Molecular diagnostics in medicine and veterinary
ABSTRACTS OF LECTURES
of the discipline: "Recent developments of Biotechnology in Veterinary Medicine and Animal Husbandry
Lecture №1
Biotechnology: History, Status and Prospects
Since the second half of the twentieth century, scientific progress obtains rapid development. During this period, two technologies have been created which radically changed the world. This is - nuclear technology and electronics. Over the past three decades in our lives enters the third new technology - modern biotechnology-based on discoveries in microbiology, immunology, biophysics, molecular biology, genetics, bioorganic chemistry, and such sciences as physics, chemistry and technology.
The background of biotechnology goes to the distant past and it is associated with the bakery, wine and other ways of cooking known to man since ancient times. For example, a biotechnological process involving the fermentation of microorganisms has been known and widely used in ancient Babylon, as evidenced by a description brewing of beer that has come down to us in the form of writing on a tablet found in 1981 during excavations of Babylon. The history of the formation of biotechnology conventionally divided into five major periods:
1. Period before Pasteur (until 1865). During this period
beer, wine, cheese, bread, yogurt, kefir and other different kinds of fermented foods were got by biotechnological methods;
2. Pasteur period (1865-1940). Microorganisms-producers became known , and it is allowed to receive ethanol, butanol, acetone, glycerol, citric acid, many vaccines, to organize the process of biological wastewater treatment by aerobic microorganisms;
3. Period of antibiotics (1940-1960). Penicillin, streptomycin, and many other antibiotics were discovered, the technology of cultivation of animal cells and receiving viral vaccines, technology biotransformation of steroid hormones were developed;
4. The period of controlled biosynthesis (1960-1975). Technologies of obtaining amino acids, microbial protein on paraffin oil, as well as enzymes used in detergents were created, methods of immobilization of enzymes (fixing them on the media) to receive glucose-fructose syrups are introduced into production, the technology of anaerobic treatment of solid waste to produce biogas were developed; microbiological method of producing polysaccharides (from xanthan to increase the viscosity of oil wells to the chewing gum) were discovered; use of microorganisms to receive vitamin B3 and B12, as well as microproteins - mycelial microscopic fungus (as a substitute for meat) was started, scientists have learned to cultivate isolated plant cells, which marked the beginning of the biotechnological production of many valuable medicinal substances using the great potential of medicinal plants, the basis bio metallurgy - leaching of copper and zinc from ores with the help of bacterial was created; etc.
|
|
|
5. The period of modern biotechnology (after 1975). It is characterized by developing genetic engineering, which allowed to created microbial technology for producing human insulin, interferon, and growth hormone somatotropin, and more; hybridoma technology of monoclonal antibodies - a powerful "tool" in the development of a huge variety of diagnostic products – was designed, so-called "transgenic" plants and animals with targeted genome construction have been appeared, etc.
With the development of biotechnology, various definitions of the term were emerged.
Moreover, to date there is no clear, comprehensive definition of science "biotechnology". Nevertheless, generalizing the existing definition, biotechnology should be determined as the science of using natural and modified prokaryotic and eukaryotic cells as well as transgenic plants and animals to produce valuable products.
Genetic and cellular engineering are the most important methods (tools) underpinning modern biotechnology. Methods for Cell Engineering focused on construction of a new cell type. They can be used to reconstruct viable cells from the individual fragments of different cells, to combine whole cells belonging to different species with the formation of cells carrying the genetic material of both the original cells, and other operations. Genetic engineering methods are aimed at design of new combinations of genes that do not existing in nature. As a result of genetic engineering techniques recombinant (modified) RNA and DNA can be obtained. After a certain manipulation of these genes by introducing them to other organisms (bacteria, yeast and mammals), which having received the new gene (s) will be able to synthesize the final products with the changed, desired properties. In other words, genetic engineering makes it possible to obtain transgenic plants and animals.
