Jana Žiarovská

Changes in expression of BetV1 allergen of silver birch pollen in urbanized area of Ukraine.

The aim of the study was to determinate the level of expression of silver birch allergen Betv1 in pollen samples from different Ukraine areas by RT-qPCR SYBR Green assay. Protocol for quantifying the expression of Betv1 allergen was developed when testing of three housekeeping genes—cyclophylin, alpha-tubulin and transcription factor CBF1. Samples from urbanized area was analysed by real-time PCR when a sample from forest growth conditions was used as a calibrator. Real-time PCR based quantifying of Betv1 provides a useful method for rapid and sensitive analyses of this silver birch allergen. Our results show higher expression levels in samples from central parts of urbanized area as housing estates when compared to the samples from borders of the urbanized area.

Variability of Corylus avellana, L. CorA and profilin pollen allergens expression.

Corylus avellana is the source of inhalant allergies induced by hazel pollen as well as food allergies induced after ingestion of hazelnuts. In this study, real-time PCR approach was used to analyse expression of hazel pollen allergens on the molecular level. Relative quantity of hazelnut allergens Corylus avellana, L. CorA and Corylus avellana, L. pollen profiling in samples from different Ukraine areas were determining and comparing. Differences among the levels of both analysed allergen transcripts were found for hazel CorA and profillin. In both cases, the expression within the urbanized growth conditions was higher when compared to the sample from village area. The average expression for CorA was 0.84 times higher than for profilin and the results are very variable depending on the place of growth. Expression levels here were within the range of 2.957 up to the 52.936. Profilin expression was the highest in the sample from the polluted place of growth—cement plant area with the value of 52 times higher when compared to the sample from the village area. In this study, comparison of expression levels of hazel CorA and profiling pollen allergens was performed for the first time. Real-time PCR assay developed in this study proved the sensitivity for detection of the changes of the hazel pollen allergens expression levels and could benefit labs by fast and reproducible detection method of these allergens.

Identification of sweet chesnut pollen in bee pollen pellet using using molecular analysis.

Castanea sativa posses many characteristics that are used by human for different purposes, not only as a part of the food. One of them is the utilization of the sweet chesnut pollen for its pharmacological benefits. Actually, no information about the DNA based identification of the sweet chesnut exist. Here, an identification of Castanea sativa based on the specific DNA fragment amplification is described for the first time. Sweet chesnut identification was performed in the very complex sample of bee pollen pellets that were identified as to contain sweet chesnut pollen grains by morphological analysis. First, bioinformatic analysis was performed to find a Castanea sativa conservative part of galactol synthase gene. BLAST alignment of the CDS of GolS1 gene was performed by BLASTtn against plants nucleotide sequences in the NCBI database to ensure for the specifity or existing nucleotide differences. Then, specific primers were subsequently designed and PCR amplification was performed. All the PCRs have run in duplicates for pollen pellet sample and two independent samples of Castanea sativa pure pollen. Restriction cleavage of the PCR amplified fragment was performed to confirm the specifity of the obtained PCR product with the positive confirmation as the predicted three restriction fragments were obtained that fully correspond by the length to those from virtual clevage. Restriction endonuclease Hpy166II was used in restriction cleavage analysis. Castanea sativa pollen grains were confirmed reliable in multifloral pollen pellet by PCR and this approach has the potential to be used effectively for the authentication purposes of sweet chesnut.

