S. K. Zavriev

Two-dye and one- or two-quencher DNA probes for real-time PCR assay: synthesis and comparison with a TaqMan™ probe.

AbstractA typical TaqMan™ real-time PCR probe contains a 5′-fluorescent dye and a 3′-quencher. In the course of the amplification, the probe is degraded starting from the 5′-end, thus releasing fluorescent dye. Some fluorophores (including fluorescein) are known to be prone to self-quenching when located near each other. This work is aimed at studying dye–dye and dye–quencher interactions in multiply modified DNA probes. Twenty-one fluorogenic probes containing one and two fluoresceins (FAM), or a FAM–JOE pair, and one or two BHQ1 quenchers were synthesized using non-nucleoside reagents and “click chemistry” post-modification on solid phase and in solution. The probes were tested in real-time PCR using an ~300-bp-long natural DNA fragment as a template. The structural prerequisites for lowering the probe background fluorescence and increasing the end-plateau fluorescence intensity were evaluated and discussed. FigureFluorogenic TaqMan probes with various modifications for real-time PCR

Defense peptides from barnyard grass (Echinochloa crusgalli L.) seeds.

A number of defense polypeptides from latent seeds of weed cereal barnyard grass (Echinochloa crusgalli L.) has been isolated and characterized using an acidic extraction and high performance liquid chromatography methods in combination with MALDI-TOF mass spectrometry and Edman sequencing. Members of three antimicrobial peptide families and two protease inhibitor families were found to be localized in barnyard grass seeds. Their biological activity concerning to Gram-Positive and Gram-Negative phytopathogenic bacteria, as well as oomycete Phytophthora infestans, has been investigated. Diversity of barnyard grass defense peptides is a significant factor that provides a resistance of E. crusgalli seeds to germination and latent phases.

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.

Identification of a Novel Small Cysteine-Rich Protein in the Fraction from the Biocontrol Fusarium oxysporum Strain CS-20 that Mitigates Fusarium Wilt Symptoms and Triggers Defense Responses in Tomato

The biocontrol effect of the non-pathogenic Fusarium oxysporum strain CS-20 against the tomato wilt pathogen F. oxysporum f. sp. lycopersici (FOL) has been previously reported to be primarily plant-mediated. This study shows that CS-20 produces proteins, which elicit defense responses in tomato plants. Three protein-containing fractions were isolated from CS-20 biomass using size exclusion chromatography. Exposure of seedling roots to one of these fractions prior to inoculation with pathogenic FOL strains significantly reduced wilt severity. This fraction initiated an ion exchange response in cultured tomato cells resulting in a reversible alteration of extracellular pH; increased tomato chitinase activity, and induced systemic resistance by enhancing PR-1 expression in tomato leaves. Two other protein fractions were inactive in seedling protection. The main polypeptide (designated CS20EP), which was specifically present in the defense-inducing fraction and was not detected in inactive protein fractions, was identified. The nucleotide sequence encoding this protein was determined, and its complete amino acid sequence was deduced from direct Edman degradation (25 N-terminal amino acid residues) and DNA sequencing. The CS20EP was found to be a small basic cysteine-rich protein with a pI of 9.87 and 23.43% of hydrophobic amino acid residues. BLAST search in the NCBI database showed that the protein is new; however, it displays 48% sequence similarity with a hypothetical protein FGSG_10784 from F. graminearum strain PH-1. The contribution of CS20EP to elicitation of tomato defense responses resulting in wilt mitigating is discussed.

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.

Structural evolution of the 4/1 genes and proteins in non-vascular and lower vascular plants

The 4/1 protein of unknown function is encoded by a single-copy gene in most higher plants. The 4/1 protein of Nicotiana tabacum (Nt-4/1 protein) has been shown to be alpha-helical and predominantly expressed in conductive tissues. Here, we report the analysis of 4/1 genes and the encoded proteins of lower land plants. Sequences of a number of 4/1 genes from liverworts, lycophytes, ferns and gymnosperms were determined and analyzed together with sequences available in databases. Most of the vascular plants were found to encode Magnoliophyta-like 4/1 proteins exhibiting previously described gene structure and protein properties. Identification of the 4/1-like proteins in hornworts, liverworts and charophyte algae (sister lineage to all land plants) but not in mosses suggests that 4/1 proteins are likely important for plant development but not required for a primary metabolic function of plant cell.

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.

Four-locus phylogeny of Fusarium avenaceum and related species and their species-specific identification based on partial phosphate permease gene sequences

The fungus Fusarium avenaceum and its closest relatives are responsible for contamination of agricultural plants and their products by mycotoxins such as enniatins and moniliformin. Precise identification of mycotoxin producers is necessary for estimation of the accumulation risk of those compounds and for preventing the consumption of highly contaminated products. Nucleic acids amplification-based techniques proved to be the most rapid and reliable approach for pathogen diagnostics and identification. In this study partial phosphate permease gene (PHO) sequences were determined for Fusarium avenaceum (including one isolate identified as F. arthrosporioides), F. tricinctum, F. acuminatum and F. torulosum. Phylogenetic analysis of 40 isolates of those species from different climates and geographical regions of Russia and some neighboring countries based on sequences of PHO, translation elongation factor 1 alpha (TEF1α), beta-tubulin (β-TUB), enniatin synthetase (Esyn1) genes and combined data set demonstrated that the PHO gene possesses the highest rate of variability among them and can be considered as an informative marker for phylogenetic studies of these species. According to the combined data set phylogeny, the isolates of each species formed clusters with a high bootstrap support. Analysis of PHO sequences revealed a high intraspecific variability of F. avenaceum: there were 5 independent clusters on the dendrogram, including one cluster which was closer to F. torulosum than to other F. avenaceum isolates. Variable sites in PHO sequences have been used for the design of species-specific primers and a fluorescent hydrolysis probe. The specificity of the assay was shown for DNA samples extracted from 68 isolates of 23 Fusarium species. Quantitative PCR approach was applied to estimate the contamination rate of 17 naturally infected oat and barley samples, previously characterized by microbiological procedures.