|
|
|
People have always thought about how to learn to control nature, and searched for ways to get, for example, plants with improved properties: a high yield, a large and delicious fruit, or with increased cold tolerance. Since ancient times, the main method used for this purpose was selection. It is widely used to date, and it is aimed at creating new and improving existing varieties of cultivated plants and breeds of domestic animals, strains of microorganisms with valuable features and properties for a man. Breeding is based on the selection of animals (plants) with distinct symptoms and further favorable crossing of such organisms, whereas genetic engineering allows direct intervene in the genetic apparatus of cells. It is important to note that during the traditional breeding for obtain hybrids with the desired combination of useful features is very difficult because very large fragments of the genome of each parent are transmitted to the offspring, whereas the genetic engineering techniques allow to work more often with one or several genes. As a result, one or more of the useful features that is very valuable for the creation of new varieties and new forms of plants can be added, without losing other useful properties of plants. It became possible to modify in plants, for example, their resistance to climate and stress, or susceptibility to insects or disease widespread in some regions, drought, etc. There is considerable research to improve the nutritional value of various crops such as corn, soybeans, potatoes, tomatoes, peas, etc. Historically, "three waves" in the creation of genetically modified plants were distinguished. The first wave (late 1980s) is the creation of plants with novel properties of resistance to viruses, parasites, or herbicides. In plants of the "first wave" only one additional gene was introduced, and it was forced to "work" that is, to synthesize an additional protein. "Good" genes "were taken" from plant viruses (for the formation of resistance to this virus) or from soil bacteria (to form a resistance to insects, herbicides). The second wave (early 2000s) is the creation of plants with new consumer properties: oilseeds with a high content of reformulated oils, fruits and vegetables with high content of vitamins, more nutritious grains, etc. Today, scientists create plants of "third wave", which in the next few years appear on the market. They are plant vaccines, plant bioreactors for the production of industrial products (components for various types of plastics, dyes, industrial oils, etc.), plant –factory for medicines, etc.
|
|
|
Genetic engineering in livestock have another problem. Technology which is achievable at the present level is the creation of transgenic animals with a specific target gene. For example, transgenic goats, by the introduction of corresponding gene can produce a specific protein - factor VIII, which prevents bleeding in patients with hemophilia, or an enzyme - trombokinasa that promotes resorption of the thrombus in blood vessels, which is important for the prevention and treatment of thrombosis in humans.
Transgenic animals produce these proteins are much faster and much cheaper than the very method of the traditional.
Another no less important direction of modern biotechnology is the "cell engineering", engaged in the acquisition of new cells with desired properties by merging the parental cells. Development of methods for somatic cell fusion made it possible for researchers to seek new ways of solving actual problems in medicine, veterinary medicine and agriculture. In particular, a new approach to obtain homogeneous (homogeneous) antibodies against infectious agents or other antigens that have great diagnostic value. A method for obtaining homogeneous of antibodies is called hybridoma technology.
Its essence is as follows. In the cause of entering organism specific foreign substance (antigen) protective body - antibodies which are heterogeneous in their physicochemical and biological properties are formed. Antibodies are produced by different lines of B-lymphocytes and they are directed to different regions (determinants) of antigen. If single cell of lymphocyte could be isolated and cultivated in vitro, then the resulting clone would have produced one type of antibody - a monoclonal antibody. However, lymphocytes can not grow outside the body. At the same time, there are cancers - myeloma cells that synthesize a large number of abnormal immunoglobulins. They are capable of unlimited growth and produce immunoglobulins which are similar in all respects. Thus, studies to obtain cell hybrids capable of producing monoclonal antibodies in vitro conditions have had a theoretical basis. At the end of 1974 hybridoma by fusion of myeloma cells and spleen lymphocytes from mice immunized with sheep erythrocytes was obtained by Koehler and Milstein. They were able to isolate clones of cells producing a specific type of antibody molecules and grow in the medium outside the body. Cell chimeras, called hybridomas, inherit the capacity for unlimited growth in culture and at the same time to produce antibodies of identical specificity, ie, monoclonal antibodies. The latest become a powerful tool in the development of efficient methods for diagnosis and treatment of diseases.
|
|
|
Another method of cell engineering is cloning which finds its application in animal husbandry. Cloning is a method of producing identical offspring by asexual reproduction. Otherwise, the cloning can be defined as the reproduction of genetically identical copies of a single organism. In nature, cloning is widespread among different organisms. In plants, natural cloning is done with various methods of vegetative reproduction in animals - in various forms of parthenogenesis and polyembryonesis (polyembryone from "poly" and Greek word embrion - «germ" - the formation of multiple embryos of animals (twins) from the same zygote as a result of an incorrect division due to the impact of random factors).
As for human example of polyembryony can serve the birth of identical twins, which are natural clones. There is a widespread clonal propagation among the crustaceans and insects.
