Use Of Molecular Genetic Methods Research Articles

Optimization of Molecular Genetic Method for Identification of Dairy Raw Materials

Introduction: This article addresses the pressing issue of ensuring the quality and safety of dairy products by combating adulteration, a challenge that continues to plague the dairy industry. Adulteration, often driven by cost-cutting motives, involves altering the composition, quality, or origin of dairy products, even in the face of tightened control measures and improved monitoring systems. Specifically, this study hones in on the problem of species-specific adulteration, particularly the substitution of goat milk with cow milk due to the significant cost differential. Detecting and identifying such adulteration is crucial, and while traditional methods like electrophoresis and chromatography have been used, they are often expensive and labor-intensive. The article explores the use of molecular genetic methods, particularly polymerase chain reaction (PCR), as a more efficient and accurate means of identifying species-specific milk adulteration. Methods like PCR offer high specificity, sensitivity, speed, and the ability to perform quantitative and multiplex analyses.Purpose: The objective of this study is to optimise a method for species identification of dairy products by PCR-based screening using DNA isolated from cow’s and goat’s milk comatic cells.Materials and Methods: This research conducted at the Central Laboratory of Microbiology, All-Russian Research Institute of Dairy Industry, focuses on milk species identification of cattle and small ruminants using a specific set of primers targeting the polymorphic amylogenin gene. The study involves PCR screening with DNA extracted from cow’s and goat’s raw milk utilizing a MiniAmp instrument for the analysis. Qualitative and quantitative assessments of DNA preparations were performed, measuring DNA concentration with a Qubit 4 fluorimeter and Qubit dsDNA BR Assay Kit. PCR analysis was carried out in 25 μl reactions, including key components such as 5xScreen Mix, specific primers, DNA samples, and mQ H2O. The amplification program comprised denaturation, annealing, and elongation steps over a series of cycles.Results: The experiment was aimed at assessing the suitability of primers SE47 and SE48 for amplification of nuclear DNA of milk somatic cells of cattle (Bos taurus) and goats (Capra hircus). Initially, a calculated annealing temperature of 56 °C was used in the PCR reaction, yielding in numerous nonspecific fragments appeared on the electropherogram. To solve this problem, we gradually increased the annealing temperature, which resulted in a significant decrease in the nonspecific fragments number and their complete absence at annealing temperature of 70°C.Conclusion: We succeeded in optimizing a PCR-based detection system for the milk species identification of cattle and small ruminants. The results obtained confirm the possibility of using genomic DNA of milk somatic cells for the successful amplification of species-specific nuclear markers, but there remains a need for further research to determine the sensitivity of the PCR system and the possibility of its use in the analysis of milk processing products.

Ciliates from the faeces of the free-ranging dromedary from Oman: Morphology and molecular phylogeny

Ciliates Infundibulorium cameli from the faeces of the free-ranging dromedary from Oman were studied using a set of methods of the light and immunofluorescence microscopy and molecular phylogeny. With the use of molecular genetic methods, it was confirmed that the cysts found in the samples simultaneously with trophozoites actually belong to the species I. cameli. Tubulin cytoskeleton organization of trophozoites and cysts of this species were described for the first time. A striking morphological similarity between species I. cameli and Buxtonella sulcata was demonstrated, including the organization of ciliature. Different isolates of I. cameli and B. sulcata formed a common clade on the phylogenetic tree. The level of evolutionary divergence between the 18 S rRNA sequences of I. cameli, B. sulcata and species closest to them according to the results of molecular phylogenetic analysis was estimated. It was demonstrated that the divergence between I. cameli and B. sulcata is extremely low compared to members of other genera included in the analysis. Taxonomic position of I. cameli and B. sulcata was discussed in according to the data of comparative morphology and molecular phylogeny

Comparative Analysis of Gene Expression Profile in Tumor and Healthy Tissue in Patients with Colorectal Cancer

