D. Yu. Ryazantsev

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 (ITS1) 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.

Detection of Staphylococcus aureus toxins using immuno-PCR

A highly sensitive test system, based on the immuno-PCR method, 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 to obtain supramolecular complexes of bisbiotinylated oligodeoxynucleotides and streptavidin, which were to be 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 lower than 10 pg/mL.

An efficient diagnostic method for the identification of potato viral pathogens

Potato is commonly affected by various pathogens of viral, bacterial, and fungal origin; therefore, simple and accurate diagnostic and identification techniques are of key importance both for the production of virus free planting material and for the monitoring of the phytosanitary state of planting areas. The newly developed test systems based on qualitative fluorescent amplification-based specific hybridization (FLASH-PCR) enable fast and accurate diagnostics of the major potato pathogens, i.e. potato viruses A, Y, X, M, and S, potato leaf roll virus, potato mop top virus, as well as potato spindle tuber viroid, while minimizing the risk of the working zone contamination.

FLASH-PCR diagnostics of toxigenic fungi of the genus Fusarium

A test system for the diagnostics and identification of seven toxigenic fungi causing Fusarium head blight (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-1α) 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.

Phosphate permease gene as a marker for the species-specific identification of the toxigenic fungus Fusarium cerealis

A PCR-based system has been developed for highly specific identification of the phytopathogenic fungus Fusarium cerealis on the basis of the nucleotide sequence of the phosphate permease gene. Sequencing and the following analysis showed that this gene demonstrated a high degree of polymorphism and can be used for both phylogenetic studies and as a marker for specific primer design. Investigation of the specificity of the designed primers confirmed the lack of cross-reactivity with DNA of closely related fungi species of the Fusarium genus. The qPCR assay demonstrated high sensitivity of the test system, which was 10 pg of the specific DNA per reaction.

Expression of house dust mite allergens Der f 1 and Der f 2 in leaves of Nicotiana benthamiana

In test systems for diagnostics of type I allergy, recombinant allergens in native conformation are used, because immunoglobulins E (IgE), responsible for the development of this type of allergy, recognize conformational epitopes of protein allergens. To obtain recombinant allergens of the Dermatophagoides farinae house dust mites (HDM), Der f 1 and Der f 2, two systems of heterological expression were used: Escherichia coli and Nicotiana benthamiana plants. IgE from sera of children allergic to HDM were shown to recognize the recombinant Der f 2 protein synthesized in both E. coli and N. benthamiana plants, as well as the recombinant Der f 1 protein produced in N. benthamiana plants, while mature form of Der f 1 produced in E. coli did not interact with IgE.

Caspases participation in cell death induced by the GD2-specific antibodies

Contribution of the main caspases in cytotoxic effects induced by the monoclonal antibody 14G2a specific to the tumor-associated ganglioside GD2 was studied in the EL-4 mouse lymphoma cells. The constitutive expression of the procaspase genes was found in the EL-4 cells. Incubation of the cells with the 14G2a antibodies did not result in increasing of the procaspase synthesis. We also demonstrated with the use of fluorescently labeled substrates of caspases that the procaspase enzymatic activity was not enhanced. At an equal level of cell death, activities of caspase-3 and caspase-9 in the cells which were incubated with the 14G2a antibodies were 7.5 and 3 times lower, respectively, than those in the staurosporine-treated cells. The pan-caspase inhibitor (Z-VAD-FMK) and the caspase-3 inhibitor decreased the cytotoxic effects induced by the 14G2a antibodies by 9–16 and 6–13%, respectively. The staurosporine-induced level of the apoptosis decreased by 55–65% under the same conditions. Inhibitors of the initiation caspase-8 and caspase-9 had no influence on the antibody-induced cell death. The inhibitory analysis also demonstrated that the caspases were not involved in the triggering of the initial stages of the antibody-induced cell death such as apoptotic volume decrease and permeabilization of the cell plasma membrane. Thus, caspases did not play a key role in the cell death induced by the anti-GD2 antibodies, and their slight enzymatic activity did not determine the main mechanism of cell death mediated through the tumor-associated ganglioside GD2.

Effect of cocksfoot mottle virus defective RNA on accumulation of virus capsid protein in wheat plants

The functional role of defective cocksfoot mottle virus RNA in regulating the expression of the viral genome was investigated for testing the hypothesis of its possible participation in transactivation of the synthesis of subgenomic RNA encoding the virus coat protein gene.

Determination of Specific Class E Immunoglobulins to Bet v 1 Birch Allergen by the Immuno-PCR Method

The aim of the work is the development of a method of detection of specific class E immunoglobulins (IgE) to the main Bet v 1 birch allergen based on immuno-PCR (iPCR). The recombinant Bet v 1 allergen was obtained in E. coli cells. Its ability to bind to specific IgE was confirmed by enzyme-linked immunosorbent assay (ELISA) using previously characterized sera of individuals with an allergic reaction to birch pollen and control sera in individuals, in which the reaction to this allergen is absent. Based on the obtained recombinant protein, the method of iPCR analysis of specific IgE to Bet v 1 was developed. It was demonstrated that iPCR sensitivity is comparable to ELISA sensitivity, and the titration curves of specific sera in iPCR (unlike those in ELISA) demonstrate a linear dependence; this makes the developed method preferable for quantitative estimation of specific IgE in sera as compared with ELISA.

Immuno-PCR Assay for Quantitation of Antibodies to Epstein–Barr Virus

Successful disease prevention and therapy critically depend on timely diagnosis of infections. Quantitative immuno-PCR (qiPCR) technology improves the sensitivity in the detection of antibodies to pathogens. A qiPCR-based assay was developed to determine IgG antibodies to Epstein–Barr virus (EBV) in the human blood serum. EBV nuclear protein 1 fragment (pEBV) was expressed in Escherichia coli. A synthetic single-stranded deoxyribonucleotide was conjugated to streptavidin, and the conjugate was used to detect рEBV–IgG1–biotin complexes by qiPCR. The IgG1 titers determined by qiPCR were compared to the results of enzyme-linked immunosorbent assay (ELISA). The sensitivity of qiPCR was one order of magnitude higher than that of ELISA. Thus, a highly sensitive qiPCR-based assay was developed to quantitate antibodies specific to the recombinant EBV antigen.