The first artificially cloned multicellular organism was the sheep Dolly in 1997. The essence of the technique of "nuclear transfer" used in cloning is the replacement of cell nucleus of a fertilized egg to the nucleus extracted from the cell body, an exact genetic copy of which is scheduled to receive. To date currently developed not only the methods of reproduction of the organism from which the cell was taken but also those from which the genetic material was taken. There was potential to reproduction a dead body, even when it remain a minimum of parts - only necessary that one could isolate DNA. Cloning opens new prospects in the agriculture and animal husbandry. Animals with high productivity of eggs, milk, wool, or animals that produce the necessary enzymes to man (insulin, interferon, etc.). By combining the techniques of genetic engineering with cloning, we can derive transgenic agricultural plants that are able to protect themselves from pests or to be resistant to certain diseases. Here are listed just some of the opportunities opening through the use of this new technology. However, with all its advantages and prospects, so important for solving many problems of humanity, cloning is one of the most discussed areas of science and medical practice. This is due to the unresolved whole complex of moral and ethical and legal aspects of manipulation with sexual and stem cells, fate of embryonic and human cloning.
Cloning of organisms can be complete or partial. A full cloning recreates the whole organism as a whole, and the partial - recreated only certain tissues. Technology recreating the whole organism is extremely perspective in the case of the need to preserve rare species of animals or for the restoration of extinct species. Partial cloning - could become a major trend in medicine, because the cloned tissues can compensate for the deficiency and defects of the human body's own tissues, and, most importantly, they are not rejected during transplantation. Such therapeutic cloning did not initially involves getting the whole organism. Its development is consciously stopped in the early stages, and the resulting cells which are called embryonic stem cells (embryonic or fetal stem cells are the most primitive cells that arise in the early stages of embryo development, can develop into all cells of the adult organism) are used to generate the desired tissue or other biological products. Experimentally proved that therapeutic cloning can also be successfully used to treat certain human diseases, which are still considered incurable diseases (Alzheimer's, Parkinson's disease, heart attack, stroke, diabetes, cancer, leukemia, etc.), to avoid the birth of children with the Down syndrome and other genetic diseases. Scientists see the possibility of successful use of cloning techniques in the fight against aging and increasing longevity. The most important application of this technology is the area of reproduction, for example, case of sterility, both female and male.
In his lecture only a few of the many problems that arise in connection with the rapid development of biotechnologies and their invasion into human life are given. Of course, the progress of science can not stop and its problems poses faster than society can find answers to them. To cope with this situation we can only understand how is important discussing in society, ethical and legal issues that arise with the development of biotechnology. The presence of enormous ideological differences on these problems raises a serious need for consciously necessity of state regulation in this area.
The proposed discipline consider contemporary issues of Veterinary Medicine and Animal husbandry (immuno-and gene diagnostics, a new generation of vaccines, pharmaceuticals and food products, the creation of transgenic animals embrio- engineering, biotechnology, and biosecurity), which can be successfully solved using the techniques of modern biotechnology.
Test questions: 1.List the main periods of biotechnology 2. Define the classical and modern biotechnology; 3.Iist the areas of modern biotechnology, 4. What tasks set themselves genetic engineering of livestock? 5. Perspective tasks of cell engineering in veterinary medicine and animal husbandry.
Lecture №2
Molecular diagnostics in medicine and veterinary
One of the urgent problems of modern medicine and veterinary medicine is the development of methods for rapid detection of infectious diseases in human and animals, as well as water, food, feed, soil and other environmental objects. Prevention and treatment of infectious diseases greatly facilitates early and accurate diagnosis. Each of the methods used in human and veterinary medicine to identify the pathogen, has certain disadvantages. For example, the microscopic method, although it is a simple and affordable, does not allow to differentiate similar microscopic organisms, isolation of pure culture of the causative agent by means of bacteriological methods is indisputable proof of an infectious disease, but it takes a long time and do not identify those agents that do not grow on artificial media either do not amenable to cultivation, serological method for diagnosing the disease based on the detection in the blood antibodies to the pathogen-specific are not always specific. In this regard, the approaches of molecular genetic diagnosis, based on the methods of detection of specific DNA of the agent in the material, are aimed at eliminating these fundamental limitations, and, therefore, of great practical significance. Every effective diagnostic test should be: 1) a highly specific with respect to the target molecule, and 2) sufficiently sensitive to detect small amounts of the target, and 3) fairly simple that let you easily obtain unambiguous results. There are two types of molecular diagnostic techniques: one based on the affinity of antibodies to a particular antigen (immunological methods), the other is to identify specific nucleotide sequences by hybridization.
The most common immunological test is enzyme linked immunosorbent assay (ELISA). In this test system enzymatic label can be introduced in anti species or specific antibodies or antigen, depending on the scheme of setting analysis.