INTRODUCTION: Colorectal cancer (CRC) is a sure leader among malignant neoplasms (12.3%), without taking into account gender. Five-year survival rate in stage I CRC is 91%, in stage IV 14%. The currently existing treatment methods are helpless to significantly reduce mortality the approaches should be personalized and include the use of molecular genetic methods.
 AIM: To perform a comparative evaluation of expression profile of samples of tumor and healthy colon tissue in CRC.
 MATERIALS AND METHODS: The material for the study was 19 samples of tumor tissue taken from the pathologically altered colonic mucosa of 19 patients with CRC, and 7 samples of healthy tissue taken 10 cm12 cm distally or proximally from the visual boundary of the tumor. Biopsy materials were homogenized using a mechanical method. The quality and quantity of ribonucleic acid in the eluted solution were evaluated using IMPLEN nanospectrophotometer (Germany). Gene expression was evaluated using microchip kit SurePrint G3 HumanGeneExpv3 ArrayKit (Agilent, USА). Microchips were scanned on InnoScan 1100 AL apparatus (США) with subsequent image processing in Mapix Software program (USA).
 RESULTS: The analysis of expression profile demonstrated 505 differentially expressed genes, 337 of which showed reduced expression and 168 enhanced expression in the tumor material. The highest expression was demonstrated by genes bound with miRNA: hsa-miR-29b-3p and hsa-miR-1-5p, and also genes Н19, FOXQ1, INHBA, MMP1, CDH3, CXCL2, MDFI, THBS2. On the contrary, genes TMIGD1, GUCA2B, ZG16, AQP8, SLC4A4, CDKN2B-AS1, CA4, СА1 demonstrated a low expression in the tumor material. Expression of genes responsible for functioning of signal pathways: IL-17, NF-kappa B, TNF, was increased in tumor samples. Genes responsible for signal pathways Fatty acid degradation, Drug metabolism cytochrome P450, Metabolic pathways, Fatty acid metabolism and Steroid hormone biosynthesis, showed reduced expression.
 CONCLUSION: Significant differences were found in the expression profile of tumor and healthy tissue in patients with CRC. A comparative analysis of gene enrichment and the data of the international databases permitted to identify a number of terms, genes, clusters that can be used in future in search for predictors of prognosis and of response to treatment.

ВИДОВОЙ СОСТАВ ТРИПСОВ (INSECTA: THYSANOPTERA) НА СЕЛЕКЦИОННЫХ ПОСЕВАХ ЯРОВОЙ ПШЕНИЦЫ В РГАУ-МСХА ИМЕНИ К.А. ТИМИРЯЗЕВА

Cereal thrips are ubiquitous, widespread and strong pests of grain crops. Imago and larvae of thrips can damage leaves, ears, flowers and grains by injecting and sucking plant juices. Thrips reduce not only the weight but also the sowing qualities of grain, posing a threat to breeding and seed crops. Cereal thrips are a systematically and biologically diverse group of species that are diffi-cult to diagnose and study. Morphological diagnosis of thrips is carried out according to the adult phase; diagnosis of larvae of closely related species requires the use of molecular genetic methods. The wheat thrips are usually the main focus; other species are less known. In the experimental crops in Russian State Agrarian University – Moscow Timiryazev Agricultural Academy, cereal thrips are one of the main pests. In 2020, on spring wheat, their share in the number of insect collections by mowing with a net reached 14 in 2022, it reached 29%. However, the species composition of thrips has not been previously studied here. In the genetic collection of spring wheat (45 varieties) and the collection of endemic wheat (18 varieties) of the breeding experimental station of the Russian State Agrarian University – Moscow Timiryazev Agricultural Academy, censuses and diagnostics of the species composition of thrips were carried out. A total of 993 adults of thrips were analyzed and micro preparations were made. Seven species of herbivorous and one species of predatory thrips were identified. The species composition is dominated by three species of cereal thrips: Haplothrips aculeatus, Haplothrips tritici, and Frankliniella tenuicornis. The rarest among them is the rye thrips Limothrips denticornis. Some differences between thrips species are noted in terms of appearance on crops and the period of the main damage. For the most widespread, closely related and outwardly similar species, the main diagnostic features are indicated.

