Reverse transcription quantitative PCR (RT-qPCR) is already an established tool for mRNA detection and quantification. Since recently, this technique has been successfully employed for gene expression analyses, and also in individual cells (single cell RT-qPCR). Although the advantages of single cell measurements have been proven several times, a study correlating the expression measured on single cells, and in bulk samples consisting of a large number of cells, has been missing. Here, we collected a large data set to explore the relation between gene expression measured in single cells and in bulk samples, reflected by qPCR Cq values. We measured the expression of 95 genes in 12 bulk samples, each containing thousands of astrocytes, and also in 693 individual astrocytes. Combining the data, we described the relation between Cq values measured in bulk samples with either the percentage of the single cells that express the given genes, or the average expression of the genes across the single cells. We show that data obtained with single cell RT-qPCR are fully consistent with measurements in bulk samples. Our results further provide a base for quality control in single cell expression profiling, and bring new insights into the biological process of cellular expression.

• MeSH
• analýza jednotlivých buněk * MeSH
• gliový fibrilární kyselý protein genetika   MeSH
• messenger RNA genetika   MeSH
• myši transgenní MeSH
• myši MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• stanovení celkové genové exprese * MeSH
• zelené fluorescenční proteiny genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• antiinfekční látky farmakologie   MeSH
• Bacteria účinky léků   MeSH
• bakteriální léková rezistence MeSH
• mikrobiální testy citlivosti MeSH
• mléko mikrobiologie   MeSH
• prevalence MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH

• MeSH
• analýza potravin MeSH
• kontrola potravin MeSH
• krmivo pro zvířata mikrobiologie   normy   MeSH
• mikrobiologické techniky normy   MeSH
• potravinářská mikrobiologie MeSH
• potraviny normy   MeSH
• potravní řetězec MeSH
• Publikační typ
• směrnice MeSH

• Konspekt
• Metrologie. Standardizace
• NLK Obory
• mikrobiologie, lékařská mikrobiologie
• zemědělství a potravinářství
• NLK Publikační typ
• brožury

Enterotoxigenic Staphylococcus aureus in raw milk poses a potential health hazard to consumers, and the identification of such strains should be used as part of a risk analysis of milk and milk products. The primary purpose of this study was to investigate the occurrence of enterotoxigenic S. aureus strains in raw milk supplied for dairy processing in the Czech Republic. A further aim was to compare the production of staphylococcal enterotoxins (SEs) with the presence of the corresponding genes. This was undertaken using multiplex polymerase chain reaction (PCR) and reversed passive latex agglutination (RPLA). Out of 440 bulk tank milk samples from 298 dairy herds, 70 proved positive for S. aureus (15.9%). Staphylococcal enterotoxin genes (ses) were detected in 39 (55.7%) isolates. The genes most commonly detected were sei (38.6%), seg (31.4%) and sea (27.1%). Genes seb, seh, sed, sej and sec were observed in 10%, 4.3%, 2.9%, 2.9% and 1.4% of strains respectively. Genes see and sel did not occur. The most frequently detected genotypes were seg, sei at 11.4%; sea at 10.0%; and sea, seg, sei at 8.6%. Toxin production was observed in nine (12.9%) S. aureus isolates. Seven strains were detected as SEB- (10%) and two as SED- (2.9%) producing. A relatively high number (32%) of discrepancies between the results with multiplex PCR and RPLA assays was obtained, particularly on account of SEA. Nineteen strains were sea positive by PCR but SEA negative by RPLA, and one strain was sec positive and SEC negative. The results of both methods were identical concerning SEB and SED. It was concluded that detection of ses by PCR was a useful additional tool to support identification of enterotoxigenic strains.

• MeSH
• amplifikace genu MeSH
• antigeny bakteriální analýza   genetika   MeSH
• DNA bakterií genetika   chemie   MeSH
• enterotoxiny analýza   genetika   MeSH
• genotyp MeSH
• kontaminace potravin analýza   MeSH
• latex fixační testy MeSH
• lidé MeSH
• mléko MeSH
• počet mikrobiálních kolonií MeSH
• polymerázová řetězová reakce metody   MeSH
• potravinářská mikrobiologie MeSH
• prevalence MeSH
• sekvence nukleotidů MeSH
• skot MeSH
• spotřebitelská bezpečnost produktů MeSH
• Staphylococcus aureus izolace a purifikace   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Geografické názvy
• Česká republika MeSH

