The main goal of the study was to develop and test an effective technology for cattle genotyping by the CSN3 gene based on real-time PCR with hybridization-fluorescence detection. There was developed a method for real-time PCR for cattle genotyping by A and B alleles of the CSN3 gene in the format of hybridization-fluorescence detection, involving the use of two 5/-fluorescence-labeled forward allele-specific primers, one reverse common primer, and one anti-primer labeled with a fluorescence quencher at the 3/-end of the oligonucleotide. As a result of practical studies aimed at testing the developed method, we obtained the technical result provided by the proposed technology, expressed in the effective identification of the desired genotypes due to correct interpretation of these curves of increasing fluorescence intensity, the results reliability of which was also confirmed by the well-known PCR-RFLP analysis technique for Bos taurus genotyping for similar allelic variants of the kappa-casein gene.
DNA authentication of wines is a process of verifying their authenticity by genetic identification of the main plant component. The sample preparation of experimental and commercial wines was carried out by precipitation of wine debris by centrifugation with preliminary exposure with precipitators and co-precipitators, including developed macro- and micro- volume methods applicable to white or red wines, using polyvinylpyrrolidone as a co-precipitator. Addition of 2-mercaptoethanol and proteinase K to the lysing solution made it possible to adapt the technology for DNA extraction from the precipitated wine debris. The additionally tested technique of DNA extraction from wine debris by dimethyl sulfoxide (DMSO) lysis had fewer stages and, consequently, a lower risk of contamination. The results of further testing of one of the designed primer pairs (UFGT-F1 and UFGT-R1) in conjunction with the tested methods of wine material sample preparation and nucleic acid extraction, showed the advantage in the given set of oligonucleotides over previously used ones in terms of sensitivity, specificity and reproducibility. The developing strategy for genetic identification of grape varieties and DNA authentication of wines produced from them based on direct sequencing of polymerase chain reaction (PCR) products is implemented by interpreting the detected polymorphic positions of variable Vitis vinifera L. UFGT gene locus with distribution and split into 13 UFGT gene-associated groups.
Molecular genetic research methods make it possible to evaluate the genetic diversity of bovine leukemia virus (BLV) and are the most informative approaches to its genetic identification. Molecular genetic research methods work well for the phylogenetic analysis of sequenced nucleotide DNA sequences of the provirus, as well as for the polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) according to the phylogenetic classification of the pathogen. The purpose of the research was to study the scientific and methodological approaches to the genetic identification of bovine leukemia virus, integrated into the molecular monitoring of infection of cattle with BLV genotypes. The authors used PCR-RFLP-genotyping and comparative phylogenetic analysis of aligned nucleotide sequences of the env gene fragment of the BLV provirus isolates to detect the genotypic affiliation of the cattle from twenty-one livestock farms of the Republic of Tatarstan. As a result, isolates of four out of ten BLV genotypes were found in the Tatarstani cattle, namely genotypes 1, 4, 7, and 8. The research involved a comparative analysis of 505 nucleotide sequences of a fragment of the BLV env gene, including those deposited in GenBank NCBI. The analysis confirms the inconsistency of several earlier PCR-RFLP typing strategies with the current approach in assessing the genotypic diversity by phylogenetic analysis. The improved strategy of PCR-RFLP genotyping of BLV corresponds with its modern phylogenetic classification. The strategy makes it possible to identify all the known genotypes of the viral pathogen. Its validity has been proved by in silico modelling of restrictogrammes and a phylogenetic analysis of the env gene fragment of 57 reference isolates of ten BLV genotypes that generate 57 genotype-associated combinations of diagnostically significant PCR-RFLP profiles.
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