Experience in the use of phylogenetic analysis in the investigation of cases of HIV-infected patients

Objective. To establish a probable source of infection using phylogenetic analysis in the investigation of cases of HIV infection. Materials and methods. HIV genome sequencing was carried out using the kit ViroSeq (Celera). The phylogenetic tree was constructed using the maximum likelihood method in the Mega 6 program. As a comparison group, 40 samples of HIV-infected patients from the Krasnoyarsk region were used, as well as 25 nucleotide sequences of various HIV subtypes taken from the international GenBank database. The reliability of the resulting tree was evaluated in bootstrap analysis, a branch rotation test for a thousand random constructions.Results and discussion. The paper presents five cases of epidemiological investigations with a probable nosocomial and occupational mechanism of HIV infection transmission in the Krasnoyarsk region. In 2013, a study was conducted on the infection of three persons treated in the same hospital with an HIV-infected patient. A close genetic relationship of HIV was shown in newly identified individuals, the probable source could not be examined due to death. Second epidemiological investigation in 2013. The association of cases of infection between a newly diagnosed woman with HIV infection and a patient with a previously established HIV status is shown. Both persons were simultaneously in the same medical institution. To determine the direction of transmission of the virus, a test for the duration of infection was used. In 2014, after receiving medical care in four medical organizations, the patient was confirmed to be HIV positive. In all hospitals there were persons with a confirmed diagnosis of HIV infection. Three of them have been examined. The absence of a phylogenetic relationship was confirmed, the fourth died, the material could not be obtained from him. In 2015, a nurse was injured while caring for an HIV-positive patient. Phylogenetic analysis showed a high degree of genetic relationship between these cases of infection. In 2016, after a car accident, two patients were hospitalized in the intensive care unit. After some time, both were diagnosed with HIV infection. Four HIV-infected patients were in intensive care at the same time. Phylogenetic analysis was carried out in two newly identified individuals and one putative source. The association of infections in newly identified patients was shown, the alleged source could not be established.Conclusion. The use of molecular genetic methods in epidemiological investigations significantly improves the quality and accuracy of deciphering epidemic chains of HIV infection.

Current status and prospects of raspberry breeding

Raspberries are among the most popular berries on the market; therefore, interest in this crop is constantly growing. Raspberries are cultivated in more than 50 countries around the world, and this number is increasing. The steady expansion of raspberry cultivation and yields is due to the scientific support of the industry with new cultivation technologies and varieties. The present study was aimed at revealing difficulties and problems in raspberry breeding and identifying current tendencies and development trends. The paper provides a brief overview of raspberry production in the world, as well as an analysis of active breeding programs in Russia and abroad. The features of the breeding tasks are considered depending on the regions of cultivation. The main problems, methods used, source materials and breeding achievements are presented. It is revealed that the overall purpose of breeding programs is to obtain productive varieties with high adaptation to abiotic and biotic stressors, suitable for various cultivation technologies and having excellent fruit quality. It was found that despite the successes achieved in raspberry breeding, at present there is still lack of varieties with high adaptation to the environment and very early or late-ripening varieties, allowing to extend the period of consumption of fresh berries, as well as lack of high-tech industrial varieties suitable for contemporary cultivation technologies, and varieties with required commodity and consumer properties. The prospects of raspberry breeding for the coming years are considered. It is shown that in order to achieve the strategic objectives in breeding, it is necessary to enrich the cultivated gene pool of raspberry with new inclusions of wild species of Rubus. This requires the cooperation and coordination of all breeding programs for the expansion and exchange of genetic resources. In order to accelerate the breeding process, it is necessary to actively use a large set of biotechnological methods tested and well-proven on berry crops. Wider use of molecular-genetic methods is essential to significantly increase the speed and accuracy of breeding.