Bacteria and yeasts isolated from different grape varieties

Patasiova Martina 14.00 The aim of this study was to isolate and identify bacteria and yeasts in different grape samples. The samples were collected in September 2017. Used 13 grape samples in this study (9 white and 4 red) were from the local Slovak winemakers. Alibernet, Irsai Oliver, Dornfelder, Blue Frankish, Feteasca regala, Green Veltliner, Palava, Mūller Thurgau, Rhinriesling, Cabernet Savignon, Pinot Blanc, Savignon Blanc and Welschriesling. Two cultivation media were used for detection of bacteri and yeasts in grape samples. Malt extract agar base (MEA) and Tryptone Soay agar (TSA) were used for the cultivation of bacteria and yeasts. Cultivation was performed by spread plate method. Ethanol/formic acid extraction procedure was used for preparation of samples. MALDI-TOF Mass Spectrometer (Microflex LT/SH) (Bruker Daltonics, Germany) was used for identification of bacteria and yeasts. In total, 8 genera of yeasts, 8 genera of Gram-negative bacteria and 10 of Gram-positive bacteria were identified. Together 333 isolates, yeasts, Gram-negative and Gram-positive bacteria were identified. Normal 0 21 false false false EN-US X-NONE X-NONE

Effect of different reverse transcription aproaches in Pru p 3 transcripts semiquantitative amplification

Normal 0 21 false false false MicrosoftInternetExplorer4 Reverse transcriptase transcribes the cDNA based on its previous extraction and standardization. Reverse transcription step is considered to be critical in the workflow of quantification of transcribed genes. The aim of the study was to extract total RNA by different methods and to analyse the results of the subsequent reverse transcription reaction when different commercial RT kits were used to process RNA extracted from pulp of matured peach fruit. Mature peach pulp was used in the study. The fruit of variety Vistarich was collected in summer 2017 in the orchard of Dvory nad Žitavou. Two RNA extraction methods, TRIzol® Reagent and GeneJET Plant RNA Purification Kit, were tested in to determine the suitable method for peach fruit RNA extraction. Three different cDNA reagent sets were used to transcribe 115 ng/500 ng total RNA or 11 ng/115 ng, respectively. Both variants of the primers, random hexamers as well as oligo (dT) 18, were used to anneal the target mRNA of Pru p 3 allergen following the manufacturer instructions. No specific effect was obtained in the case of peach fruit when using ethanol-extracted tissue treatment and the effect of the used extraction method was more significant. The A260/230 ratios were similar for three from four tested methods. In the case of these three methods, the A260/A230 ratios for all the extracted samples were higher than 1.9 which indicates high purity without contamination by polyphenols and polysaccharides. The specificity of obtained amplicons was proved by restriction cleavage using Tse I restriction endonuclease. This provided the cleavage of the 179 bp long product in all amplicons. Working with mature fruit meet a specific situation in the field of RNA extraction and subsequently all the downstream applications. That is, why choosing the most fitting methods and kits is a crucial step. Here, the method for the semi-quantitative analysis of the Pru p 3 allergen expressions was set up in the way that will be directly applicable for Pru p 3 expression analyses.

Effect of DNA extraction in the Rosa canina L. identification under different processing temperature

Rosa canina, L. is widely used for medicinal purposes as well as in food industry where it is a valuable source, bioactive compounds and natural colorants. Actually, no specific method is recommended as suitable one for DNA extraction from rose hips. The aim of the study was to compare three commercial and three non-commercial methods to extract total genomic DNA from rose hips hyphanthium. Four methods are based on the precipitation in principle and two methods are based on resin-binding. Extracted DNA was proved for the effectivity in following PCR. In total, six different DNA isolations was performed for differently heat processes rose hips - fresh hyphanthium, 2-weeks frozen hyphanthium, dried hyphanthium (50 °C) and boiled hyphanthium (100 °C). The amplification parameters of 500 bp chloroplast gene amplicon were evaluated. Obtained amounts of extracted DNA was very variable not only for every individual method used but for individual treatment of samples, too. In general, non-commercial method provided higher amount of extracted DNA, but the A260/280 ratio was lower. When regarding the processing treatment of the samples, high differences were found among the samples untreated by heat and those that were dried or boiled for three of the used extraction methods. All the samples were positive for amplification, but different amounts of amplified product were obtained. The comparison of data for concentrations of extracted DNA and concentrations of amplified product showed large differences when regarding the achieved purity of DNA in extraction.