There are several options for setting ELISA. Method in which specific antibody in the sample are detected by enzyme-labeled antispecies immunoglobulins (conjugate) has found wide application. ELISA principle in this case is a specific interaction of conjugate with the antibodies to the test antigen, which results in indication of the resulting antigen-antibody complex. Anti species immunoglobulins conjugated to an enzyme is a globulin fraction isolated from the antiserum, prepared by immunizing animals- producers with the immunoglobulins of bovine, rabbit, mouse, and other animals, labeled with enzymes. As a solid phase are usual used polystyrene plates.
Antigen in ELISA can also be found by using the labeled specific antibodies. This method is used to identify antigens that have multiple determinants. The possibility of simultaneous binding of antigen with the antibodies of both types depends on whether it is bivalent antigen. But if it is monovalent, the first and second antibody should have specificity for different epitopes of the same antigen molecule. Name of the method "sandwich" is because in process of analysis antigen "pinched" between two antibodies. The method essence consists in the following. To the solid phase with immobilized antigen antibodies are added. Then unbound components are washed from the carrier and enzyme-labeled immunoglobulins are included. After removing the excess of the of conjugate a concentration of label associated with solid phase is determined by using substrate for the enzyme. In "sandwich" method anti species conjugates can be also used. In this case, antigens associated with adsorbed antibodies react with specific unlabeled immunoglobulins, which are then detected with the help of anti species conjugate, ie research process is increased by one step. In setting up this scheme of ELISA antibodies derived from different species of animals should be tested in order to prevent the reaction of immobilized antibodies with anti species conjugate.
"Sandwich" method can be also used to detect crossing antibodies. For this purpose at first wells are coated with antigen, then antibodies are added that specifically bind to them. Excess antibodies are washed and antigen linked to enzyme is added. If the bound antibodies cross-react with antigen then antigen bound to the enzyme will also adsorbed on the solid phase. Quantitatively, it is defined as an increase in absorption as a result of enzyme-catalyzed reactions. The presence in a "sandwich" of additional layers can increase the sensitivity, but it causes an increase of the background and the variability of the results.
The next option, called competitive ELISA can detect antigen in the sample using the same antigen-labeled enzyme. Essence of the method is as follows. To the immobilized antibody investigated sample antigen and its complex with enzyme are added. Thus there is a competition among investigated and labeled antigens for antideterminants of antibodies. After a while conjugate is redistributed between the solution and carrier. Concentration of label measured at solid phase is proportional to the initial content of test antigen. Method is simple and quickly done, but there is difficulty in obtaining the antigen-enzyme conjugates.
In some cases, the chemical modification of the enzyme is accompanied by a deterioration of its activity and antigen modification can decrease of its ability to form a stable complex with antibodies. In such cases, specific interest is the ELISA that uses non-covalent complexes of the enzyme-marker with the antibodies. The most effective approach is the inclusion of enzyme label in the avidin-biotin complex. It is based on the use of avidin that is component of egg protein (molecular weight 66,000) and biotin, i.e. vitamin (molecular weight 228) for forming the complex, binding constant of which the tens of thousands of times greater than the bond strength of antigen-antibody complexes. Adsorbed antigen is incubated with a sample containing antibody, the wells are washed and antispecies immunoglobulins covalently linked to biotin are included. Then avidin labeled with peroxidase is added, which forms a stable complex with biotin. Excess of conjugate is removed and concentration of antigen is determined after introduction of enzyme substrate.
One of the practical and highly sensitive options of ELISA is Peroxidase test. Substance of the method lies in a combination immunoperoxidase conjugate with a specific antigen, and determining the formed complex by diaminobenzidin reagent. In veterinary medicine, it is more widely used in the diagnosis of viral infections (rabies, foot and mouth disease, etc.). Peroxidase test as ELISA carried out in two ways, direct and indirect. The last sensitive than the first and does not require a set of specific immunoglobulins labeled with peroxidase. The reason for choice of peroxidase as the label of antibodies is its molecular weight that less than that of other enzymes used in ELISA. Therefore immunoperoxidase conjugate better penetrate the cell membrane.
Method of molecular hybridization is the first test of genetic testing based on the use of so-called DNA probes labeled with isotope, fluorochrome or enzyme. Last is a small piece of DNA (or RNA), the nucleotide sequence of which is specific to a certain type of microorganism. When adding a labeled probe to the sample, which contains DNA of investigated microorganism complementary connection sequences is occured. The resulting complex, depending on the type of label used is determined by radioisotope analysis, or ELISA, or immunofluorescence. It should be noted that method of DNA probes can detect nucleic acids of microorganisms in their relatively high concentration.