A rapid procedure for the determination of 2-aminoisobutyric acid in enzalutamide bulk drug substance based on hydrophilic interaction chromatography with fluorescence detection was developed. Fluorescence detection after postcolumn derivatization with o-phthaldialdehyde/2-mercaptoethanol was carried out at excitation and emission wavelength of 345 nm and 450 nm, respectively. The postcolumn reaction conditions such as reaction temperature, mobile phase and derivatization reagent flow rate and the reagents concentrations were studied and optimized due to steric hindrance of amino group of 2-aminoisobutyric acid. The derivatization reaction was applied for the hydrophilic interaction chromatography method which was based on COSMOSIL HILIC column with a mobile phase consisting of a mixture of 25 mmol/L acetic acid adjusted to pH 5.5 (using 1 mol/L potassium hydroxide) and acetonitrile using an isocratic elution (28:72, ν/ν). The benefit of the reported approach consists in a simple sample pretreatment and a quick and sensitive hydrophilic interaction chromatography method. The developed method was validated in terms of linearity, limit of detection, limit of quantification, accuracy, precision and selectivity according to the International Conference on Harmonisation guidelines. The developed method was demonstrated to be applied for the analysis of 2-AIBA in routine quality control evaluation of commercial samples of enzalutamide bulk drug substance.

• MeSH
• acetonitrily chemie   MeSH
• antagonisté androgenních receptorů analýza   chemie   MeSH
• fenylthiohydantoin analogy a deriváty   analýza   chemie   MeSH
• fluorescenční spektrometrie přístrojové vybavení   metody   MeSH
• hydrofobní a hydrofilní interakce MeSH
• kontaminace léku prevence a kontrola   MeSH
• kyseliny aminoisomáselné analýza   MeSH
• limita detekce MeSH
• merkaptoethanol chemie   MeSH
• o-ftalaldehyd chemie   MeSH
• reprodukovatelnost výsledků MeSH
• řízení kvality * MeSH
• vysokoúčinná kapalinová chromatografie přístrojové vybavení   metody   MeSH
• Publikační typ
• časopisecké články MeSH
• validační studie MeSH

The possibility that Mycobacterium avium subsp. paratuberculosis (MAP) plays some role in the development of Crohn's disease in humans is attracting attention to milk and milk products originating from infected animals. In this study, we focused on the detection of MAP in 220 bulk tank milk (BTM) samples from all dairy cattle herds in Cyprus. In total, 63 (28.6%) BTM milk samples were found to be positive for MAP using quantitative real-time PCR assays for IS900 and F57. The presence of MAP in BTM was low, and was assessed to be several tens of MAP cells per one ml of BTM. Milk samples examined by cultivation were found to be negative for MAP in all 220 BTM. In two BTM samples cultivation and subsequent sequencing of 16S rRNA revealed two isolates of M. fortuitum.

• MeSH
• bakteriální RNA analýza   MeSH
• kontaminace potravin analýza   MeSH
• lidé MeSH
• mléko mikrobiologie   MeSH
• Mycobacterium avium subsp. paratuberculosis izolace a purifikace   MeSH
• nemoci skotu diagnóza   přenos   MeSH
• paratuberkulóza diagnóza   přenos   MeSH
• plošný screening veterinární   MeSH
• počet mikrobiálních kolonií veterinární   MeSH
• polymerázová řetězová reakce veterinární   MeSH
• prediktivní hodnota testů MeSH
• RNA ribozomální 16S analýza   MeSH
• senzitivita a specificita MeSH
• skot MeSH
• zoonózy MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Geografické názvy
• Česká republika MeSH

Determination of an antihyperlipidemic drug simvastatin (SIM) was carried out using a carbon paste electrode bulk-modified with multiwalled carbon nanotubes (MWCNT-CPE). Scanning electrochemical microscopy (SECM), scanning electron microscopy (SEM), and atomic force microscopy (AFM) were used for the characterization of the prepared electrodes. Different electrodes were prepared varying in mass percentage of MWCNTs to find out the optimum amount of MWCNTs in the paste. The MWCNT-CPE in which the mass percentage of MWCNTs was 25% (w/w) was found as the optimum. Then, the prepared electrode was used in a mechanistic study and sensitive assay of SIM in pharmaceutical dosage form and a spiked human plasma sample using differential pulse voltammetry (DPV). The prepared electrode shows better sensitivity compared to the bare carbon paste and glassy carbon electrode (GCE). The detection limit and the limit of quantification were calculated to be 2.4 × 10-7 and 8 × 10-7, respectively. The reproducibility of the electrode was confirmed by the low value of the relative standard deviation (RSD% = 4.8%) when eight measurements of the same sample were carried out. Determination of SIM in pharmaceutical dosage form was successfully performed with a bias of 0.3% and relative recovery rate of 99.7%. Furthermore, the human plasma as a more complicated matrix was spiked with a known concentration of SIM and the spiking recovery rate was determined with the developed method to be 99.5%.