MYCOPLASMA CONTAMINATION OF BULL SEMEN USED FOR ARTIFICIAL INSEMINATION

Technologies of artificial insemination of cattle are actively used in livestock farms in Russia. At the same time, quality control of semen production does not involve testing the bull semen for microorganisms of the genus Mycoplasma. The active use of molecular genetic methods allows for the rapid determination and species identification of pathogenic mycoplasmas; however, it does not indicate the viability of the identified pathogen. The paper aims to study mycoplasma contamination of semen products presented on the Russian market and determine the conformity of the results of the Polymerase Chain Reaction [PCR] method and the traditionally used microbiological method. The paper presents the results of the first Russian study of the occurrence and viability of pathogenic species Mycoplasma bovis, M. bovigenitalium, M. californicum, and Ureaplasma diversum in semen production. We investigated 447 samples of cryopreserved semen of bulls-producers of various breeds from Russian breeding centers and breeding farms of the USA, Great Britain, and the Netherlands, supplying these products to the Russian market. The analysis shows a high frequency of occurrence in the bull semen of DNA of microorganisms of the genus Mycoplasma (up to 70.9%). Besides, M. bovigenitalium appeared to be the most frequently detected type of mycoplasma from those tested. Cases of identifying several types of mycoplasmas in one sample were revealed: in 58.8% of the studied samples of semen products from Russian farms and 15% of semen samples from international breeding centers. The viability of mycoplasmas that were positive according to PCR analysis when testing 65 samples was demonstrated using the microbiological method for 94% of samples. The correspondence of the results of microbiological and molecular genetic detection of mycoplasmas and ureaplasmas was 88.3%. The results obtained indicate the prevalence of mycoplasmas in semen production and the need to improve the control system of breeding material in order to prevent the spread of mycoplasma infections of cattle using artificial insemination technologies.

Use of Molecular Genetic Methods to Reduce the Risk of Incorrect Identification of Fish Strains in Brazilian Aquaculture.

The identification of fish species using traditional methods is generally based only on morphological characteristics and these methods are currently under review. This kind of identification of hybrid fishes solely based on their morphologies may be unreliable, especially when the samples include juveniles and post-F1 lineage fishes. Therefore, in the present study, we used molecular markers to accurately identify the fish species of economic interest that are used as materials in the projects developed in research institutions. We evaluated six lots of fishes sampled from different research centers, containing a total of 84 specimens acquired from private fish farms that were considered to be the representatives of pure species. Genetic analyses of all the specimens revealed that, globally, 22 samples (26.2%) were interspecific hybrids, while 20 (90.9%) samples were surprisingly characterized as post-F1 hybrids. This result confirms that hybrids are sold in markets without adequate labeling and also indicates the lack of proper control of the commercialization and management of stocks and products in fish farms. In addition, we determined that molecular diagnosis was an extremely effective and necessary method to test the reliability of biological materials currently used in scientific research.

Features of Microbiocenoses of Various Biotopes in Women as Potential Miscarriage Risk Factor

Relevance. Miscarriage is one of the most common obstetric pathologies that determine the spontaneous death of the fetus. The role of changes in the microbiome of various biotopes is considered as a risk factor of this disease.Aim of the research is to assess the microbiocenoses structure of the reproductive system and the intestine associated with the development of spontaneous miscarriages in early pregnancy.Materials and methods. A case-control study was conducted and organized. The group of «cases» included 23 women with spontaneous miscarriage at 5–14 weeks of pregnancy and the control group included 23 women with normal pregnancies Based on the data of metagenomic sequencing of V3–V4 regions of the 16S rRNA gene, a comparative assessment of the structure of the vaginal and intestinal microbiome in these groups was carried out.Results. As a result of the study, a number of taxonomic groups of microorganisms in the vagina and intestines associated with miscarriage. It was found that the decrease in the proportion of Lactobacillus spp. in the structure of the vaginal microbiome, less than 90% of the total number of identified taxa is associated with this pathology (OR = 5.28 (95% CI = 1.2-23.2)). The gut microbiocenoses of women with spontaneous miscarriage are characterized by less taxonomic diversity than the gut microbiome of women with advanced pregnancy. At the same time, some representatives of the intestinal microbiome (Akkermansia sp., Faecalibacterium sp., Bifidobacterium sp., Methanobrevibacter sp., Lactococcus sp.) predominate in women with a normal pregnancy.Conclusion. The study demonstrated a significant role of changes in microbiocinosis of the reproductive tract and intestines in miscarriage. There is a potentially protective function of lactobacilli. The obtained data substantiates the need for active use of molecular genetic methods focused on the assessing the structure of the vaginal and intestinal microbiota in assessing the risks of reproductive failures.