The specificity of the probes can be different. For example, they can differentiate two or more species, some strains within the same species or different genes. Depending on the situation, the probes can be represented by DNA or RNA molecules, they can be long (more than 100 nucleotides) or short (less than 50 nucleotides). Probes obtained in different ways. One of them is the following. DNA of Pathogen is cleaved with endonucleases and is cloned into a plasmid vector. Then recombinant plasmids are screened using genomic DNA of pathogenic as well as saprophytic strains. Those plasmids that contain sequences hybridizing with the DNA of only pathogenic strain form the basis of species-specific probes. After that series of additional hybridization of isolated DNA with DNA from different organisms are carried out to make sure that the probes do not give with them cross-hybridization. To determine the sensitivity of the method each of the probes are also verified at the model samples, including those in mixed cultures (B.Glik, Dzh.Pasternak, 2002). DNA probes are also prepared by chemical synthesis of nucleotide sequences which are responsible for a certain sign of the microorganism.
One of the examples using DNA probes for diagnosing diseases is detection procedure of Plasmodium falciparum. This parasite causes malaria, a disease that threatens about a third of the population. To detect pathogen DNA as the basis of the test highly repeated DNA sequences of Plasmodium falciparum are used. It can only detect 10 pg of purified DNA of the parasite, or 1 ng of the same DNA in blood. Through hybridization practically all pathogens can be detected. To date, DNA probes of most pathogens, viruses, and protozoa are obtained and characterized. Among the first agents which "earn" their DNA probes are Legionella pneumophila (respiratory disease), Salmonella typhi (food poisoning), Campylobacter hyointestinalis (gastritis) as well as strain of Enterotoxic Esherichia coli, Mycobacterium tuberculosis (the causative agent of tuberculosis) Brucella abortus (the causative agent of brucellosis) etc.
Molecular hybridization is usually performed on the starting material, without its culture or extraction of nucleic acids. For example, it is possible to carry out hybridization with the DNA molecules of agent which are present in samples of feces, urine, blood, milk, and other materials, as well as in the tissues without preliminary cleaning them. If the concentration of the target sequence in the material is too small, it can be increased considerably. The application of molecular genetic approaches based on the detection of pathogen DNA by amplification (multiplication) of their specific areas is a new direction in the diagnosis of infectious diseases. In 1983 K.Mulles from biotechnology company «Cetus Corporations» suggested to multiply DNA of detecting microorganisms in the sample, and then conduct identification of the DNA-DNA. This approach was later called the polymerase chain reaction (PCR).
At the heart of PCR lying multiple repetition of cycles of DNA replication. Each cycle consists of three stages with different temperature regimes. At the first stage at a temperature of 93-95 C the separation of complementary chains of DNA (denaturation) takes place. The second stage consists in adding pair of primers to sample containing DNA of detecting microorganism. One of primers is complementary to one chain and the other – to the opposite. Primers are artificially synthesized single-stranded desoxioligonukleotides consisting usually from 20-27 base pairs which are terminal sequences of required fragment of DNA. At a temperature of 50-65 C synthesis of polynucleotide chains by primer extension using desoxioligonukleotidethreephosphate (dNTP) in the presence of a thermostable DNA polymerase is taking place. During the reaction primers annealed at these chains and initiate enzymatic synthesis of DNA of towards each other.
At the end of the synthesis new chains of double-stranded DNA is melted, the same primers are annealed and once again DNA is synthesized. Repeating the three stages for 30-40 times during 1.5-3 hours millions of copies of specific DNA or RNA of certain microorganism are obtained, ie nucleic acid in amount sufficient to visualization it by agarose gel electrophoresis without the use of radioisotopes is accumulated.
PCR has two main drawbacks: false-positive reactions caused by contamination of the DNA fragments from the previously obtained products (amplicons), and false negative, as a result action of inhibitors that are present in biological fluids and tissues on the Taq-polymerase. In addition, to the disadvantages of the test should be referred high cost of reagents and equipment which are used in its production.
In 1989, A. Brisson-Noel et al. for the first time published the results of experiments on working off rapid diagnosis of tuberculosis by PCR. They were examined 35 clinical samples (sputum, stomach contents, the contents of the abscess, peripheral blood), only two of which positive in PCR failed to meet the negative results obtained by conventional methods (microscopy, bacteriological analysis).
