A Novel Fluorescent GFP Chromophore Analog-Based Dye for Quantitative PCR.

This is the first report describing the possibility of using a green fluorescent protein chromophore synthetic analog, P-HOBDI-BF2, as a fluorescent dye for a linear hydrolysis probe used in qPCR. The study was carried out on a system for detection of the plant pathogenic fungus Fusarium avenaceum using a plasmid containing translation elongation factor 1α fragment as a template. To estimate fluorogenic properties of P-HOBDI-BF2, 6-FAM- and BDP-FL-labeled probes were used. It was demonstrated that a synthetic dye based on the P-HOBDI-BF2 chromophore can be used for labeling hydrolysis probes for qPCR, but fluorescence increase levels for P-HOBDI-BF2-labeled probes were slightly lower than those for 6-FAM-labeled ones. At the same time, the sensitivity of P-HOBDI-BF2-based assays remained high, and this fact together with acceptable fluorescence levels suggests that this dye can be considered as an efficient alternative for reporters traditionally used for fluorescence detection in the FAM channel.

Phylogenetic Analysis and Molecular Typing of Trichothecene-Producing Fusarium Fungi from Russian Collections.

We performed a three-locus phylogenetic analysis of Fusarium strains presumably capable of trichothecene production, which were deposited in the Russian national collections. The intra- and interspecific polymorphism of partial sequences of the translation elongation factor 1 alpha (TEF1α) gene and two genes from the trichothecene cluster TRI5 and TRI14 was studied. A study of 60 strains of different origins using DNA markers confirmed, and in the case for several strains, clarified their taxonomic characteristics. As a result, a strain of F. commune (F-900) was identified in Russia for the first time. Furthermore, the strain F-846 proved to be phylogenetically distinct from any of the known Fusarium species. F. equiseti strains from Northwest Russia were found to belong to the North European group (I), whereas a strain from the North Caucasus - to the South European one (II). Partial TRI14 sequences from 9 out of 12 species were determined for the first time. Their comparative analysis demonstrated a relatively high level of intraspecific variability in F. graminearum and F. sporotrichioides, but no correlation between the sequence polymorphism and the geographic origin of the strains or their chemotype was found. Specific chemotypes of trichothecene B producers were characterized using two primer sets. The chemotyping results were verified by HPLC.

[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.

Corneal power determination after previous corneal refractive surgery for intraocular lens calculation.

PURPOSE: To assess the accuracy of different corneal power determination methods in patients who had undergone myopic laser in situ keratomileusis (LASIK), photorefractive keratectomy (PRK), and radial keratotomy (RK). METHODS: The results for 208 eyes of 116 patients who had had corneal refractive surgery (LASIK, PRK, RK) for myopia were analyzed retrospectively. Keratometry measurements, i.e., autokeratometry readings (K(meas)), simulated keratotopography readings (Sim-K), and topographically measured average central corneal power in a 3-mm zone (ACP) were compared with calculated refraction-derived keratometric value. Correction factors based on the difference between measured and calculated keratometric powers were rated. RESULTS: Direct power measurements with standard keratometers and planokeratotopography systems overestimate corneal power after myopic PRK and LASIK. The average K(meas) and K(topo) were significantly greater than the average calculated refraction-derived keratometric values. Corneal power overestimation correlated significantly with the spherical equivalent change after refractive surgery (p < 0.001). After RK, there is no significant correlation of the difference between all measured K values and refraction-derived power with the spherical equivalent change. In these cases, the Sim-K value seems the most accurate among measured keratometric powers. The precision of measurement significantly depends on the parameters of the autokeratometer (i.e., measurement place, number of measurement points, keratometric index of refraction). CONCLUSIONS: To avoid underestimation of intraocular lens power after cataract surgery in the eyes that had previously undergone myopic corneal refractive surgery, the measured corneal power must be corrected. Although correction factors may be calculated for cases after PRK and LASIK, there are no universal and absolutely reliable methods to determine corneal power in these cases. More than one accessible method should be used, and the lowest, most reliable data should be used.