[Detection of Staphylococcus aureus toxins using immuno-PCR].

A highly sensitive test system, based on the method of immuno-PCR, was developed for the detection of two staphylococcal toxins: enterotoxin A (SEA) and toxic shock syndrome toxin (TSST). A key element of the developed systems was obtaining supramolecular complexes of bisbiotinylated oligodeoxynucleotides and streptavidin, which were used as DNA tags. Specificity studies showed no cross-reactivity when determining SEA and TSST. The sensitivity of detection of these toxins in the culture supernatants S. aureus was not less than 10 pg/mL.

[Caspases participarion in the cell death induced by GD2-specific monoclonal antibody].

The participation of the main caspases in the cytotoxic effects induced by monoclonal antibody 14G2a specific against tumor-associated ganglioside GD2 was studied in the EL-4 cells. It has been found constitutive expression ofprocaspases genes in the EL-4 cells; incubation of the cells with 14G2a antibodies didnot result in increasing of the procaspases expression. Weak enzymatic activity of caspases has been shown using fluorescent labeled substrates. At the same cell death level, activity of caspase-3 and caspase-9 in the cells incubated with 14G2a was about 7.5- and 3-fold lower than in cells after incubation with staurosporine. Pan caspase inhibitor Z-VAD-FMK, and caspase-3 inhibitor reduced the cytotoxic effects induced by 14G2a at 9-16 and 6-13%, respectively. At the same conditions, pan caspase inhibitor decreased staurosporine-induced apoptosis at 55-65%. Inhibitors of other caspases had no effect on the cell death triggered by the antibodies. Inhibition analysis demonstrated also that caspases did not involved in the cell volume decreasing and permeabilization of the cell plasma membrane, which were the first stages of anti-GD2-mAb-induced cell death in the EL-4 cells. Thus, despite the slight activation of caspases during the cell death induced by antibodies directed to GD2, they do not play a key role and do not determine the mechanism of cell death triggered through the tumor-associated ganglioside GD2.

[[Expression of house dust mite allergens Der f1 and Der f2 in Nicotiana benthamiana leaves].

The class E immunoglobulins (IgE) is known to recognize conformational epitopes and therefore the native conformation of recombinant allergens is essential for their using in test-systems. Recombinant Dermatophagoides farinae house dust mite (HDM) allergens Der f1 and Der f2 were expressed in bacteria Escherichia coli and Nicotiana benthamiana plants. It has been shown that IgE in sera from children allergic to HDM recognizes Der f2 expressed both in E. coli and N. benthamiana. Mature form of Der f1 expressed in E. coli does not interact with IgE while the protein purified from N. benthamiana is able to recognize IgE as a native allergen.

[Phosphate permease gene as a marker for the specific identification of toxigenic fungus Fusarium cerealis].

We have developed phosphate permease gene sequence-based PCR detection system of Fusarium cerealis phytopathogenic fungus. Sequencing and analysis revealed that the gene displayed unique polymorphism and could serve to establish phylogenetic relations as well as a marker to design specific primers. The specificity assay has confirmed the absence of cross reactions with DNAs of closely related Fusarium species. The qPCR assay demonstrated the 10 pg detection limit of specific DNA per reaction.

[Novel DNA markers reveal for taxonomic characterization and identification of the Fusarium fungi species].

RAPD analysis for ten F. sporotrichioides strains of different geographical origin was done for DNA loci, potentially suitable as a new markers for taxonomic characterization and identification of toxigenic Fusarium fungi. Three selected monomorphic fragments--products of amplification with one of standard RAPD primers were sequenced that allowed creating SCAR markers for identification of Fusarium fungi on the species group level with similar profiles of produced mycotoxins.

[Efficient approach for potato viral pathogen sensitive diagnostic and identification].

Potato, one of the most widespread agricultural plants in Russia, is strongly affected by various pathogens of viral, bacterial, and fungal origin as well as by pests. Their simple and accurate diagnostics and identification sound rather important both for production of virus free planting material and to perform monitoring of the phytosanitary state of planting areas. Based on qualitative Fluorescent Amplification--based Specific Hybridization Polymerase Chain Reaction (FLASH-PCR) we have developed the diagnostic systems, which provided fast, careful, and with the minimum risk of contamination in the working zone by amplification products, detection of the major potato pathogens, i. e. A, Y, X, M, S potato viruses, potato leafroll virus, potato mop top virus, as well as potato spindle tuber viroids.

[FLASH-PCR diagnostics of toxigenic fungi of the genus Fusarium].

A test system for the diagnostics and identification of seven toxigenic fungi causing fusarioses of cereals (Fusarium graminearum, F. culmorum, F. poae, F. sporotrichioides, F. langsethiae, F. avenaceum, and F. tricinctum) was developed using PCR. The identification of pathogen is based on the specific amplification of a DNA fragment of the gene of translation elongation factor 1 alpha (tef-1alpha) and subsequent detection of the results by the fluorescent amplification-based specific hybridization method. The system was tested on 38 isolates of different fungi of the genus Fusarium.

[Diagnostics of phytopathogen fungi Septoria tritici and Stagonospora nodorum by fluorescent amplification-based specific hybridization (FLASH) PCR].

A PCR system in the fluorescent amplification-based specific hybridization (FLASH) format was developed for the detection and identification of two important wheat pathogenic fungi Septoria tritici (teleomorph of Mycosphaerella graminicola and Stagonospora nodorum (teleomorph of Phaeosphaeria nodorum), which cause spots on leaves and glumes, respectively. The pathogen detection system is based on the amplification of a genome fragment in the internal transcribed spacer 1 (ITS 1) region and a site encoding the 5.8S ribosomal RNA. The forward primers to ITS1 and a universal reverse primer and a Beacon type probe to the 5.8S ribosomal RNA region were chosen to provide the detection of the products in the FLASH format. This system was tested on different isolates of the pathogens, and on infected soil, leaf, and seed samples.