Clinical fnd epidemiological features of measles by adults in modern period

Positive results have been achieved during the implementation of the measles elimination program in the Russian Federation and in the Northwestern Federal District (NWFD). However, measles remains an urgent problem for some regions due to the peculiarities of this infection.Purpose of the work: to characterize the clinical, epidemiological and molecular genetic characteristics of measles in adults during the period of increasing incidence in St. Petersburg.Materials and methods: the incidence of measles in the Northwestern Federal District was analyzed in 2006–2020, and the data of 30 patients of S.P. Botkin clinical infectious diseases hospital over 18 years old (2018). The diagnosis of measles is confirmed by enzyme immunoassay. Molecular genetic studies (PCR, sequencing) were carried out at the National Scientific and Methodological Center for the Surveillance of Measles and Rubella of G.N. Gabrichevsky Moscow Scientific Research Institute of Epidemiology and Microbiology, biological material was used (nasopharyngeal washings, urine).Results: there was an increase in morbidity in some regions of the Northwestern Federal District in conditions of high coverage of the population with measles vaccinations. In the age structure in 2018–2019 the proportion of adults was 65%. 74% of patients were not vaccinated against measles. A moderate-severe course (100%) was noted, regardless of the genotypes of the virus. Clinical manifestations were characterized by febrile, catarrhal and exanthema syndrome. Diarrhea was found in 36,7% of patients, hepatomegaly in 43.4%, and an increase in transaminases in 87%. 66,7% of patients traveled outside St. Petersburg. The genotypes of the virus were identified: B3 Kabul and B3 Dublin of African origin, D8 Girsomnath of Indian origin.Conclusions: measles remains an urgent problem, in recent years, adults prevail among patients, both local and imported cases are recorded. The clinical course of the disease may be accompanied by intestinal complications and other symptoms that make it difficult to diagnose at the prehospital stage. The use of molecular genetic methods makes it possible to identify the pathogen, assess the identity of viral isolates, and improve the epidemiological surveillance of the infection.

Nomenclatural standards of modern Russian potato cultivars preserved at the VIR herbarium (WIR): A new approach to cultivar genepool registration in a genebank

VIR is creating a collection of nomenclature standards for cultivars of different crops in line with the provisions of the International Code of Nomenclature for Cultivated Plants (ICNCP). The nomenclatural standards are designed in the form of a herbarium sample in cooperation with the authors of cultivars, who select plant material for each cultivar in the experimental field of their organization and forward it to the scientific herbarium together with the accompanying documents. Registering of herbarium specimen in the scientific herbarium is followed by the publication of nomenclatural standards. The article presents a new VIR genebank strategy for registering plant material of domestic cultivars received from breeders, which includes the design of nomenclatural standards; the development of a genetic passport of a cultivar using DNA samples from the plant delivered to the scientific herbarium; and application of biotechnological methods for keeping explants (isolated from the acquired plant material as buds or meristems) alive. The proposed integrated approach makes it possible to document cultivars not only with the help of nomenclatural standards, but also with the use of molecular-genetic methods, as well as to save genotyped specimens, transferred to herbarium, in in vitro and in cryo collections. This approach is applicable to any vegetatively propagated crop. The article summarizes the first results of our proposed strategy using modern domestic potato cultivars as an example. Collection, transfer and preparation of plant material for nomenclatural standards of potato varieties are specific; this article explains our protocol for this work. The implementation of this strategy in 2018-2020 led to the designation of nomenclatural standards for 66 Russian potato varieties and elaboration of their genetic passports, which are presented in a series of articles included into issues Nos.3 and 4, Volume 3, of the journal “Plant Biotechnology and Breeding”, containing an overview of methodological approaches to registration of modern domestic cultivars in the VIR collection. DNA samples obtained from the plant material provided by the cultivar authors for nomenclatural standard designation will continue to be used as a source of information on the genetic potential of a cultivar through the expansion of the list of DNA markers and the application of new genetic technologies.

Molecular genetic testing for BRAF V600 gene mutations in melanoma in the Republic of Kazakhstan

