolated from relevant planktonic grown cells. The process of eDNA isolation consisted of biofilm cultivation, its collection, sonication, membrane filtration, dialysis, lyophilisation, and extraction of DNA separated from the biofilm matrix with cetyltrimethylammonium bromide. An amplified fragment length polymorphism (AFLP) was used for comparing eDNA and gDNA. AFLP profiles showed a significant similarity between eDNA and gDNA at the strain level. The highest similarity, with a profile concordance rate of 94.7% per strain, was observed for S. aureus, L. monocytogenes, and S. enterica exhibited lower profiles similarity (78% and 60%, respectively). The obtained results support the hypothesis that the eDNA of studied bacterial species has its origin in the gDNA.
Polymerase chain reaction (PCR) is one of the main techniques of molecular biology. Currently, there is a boom of digital PCR which allows precise, sensitive and reproducible quantification without using a calibration curve. Due to its features, digital PCR has a wide analyti-cal application in many areas (e.g. biomedical applications or food analysis). Presently, there are several types of digi-tal PCR that differ from each other particularly in the way of dispersing the sample into many aliquots of very small volumes. All platforms allow multiplex analyses based on the use of differently labelled probes and/or different con-centrations of probes labelled with one fluorescent color. This can reduce the financial and time requirements for a single sample analysis. The advantages and disadvantages of each type of digital PCR are summarized in this article.
Genetically modified organism is an organism whose genetic material has been intentionally altered using genetic engineering techniques. The number of genetically modified organisms that are being developed and used increases every year. In many countries the use of genetically modified organisms is regulated and it is therefore important to introduce legal proceedings, regulatory instructions and reliable methods for the detection and quantification of genetically modified organisms. These regulations are different for each country. Somewhere, the cultivation of genetically modified crops is allowed and the genetically modified products need not to be labelled, elsewhere, the approval process concerns every genetically modified event in European Union. Somewhere, genetically modified organisms are forbidden. With the growing number of these products, the number of opponents who are worried about genetically modified products is rising. They look for GMO-free food and this labelling is more common on the market due to adaptive marketing strategy.
Nowadays, food adulteration is a common problem. Fish meat is a typical commodity which is adulterated very often. Therefore, it is necessary to find effective methods for fish meat authentication. Molecular-biological methods based on the analysis of mitochondrial and genomic markers were proved to be suitable for this purpose. Especially, polymerase chain reaction is commonly used, but there are new methods like Loop-mediated Isothermal Amplification or Multi-Analyte Profiling with a huge potential for fish species identification. In this paper, advantages and disadvantages of a use of different markers and three molecular-biological methods for the purpose of fish fraud detection are summarized.