• MeSH
• elektrody * MeSH
• lidé MeSH
• mikroskopie elektronová rastrovací MeSH
• nanotrubičky uhlíkové chemie   ultrastruktura   MeSH
• simvastatin chemie   MeSH
• uhlík chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Similarly to many other sample extraction techniques, efficient extraction of very polar compounds with electromembrane extraction (EME) is difficult. To date, the best known strategy to improve the mass transfer of these compounds is the addition of an ionic carrier, often bis(2-ethylhexyl) phosphate (DEHP) to the supported liquid membrane (SLM). DEHP is known to work by providing ionic interactions with basic compounds, to improve the partitioning into the SLM. In this work, the behavior of DEHP during extractions was studied for the first time. Interestingly, substantial amounts of DEHP was found to leak from the SLM into the aqueous sample at pH > 4. Due to this leakage, the ion-pair formation between analytes and DEHP was moved from the sample/SLM interface (interfacial complexation) to the bulk of the sample solution (bulk-sample complexation), which improved the mass transfer of polar bases considerably. Based on this, an extraction procedure for eight polar bases with log P values from +0.7 to -5.9 was developed and optimized. The optimization demonstrated that extraction of more polar analytes was favored by bulk-sample complexation. With optimized conditions, extraction from biological samples such as urine, protein-precipitated plasma, and raw plasma were performed with recoveries >40%, except for a few analytes. In addition, the extraction system could be operated under robust conditions with relatively low current (<70 μA for plasma), and provided low variability (<16% RSD), as well as good clean-up efficiency. These findings are an important step in further scientific anchoring of EME, and development of the technique towards selective extraction of very polar substances from complex biological matrices.

• MeSH
• elektrochemické techniky * MeSH
• organofosfáty chemie   izolace a purifikace   MeSH
• Publikační typ
• časopisecké články MeSH

Animal or human protothecosis belongs to rather rare, endemic, pro-inflammatory infections. It is caused by achlorophyllous algae of the genus Prototheca. Especially, P. bovis (formerly P. zopfii genotype 2) is often inflected as a non-bacterial causative agent of dairy cattle mastitis. In this study, we present a multiplex real-time PCR (qPCR) system for rapid and exact Prototheca spp. detection and quantification. Limit of detection, diagnostic sensitivity, and specificity were determined. For the first time, specific sequences of AccD (encoding acetyl CoA reductase) for P. bovis, cox1 (encoding cytochrome C oxidase subunit 1) for P. wickerhamii, cytB (encoding cytochrome B) for P. blashkeae and atp6 (encoding transporting ATPase F0 subunit 6) for P. ciferrii (formerly P. zopfii genotype 1) were used for species identification and quantification together with 28S rRNA sequence detecting genus Prototheca. The developed qPCR assay was applied to 55 individual cow milk samples from a herd suspected of protothecosis, 41 bulk milk samples from different Czech farms, 16 boxed milk samples purchased in supermarkets and 21 environmental samples originating from a farm suspected of protothecosis. Our work thus offers the possibility to diagnose protothecosis in the samples, where bacterial mastitis is the most commonly presumed and thereby assisting adequate corrective measures to be taken.

• MeSH
• DNA chemie   izolace a purifikace   MeSH
• farmy MeSH
• klonování DNA MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• limita detekce MeSH
• mikrobiologie životního prostředí MeSH
• mlékárenství MeSH
• mléko mikrobiologie   MeSH
• multiplexová polymerázová řetězová reakce metody   MeSH
• plazmidy genetika   MeSH
• Prototheca genetika   růst a vývoj   izolace a purifikace   MeSH
• senzitivita a specificita MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH

The objective was to clarify the association between bulk tank milk somatic cell count (BTSCC) and total bacterial count (BTTBC) and coliform bacteria count (BTCBC) in a large set of data based on the currently accepted legal limit of BTSCC=400,000/ml. We analysed the database obtained from one of four laboratories offering routine estimation of microbiological indicators and counts of somatic cells in bulk tank milk samples in the Czech Republic during the year 2003 (74,174; 73,921 and 33,020 records of BTSCC, BTTBC and BTCBC estimations, respectively, in milk from 2,769 suppliers). Raw data of BTSCC (with arithmetic mean 220,000/ml; 95th percentile=502,000/ml; 99th percentile=784,000/ml) indicated that the BTSCC limit was exceeded in 12% of samples. BTSCC did not sufficiently reflect the hygiene status of particular producing herds because correlation coefficients between bulk tank milk somatic cell score (BTSCS) and log BTTBC or log BTCBC were low. Categorization of herds according to the percentage of records exceeding the BTSCC limit gave significantly higher correlation coefficients for the association between this characteristic and log BTTBC or log BTCBC (r=0.84 and r=0.68, respectively). The percentage of records exceeding the BTSCC limit was a useful tool to highlight problem herds kept in inadequate hygienic conditions in primary milk production. Likewise, the value of BTSCS>5 seemed to be a useful tool for the discrimination of problem herds.

• MeSH
• Enterobacteriaceae izolace a purifikace   MeSH
• financování organizované MeSH
• hygiena MeSH
• laboratoře normy   MeSH
• mastitida skotu mikrobiologie   MeSH
• mlékárenství normy   MeSH
• mléko cytologie   mikrobiologie   normy   MeSH
• počet buněk veterinární   MeSH
• počet mikrobiálních kolonií veterinární   MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• srovnávací studie MeSH
• Geografické názvy
• Česká republika MeSH