Along with other diagnostic methods, all current recommendations and protocols for malignant tumors’ diagnostics and treatment require mandatory molecular genetic testing at the initial diagnosis and in case of disease progression.
 The discovery of the BRAF gene mutation in skin melanoma allows the treatment with tyrosine kinase inhibitors, so-called BRAF inhibitors. This increases the probability of tumor response by half and opens up new therapeutic options.
 The study aimed to identify the BRAF V600E gene mutation frequency in patients with stage III-IV melanoma in the Republic of Kazakhstan to determine the need for targeted therapy.
 Results: 2251 persons are currently registered with melanoma in the Republic of Kazakhstan, with 355 new primary melanoma cases in 2019. Of them, 54% were diagnosed at stages III-IV and were subject to molecular genetic testing. 278 of 2251 patients were sent for BRAF mutations testing. The mutations were detected in 105 (37.7%) patients.
 Conclusion: The introduction of molecular genetic testing for malignant neoplasms under the Comprehensive Cancer Control Plan framework for 2018-2022 will increase the use of molecular genetic methods in the Republic of Kazakhstan. Molecular genetic testing is an important step in diagnosing melanoma and choosing the appropriate therapy to personalize cancer treatment.

Molecular genetic testing for BRAF V600 gene mutations
 in melanoma in the Republic of Kazakhstan

Relevance: Along with other diagnostic methods, all current recommendations and protocols for malignant tumors’
 diagnostics and treatment require mandatory molecular
 genetic testing at the initial diagnosis and in case of disease
 progression. The discovery of the BRAF gene mutation in skin
 melanoma allows the treatment with tyrosine kinase inhibitors, so-called BRAF inhibitors. This increases the probability of
 tumor response by half and opens up new therapeutic options.
 The study aimed to identify the BRAF V600E gene mutation frequency in patients with stage III-IV melanoma in the
 Republic of Kazakhstan to determine the need for targeted
 therapy.
 Results: 2251 persons are currently registered with melanoma in the Republic of Kazakhstan, with 355 new primary
 melanoma cases in 2019. Of them, 54% were diagnosed at
 stages III-IV and were subject to molecular genetic testing.
 278 of 2251 patients were sent for BRAF mutations testing.
 The mutations were detected in 105 (37.7%) patients.
 Conclusion: The introduction of molecular genetic
 testing for malignant neoplasms under the Comprehensive
 Cancer Control Plan framework for 2018-2022 will increase
 the use of molecular genetic methods in the Republic of Kazakhstan. Molecular genetic testing is an important step in
 diagnosing melanoma and choosing the appropriate therapy to personalize cancer treatment.

Герпетологические исследования в Средней Азии и Казахстане: краткая история изучения рептилий, основные достижения и перспективы в XXI веке

This review is devoted to the history and progress in herpetological research in Central Asia and Kazakhstan, a region that is positioned within Central Asia. Studies of the herpetological diversity of Central Asia have a long history and are a special field of research. Research in this area has passed through several developmental stages, the first of which was associated with early expeditions which penetrated into Asia including those of P.S. Pallas and I.I. Lepekhin. The interest of naturalists was focused on this vast unexplored territory. The list of prominent researchers-encyclopedists and great travelers to work in Central Asia included E.I. Eichwald, A.A. Kaiserling, V.A. Perovsky, A.I. Schrenk, G.S. Karelin, G.I. Radde, G.I. Sievers, E. A. Eversmann, O. A. Grim, A.I. Lehman, O. Bettger, V.D. Alenitsyn, N.A. Severtsov, M.N. Bogdanov, A.P. Fedchenko, S.N. Alferaki, P. Y. Schmidt, N.A. Zarudny, L.S. Berg, and many others. At this historical stage, herpetological studies were often not separated into a special scientific branch and were carried out as part of complex zoological works. Later, in the twentieth century, large-scale herpetological studies were launched; a preliminary inventory of the fauna was carried out, many species were described, a synopsis of the species composition was created, and a zoogeographic analysis of the arid territories of Central Asia was performed. For the first time, the outstanding herpetologist A.M. Nikolsky, whose name our Herpetological Society bears, undertook this task. The history of research in the twentieth century and the current trends of the herpetological branch of zoology, which require an analytical understanding of descriptive and comparative data accumulated over previous periods, are considered in detail in this article. The use of an integrative approach in zoological research is becoming increasingly relevant, including the results of studying diversity structures, modes of speciation, and ecogeographic trends in a phylogenetic context. An integrative approach with the use of molecular genetic methods has allowed us to approach the solution of issues that have confounded many generations of herpetologists. The horizons of comparative analyses have also been expanded due to the increased availability of new, previously unexplored territories for researchers. Data obtained are of practical importance in the preparation of proposals for the allocation of protected areas and the assessment of the conservation status of particular species of model groups, including reptiles. Examples of integrative studies in model groups of reptiles (in various groups of turtles and lizards) are therefore also presented in this article.