Highly effective PCR test systems for detecting Mycobacterium tuberculosis in a variety of tissues and body fluids were offered by researches of VGNKI (Russia)). Trials showed that PCR is able to replace all of the methodology applied to the laboratory diagnosis of the disease. The test result can be obtained in a few hours, contamination of materials with other species of mycobacteria and any other microflora does not matter. Also of interest is determining the applicability of PCR for the in vivo diagnosis of tuberculosis and detection MbT.
PCR for detection of Brucella for the first time was applied by D.Fekete et al. (1990). Later, it was used successfully for generic and specific identification of these organisms in the study of bacterial cell lysates. Shumilov KV et al. (1996) studied the sensitivity and generic specificity of PCR (in option two step reaction with nested primers complementary to the gene BCPR 31 Brucella compared with bacteriological method). Samples of milk and blood were collected as pathological material, contaminated with culture Brucella abortus 19 at a concentration of Brucella 1 billion cell/ ml; samples of spleen, liver from guinea pigs killed in 20 days after immunizations with Brucella abortus 19 and 104M at 1 billion cell/ ml. It was established that PCR confirm all the positive results of bacteriological method, and taking into account the studies of liver samples sensitivity of PCR was 2 times higher than that of bacteriological method. The authors concluded, PCR method is highly sensitive and specific and allows to determine the presence or absence of the pathogen in the material during one working.
Test system for the identification of microorganisms of genus Brucella by PCR ("GENE-BRU") was developed by Russian SRI "Microbe". A.A.Pavlov (2002) studied the effectiveness of the Diagnostic Kit in comparison with serological diagnosis of brucellosis. It was shown that the sensitivity of PCR exceeds AT and CFT in 2-2,4 times. PCR achieve maximum sensitivity when blood samples of animals were used as an diagnostic material. The possibility of detecting DNA of vaccine strain B.abortus 82 in the later period after immunization was established, which is evidence of its long-term survival in the body of the vaccinated animals. It is proved that test system can be used for detecting of Brucella DNA in milk, as a method of evaluation of sanitary conditions of food of animal origin.
D.Sklyarov et al. (2004) developed a test system "BRU-COM", based on the use of PCR, which allows to detect the DNA of all species of Brucella in milk, blood, sperm, organs and lymph nodes of infected animals. PCR Trial of test system showed that it significantly exceeds bacteriological method in sensitivity. For the diagnosis of brucellosis by PCR authors recommended to investigate a few items (about 5-6 pairs of large lymph nodes, spleen and liver), since the study of some of them may affect the reliability of the result.
N.S.Yudin et al. (2008) used PCR to identify the virus bovine leukemia in leukocytes. Primers used were specific for the envelope gene env. Matching percentage between RIA and PCR was 31%. Thus, 25 animals positive for RID were PCR-negative, while 22 RID-negative animals were PCR-positive. The authors recommend the PCR as an additional method for the early diagnosis of leukemia. N.V.Kuznetsova et al. using PCR to detect leukemia virus-infected cattle, came to the conclusion that this test can be used to determine the source of infection in herds during breeding sale, prevention, and at the final stages of infection eradication. According to the authors, in animals with a slight increase in morphological parameters of blood PCR can reliably distinguish leukemoid state of the body from an early stage of leukemic process.
V.L.Zaytsev (2005) used PCR to detect viruses of sheep pox, contagious ecthyma of sheep and goats, and mouth disease in the various virus-containing materials. Specific primers were developed that allow for differentiation of viruses of sheep pox and goat pox. Test set allowed with high sensitivity and specificity to identify virus in 2-5 pg of genomic DNA.
O.Sementsova et al. (2011) for the study of biological samples from patients and animals, as well as vectors (insects and mites) for the presence of genetic material of flavivirus diseases (encephalitis, West Niel fever, yellow fever, Japanese encephalitis) a laboratory version of the multiplex PCR (allowing to identify multiple pathogens in a single tube) was developed to the registration of the results in real time. Thus, the methods of molecular diagnostics as a highly sensitive and specific tests can find practical application in veterinary medicine for the early identification of infected animals and detecting pathogens in biological material or environmental objects.
Test questions: 1.List the advantages of molecular diagnostics in comparison with other known methods, 2. Tell us about the principles of setting different options of ELISA 3. What distinguishes the method of molecular hybridization from polymerase chain reaction (PCR)? 4. Tell us about the status and prospects of using of PCR in veterinary practice.
Lecture №3
Дата добавления: 2018-04-15; просмотров: 400; Мы поможем в написании вашей работы! |
Мы поможем в написании ваших работ!