Overview and recommendations for the application of digital PCR

The digital Polymerase Chain Reaction (dPCR), for the detection and absolute quantification of DNA, is a relatively new technique but its application in analytical laboratories is steadily increasing. In contrast to quantitative real-time PCR, DNA (fragments) can be quantified without the need for standard curves. Using dPCR, the PCR mix containing the (target) DNA is partitioned – depending on the device used – currently into a maximum of 10,000,000 small compartments with a volume as low as a few picoliters. These can be either physically distinct compartments on a chip (referred to as chamber-based digital PCR [cdPCR]), or these compartments correspond to water-in-oil droplets (referred to as droplet digital [ddPCR]). Common to both approaches, once PCR has been carried out simultaneously in all compartments/droplets, the number of positive and negative signals for each partition is counted by fluorescence measurement. With this technique, an absolute quantification of DNA copy numbers can be performed with high precision and trueness, even for very low DNA copy numbers. Furthermore, dPCR is considered less susceptible than qPCR to PCR inhibitory substances that can be co-extracted during DNA extraction from different sources. Digital PCR has already been applied in various fields, for example for the detection and quantification of GMOs, species (animals, plants), human diseases, food viruses and bacteria including pathogens. When establishing dPCR in a laboratory, different aspects have to be considered. These include, but are not limited to, the adjustment of the type of the PCR master mix used, optimised primer and probe concentrations and signal separation of positive and negative compartments. This document addresses these and other aspects and provides recommendations for the transfer of existing real-time PCR methods into a dPCR format.JRC.F.5-Food and Feed Complianc

The awareness of students of non-medical medical disciplines in the field of eye issues

Cílem bakalářské práce je poukázat na důležitost a přínos preventivních očních vyšetření v české populaci. Analyzovat informovanost studentů středních zdravotnických škol v oblasti oční problematiky. Odhalit rozdílnost v kvalitě vzdělávání na dvou středních zdravotnických školách v Pardubickém kraji a jedné střední zdravotnické škole v Královéhradeckém kraji. Zjistit, jak velký je zájem u studentů o tento obor.The aim of the bachelor thesis is the point out the importance and benefits of preventive eye examinations in Czech population. Analyze the awareness of students from two the secondary schools of nursing in Pardubice region and once the secondary school of nursing in Hradec Kralove region in the field of eye issues. Find out the differences in the quality of education at secondary medical schools in the three unnamed cities of East Bohemian region. Find out how much interest students have in this medical field.Fakulta zdravotnických studiíDoplňující otázky pro obhajobu závěrečné práce: 1. Ještě jednou charakterizujte, kdo byli respondenti a o jaké obory se jednalo? Nešlo pouze o praktické sestry? Pak by název práce měl být i v tomto směru upraven. 2. Z jakých materiálů jste čerpala v případě zpracování kapitoly 2.9. Učební osnovy ? doložte zdroj k nahlédnutí k obhajobě. 3. Proč jste ?předvýzkum? provedla u absolventů, kteří nespadají do vzorku oslovených studentů 4. ročníků? 4. Vysvětlete Vaše tvrzení v závěru práce, že informovanost je podle hodnotící skály "dostačující"? Obhajoba bakalářské práce s prezentací dobrá.Dokončená práce s úspěšnou obhajobo

The sampling methodology for GM plants unintentionally present in the environment

In this methodology, sampling of plants from agricultural crops(corn, soybean, canola and potato) is defined to assess the presence of unauthorized GM plants in the environment.The methodology is applied mainly in the field of inspection controls of the compliance with Act no.78/2004. The sampling and collected samples must represent to a maximum extent the crops of the controlled part of the field (land parcel or land parcel portion). The basic applications of the methodology include:(a) low portion of unauthorized GM plants(seed contaminated with GM plants unauthorized for cultivation), (b) monitoring of the presence of unauthorized GM plants on land parcel, where there was such an occurence recorded in previous years (for example, plants growing on the field from seeds or tubers from the so called "soil´s seed bank")

Guidance document on multiplex real-time PCR methods

This document provides guidance for the development of new qualitative and quantitative multiplex real-time methods (mpPCR) and their implementation in a routine testing laboratory for food, feed and seeds. When implementing a mpPCR method, the laboratory should perform either an (in-house) validation or a verification of the method before its use. The various method performance parameters of the specific requirements for mpPCR are described and examples are given. Several different aspects have been considered, including the selection of the fluorophores, the master mix and the real-time PCR instrument used. The technical aspects are examined and recommendations are made on the choice of the detection and quantification strategy. Finally, a separate chapter is dedicated to troubleshooting (e.g. underperforming method, no or poor signal strength, cross-talk). In addition, a collection of existing mpPCR methods with emphasis on GMO detection is presented

Impaired behavioural response to alarm substance in rainbow trout exposed to copper nanoparticles.

RCUK OA article = NE/G0018182/1To 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

Autosomal dominant ApoA4 mutations present as tubulointerstitial kidney disease with medullary amyloidosis

Sporadic cases of apolipoprotein A-IV medullary amyloidosis have been reported. Here we describe five families found to have autosomal dominant medullary amyloidosis due to two different pathogenic APOA4 variants. A large family with autosomal dominant chronic kidney disease (CKD) and bland urinary sediment underwent whole genome sequencing with identification of a chr11:116692578 G>C (hg19) variant encoding the missense mutation p.L66V of the ApoA4 protein. We identified two other distantly related families from our registry with the same variant and two other distantly related families with a ch11:116693454 C>T (hg19) variant encoding the missense mutation p.D33N. Both mutations are unique to affected families, evolutionary conserved and predicted to expand the amyloidogenic hotspot in the ApoA4 structure. Clinically affected individuals suffered from CKD with a bland urinary sediment and a mean age for kidney failure of 64.5 years. Genotyping identified 48 genetically affected individuals; 44 individuals had an estimated glomerular filtration rate (eGFR) under 60 ml/min/1.73 m2, including all 25 individuals with kidney failure. Significantly, 11 of 14 genetically unaffected individuals had an eGFR over 60 ml/min/1.73 m2. Fifteen genetically affected individuals presented with higher plasma ApoA4 concentrations. Kidney pathologic specimens from four individuals revealed amyloid deposits limited to the medulla, with the mutated ApoA4 identified by mass-spectrometry as the predominant amyloid constituent in all three available biopsies. Thus, ApoA4 mutations can cause autosomal dominant medullary amyloidosis, with marked amyloid deposition limited to the kidney medulla and presenting with autosomal dominant CKD with a bland urinary sediment. Diagnosis relies on a careful family history, APOA4 sequencing and pathologic studies