Проблема контаминации спермы быков- производителей инфекционными агентами бактериальной и вирусной природы

The article reflects the main results of the study of the veterinary and sanitary well-being of breeding products based on the use of molecular genetic methods. The possibility of transmission through the sperm of cattle of a large range of infectious agents of viral and bacterial nature, as well as protozoa, mycoplasmas and fungi, has been established. It was recorded that in the recommendations of the OIE regarding the assessment of the quality and safety of bull semen, there are no requirements, but they are mostly directed at the animal itself and at the zone of its origin. At the same time, the study of 232 series of bull semen in 47 series found an excess of the normalized value of non-pathogenic microorganisms by 1,2–1,8 times. Of particular interest are the results of the detection of Coxiella burnetii, the causative agent of Ku fever, a zoonotic disease characterized in animals by a long-term persistence of the pathogen in the chronic form of the disease, in samples of genetic material.

Meat productivity of sheep of the Altai Mountain breed of different genotypes according to the CAST and GDF9 genes

The successful development of sheep farming in the meat sector is largely determined by the use of molecular genetic methods and modern genotypes. The Altai Mountain breed of sheep optimally combines high wool and meat with unsurpassed adaptability to grazing in the highlands of the Altai Republic throughout the year. The genes of calpastatin (CAST) and differential growth factor (GDF9) are considered as promising markers of quantitative and qualitative signs of sheep meat productivity. It was established that in the Altai Mountain breed the polymorphism of the CAST and GDF9 genes is represented by three genotypes MM, MN, and NN; AA, AG and GG with a frequency of occurrence of 0.23; 0.72 and 0.05; 0.10; 0.35 and 0.55, respectively. The level of homozygosity (Ca) in the CAST gene was 73.1%, GDF9 – 60.6%. The level of effective alleles (Na) was higher in the GDF9 gene (1.65) compared with CAST (1.44). The level of factual (Ho) and theoretical (He) expected heterozygosity for the GDF9 gene was 0.538 and 0.651, respectively, for the CAST gene – 0.290 and 0.368, which indicates a certain lack of heterozygotes in both genes. The analysis of slaughter qualities established the superiority of carriers of the NN genotype over the MM genotype in the CAS gene in terms of pre-slaughter and carcass weight, meat ratio by 4.96 and 2.83 kg, respectively (P<0.05), 0.26 units (P<0.05). In the GDF9 gene, the AA-type sheep exceeded the peers of AG and GG-genotypes in the pre-slaughter weight, carcass weight and its output, meat ratio by 1.62 and 7.01 kg, respectively; 1.34 and 3.98 kg (P<0.05); 1.21 and 1.86 abs. percent; 0.16 and 0.39 (P<0.05) units.

Microbiological diagnostics of inneriotrobic infections.

Intrauterine infections - infectious diseases in which infection of the fetus occurred in the ante- or intrapartum period, accompanied by clinical manifestations. The purpose of this study was to study the information content and diagnostic significance of the microbiological research method for the etiological diagnosis of intrauterine infections of a bacterial nature. A retrospective (2011-2014) and prospective (2015-2019) analysis of the results of microbiological studies of biomaterials from puerperas and their newborns was carried out in 63 cases of early neonatal mortality with established diagnoses of intrauterine infections. In the study of the separated cervical canal, placenta samples, and amniotic fluid, seeding of coagulase-negative staphylococci was most frequently observed, among which the species Staphylococcus epidermidis dominated, Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus warneri also met. Frequent isolation of group B streptococci from the placenta and amniotic fluid was revealed in comparison with the material from the cervical canal.The information content of the microbiological study of materials from the puerpera and the newborn in terms of confirmation of the pathogen and the fact of its transmission from mother to fetus/newborn does not exceed 30%. Even with high contamination of the genital tract of the puerpera, placenta or amniotic fluid, examination of the materials from the newborn immediately after birth often does not allow to identify the causative agent, probably due to the low degree of contamination at the initial stage of development of the infectious process. An increase in the diagnostic value of microbiological research can be facilitated by an increase in the frequency of examinations, the number of samples taken; the use of techniques to increase the sensitivity of cultural research at the stage of analysis; the use of molecular genetic methods, especially in the study of materials from newborns.

