DNA metabarcoding provides great potential for species identification in complex samples such as food supplements and traditional medicines. Such a method would aid Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. A DNA metabarcoding method was developed that makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa and that facilitate the identification of species in samples containing degraded DNA. The DNA metabarcoding method was developed based on Illumina MiSeq amplicon sequencing of well-defined experimental mixtures, for which a bioinformatics pipeline with user-friendly web-interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. The advanced multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES-listed species. The method can provide improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to an enhanced quality assurance.
After induced mutagenesis and transgenesis, genome editing is the next step in the development of breeding techniques. Genome editing using site-directed nucleases - including meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system - is based on the mechanism of double strand breaks. The nuclease is directed to cleave the DNA at a specific place of the genome which is then repaired by natural repair mechanisms. Changes are introduced during the repair that are either accidental or can be targeted if a DNA template with the desirable sequence is provided. These techniques allow making virtually any change to the genome including specific DNA sequence changes, gene insertion, replacements or deletions with unprecedented precision and specificity while being less laborious and more straightforward compared to traditional breeding techniques or transgenesis. Therefore, the research in this field is developing quickly and, apart from model species, multiple studies have focused on economically important species and agronomically important traits that were the key subjects of this review. In plants, studies have been undertaken on disease resistance, herbicide tolerance, nutrient metabolism and nutritional value. In animals, the studies have mainly focused on disease resistance, meat production and allergenicity of milk. However, none of the promising studies has led to commercialization despite several patent applications. The uncertain legal status of genome-editing methods is one of the reasons for poor commercial development, as it is not clear whether the products would fall under the GMO regulation. We believe this issue should be clarified soon in order to allow promising methods to reach their full potential.
- MeSH
- editace genu metody MeSH
- endonukleasy genetika metabolismus MeSH
- genetické inženýrství metody MeSH
- geneticky modifikovaná zvířata * MeSH
- geneticky modifikované rostliny * MeSH
- genom rostlinný * MeSH
- odolnost vůči nemocem genetika MeSH
- zemědělské plodiny genetika MeSH
- zinkové prsty MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
BACKGROUND: Polymerase chain reaction (PCR) has become a common technique offering fast and sensitive analysis of DNA in food/feed samples. However, many substances, either already present in the sample or introduced during sample processing, inhibit PCR and thus underestimate the DNA content. It is therefore necessary to identify PCR inhibition in order to correctly evaluate the sample. RESULTS: We designed and validated a synthetic plasmid DNA that can be used to detect and quantify PCR inhibition. The DNA sequence, appropriate primers and probe, were designed in silico, synthesized and the sequence was inserted into a plasmid vector. The performance of the plasmid was verified via calibration curves and by performing the assay in the presence of various DNAs (crops, fungus, bacterium). The detection of PCR inhibition was assessed using six inhibiting substances with different modes of action, substances used in sample processing (EDTA, ethanol, NaCl, SDS) and food additives (sodium glutamate, tartrazine). The plasmid performance proved to be reproducible and there were no interactions with other DNAs. The plasmid was able to identify the presence of the inhibitors in a wide range of concentrations. CONCLUSION: The presented plasmid DNA is a suitable and inexpensive possibility for evaluating PCR inhibition.
To date, studies of the toxicity of engineered nanoparticles (NPs) in fish have not fully considered effects on olfactory-mediated behaviours, despite their ecological importance. In this study the effects of copper NPs (Cu NPs) on the anti-predator behavioural responses of juvenile rainbow trout (Oncorhynchus mykiss) to trout alarm substance was investigated. Individual fish were exposed for 12h to a control (no added Cu), 50μgl(-1) of Cu as Cu NPs, or 50μgl(-1) Cu as CuSO4, after which fish behaviours were analyzed in 10min periods before and after the addition of the alarm substance stimulus. The response of control fish to deionised water (negative control, no alarm substance stimulus) was also analyzed. The alarm substance elicited a behavioural response in the control fish characterized by an immediate freeze response and the slower resumption of swimming activity compared to negative controls exposed to the sham deionised water stimuli. In fish exposed to Cu NPs, the behavioural response to alarm substance was eliminated, with no significant difference in behaviours compared to negative controls. In comparison, exposure to 50μgl(-1) Cu as CuSO4 decreased, but did not eliminate the response of fish to alarm substance, which indicated a significantly greater effect of Cu NPs on olfactory mediated behaviours than of the equivalent concentration of Cu as CuSO4. Measurement of total Cu concentrations in the tissues of fish demonstrated no significant accumulation of Cu from any treatment in gill, liver or brain, confirming the effects of Cu NPs, and to a lesser extent CuSO4, on behavioural responses were mostly associated with the interaction of the materials with the external surfaces of the fish. Scanning electron microscopy revealed that Cu as CuSO4 caused a pronounced depletion of ciliated sensory and non-sensory cells in the olfactory rosette surrounding the midline raphe, whereas Cu NPs had no impact on the structure of the rosette. However, exposure to Cu NPs caused a significant increase in the ratio of oxidized to reduced glutathione in brains of fish, indicating some systemic oxidative stress that was not observed in either controls or fish exposed to CuSO4. Overall, the study showed that the olfactory mediated behaviours of fish were potentially more sensitive to Cu NPs than CuSO4 and NPs elicited effects via a mechanism that is distinct from that of the metal salt.
- MeSH
- bulbus olfactorius účinky léků ultrastruktura MeSH
- chemické látky znečišťující vodu analýza toxicita MeSH
- chování zvířat účinky léků MeSH
- glutathion metabolismus MeSH
- měď analýza toxicita MeSH
- mozek účinky léků MeSH
- nanočástice toxicita MeSH
- Oncorhynchus mykiss fyziologie MeSH
- síran měďnatý toxicita MeSH
- žábry účinky léků MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Polymerázová řetězová reakce, známá pod anglickou zkratkou PCR, se od svého objevu v 80. letech minulého století stala jednou ze základních metod detekce a kvantifikace nukleových kyselin. Díky vysoké rychlosti, citlivosti a specificitě se používá v mnoha aplikacích v oblasti molekulární biologie, přírodních věd, zemědělství i medicíny. Do reakční směsi se ale mohou ze vzorku nebo v průběhu jeho zpracování dostat látky, které mohou PCR reakci inhibovat nebo stimulovat. Výsledkem je pak nadhodnocení, resp. podhodnocení obsahu DNA ve vzorku. Inhibici PCR reakce lze rozpoznat pomocí určení efektivity reakce, spočívající v analýze sériových ředění vzorku. Rychlejší a elegantnější způsob spočívá v použití interní kontroly, což je nejčastěji exogenní DNA, která je koamplifikována spolu se vzorkem.
Since its discovery in the 1980', the polymerase chain reaction (PCR) has become one of the main methods for detection and quantification of nucleic acids. Because of its speed, sensitivity and specificity, PCR is used in many applications in molecular biology, life sciences, agriculture and medicine. However, many substances may get into in the reaction mixture from the sample or during the sample processing that can inhibit or enhance the PCR reaction. As a result, the DNA content in the sample is under- or overestimated, respectively. It is possible to indentify the inhibition by evaluating the eÇciency of the PCR reaction by analysing serial dilutions of the sample. More elegant and faster method consists in using an internal control which is a usually exogenous DNA coamplified with the target DNA.