Molecular and epidemiological characteristics of hepatitis B virus in conditions of mass vaccine prophylaxis

The urgency of the problem of hepatitis B is due to the significant economic damage, medical, social and epidemiological significance of this disease, the ubiquity, severity, high frequency of adverse outcomes of infection, active involvement in the epidemic process of persons of reproductive and working age. The socio-economic significance of hepatitis B is evidenced by the morbidity, prevalence and mortality from this infection. The decrease in the incidence of acute hepatitis B, which is observed in Russia at the present time, is not a reason for an optimistic view of the problem, since the incidence of chronic hepatitis B over the past five years is almost at the same level (9–10 cases per 100 000 population). To date, laboratory diagnosis of viral hepatitis is based on the determination of specific markers by enzyme immunoassay. The use of molecular genetic methods opens up new prospects in the study of infection, allows us to identify patterns of manifestation, mechanisms of regulation of the epidemic process, including the study of the variability of the pathogen in space and time. The use of molecular genetic research methods should be considered not as an alternative, but as a mandatory addition to the regulated diagnostic schemes, which will effectively identify the pathogens of viral hepatitis, assess the identity of viral isolates and on this basis improve epidemiological surveillance and control of these infections.

Assessment of the occurrence of gene polymorphisms CAPN316 and UoGCAST in the population of cattle

To solve the problem of increasing the rate of breeding, but reduce the breeding work with farm animals, it is necessary to form herds with the desired level of productivity, that are adapted to specific regions of breeding, resistant to various diseases, with a decrease in the time for the breeding process. The active use of molecular genetic methods has contributed to the expansion of the list of DNA markers for farm animals, which are candidate genes for economically useful traits. Among these genes are widely known members of the calpain-calpastatin system, which is associated with postmortem proteolysis and tenderisation of muscles. The calpain system consists of an actively expressed μ-calpain (CAPN1) and m-calpain (CAPN2) and a single endogenous inhibitor, CAPN1 and CAPN2-calpastatin (CAST). The study of polymorphisms of these genes contributes to the expansion of marker characterisation in breeding. DNA samples (n=139) obtained from the blood of cattle were used in the work. Real-time PCRs were carried out using a ANK-32 programmable thermocycler (Synthol, Moscow, Russia). The CAPN316 gene polymorphism was present in 15% of animals tested, with allele G being found in 85% of animals. Similar calculations on the occurrence of the UoGCAST gene polymorphism in this sample of animals found the desirable allele G in 22% of animals and allele C in the remaining 78%. From the analysis of the occurrence of both genes and their polymorphisms in the study population, animals that had a combination of both desirable genotypes (CC*GG*) of CAPN316 and UoGCAST genes were not identified. There were also no animals that had the desired CAPN316 CC genotype and the heterozygous state of the UoGCAST gene (CC*GC). While 7.6% of the animals were CC*GG for the desirable expression of gene CAPN316, they contained the homozygous form of the gene UoGCAST.

Current State and Prospects of DNA Barcoding and DNA Fingerprinting in the Analysis of the Quality of Plant Raw Materials and Plant-Derived Drugs

Abstract—This paper reviews the use of molecular genetic methods (DNA barcoding and DNA fingerprinting) for the authentication of plant raw materials and herbal medicinal products. ITS2 is shown to be the most effective DNA barcode for plants. This marker allows researchers to identify even highly degraded DNA, which is especially important in the case of herbal preparations and biologically active additives. The main problem of traditional DNA barcoding via Sanger sequencing is that it is impossible to read a barcode without preliminary cloning of an amplificate when a plant mix includes a large amount of various adulterants and excipients of plant origin, since species-specific chromatograms are superimposed on each other. Thus, NGS sequencing of ITS2 amplicons is a preferable method for the DNA barcoding of plant materials. This method can be used to analyze highly degraded DNA in multicomponent plant mixes and can be used to identify all their ingredients. The review also covers such DNA fingerprinting techniques as RFLP, RFLP-PCR, RAPD, RAPD-SCAR, AFLP-PCR, and ISSR.