Achievements andprospects of developmentof molecular diagnostics
One of the urgent problems of modern medicine and veterinary medicine is the development of methods for rapid detection of pathogens of infectious diseases in humans or animals, as well as in water, food, feed, soil and other objects of the environment. Prevention and treatment of infectious diseases is greatly facilitated by early and accurate diagnosis. Each of the methods used in medicine and veterinary medicine to identify a pathogenic agent has certain drawbacks. Thus, for example, the microscopic method, although simple and accessible, does not allow us to distinguish similar microorganisms under a microscope; isolation of pure culture of the pathogen by means of a bacteriological method is an indisputable proof of an infectious disease, but it takes a long time and does not reveal those pathogens that grow poorly on artificial nutrient media or are not culturable at all; The serological method that diagnoses the disease on the basis of the detection of antibodies specific to the causative agent is not always specific. In this regard, the approaches of molecular genetic diagnostics, based on the methods of detecting the specific DNA of the pathogen of the disease in the material under investigation, are aimed at eliminating the indicated principal limitations, and, therefore, have great practical significance.
Any effective diagnostic test should be: 1) highly specific to the target molecule; 2) sensitive enough to detect a small amount of a target;
3) simple enough, making it easy to get unambiguous results. There are two types of molecular diagnostic methods: one based on the affinity of antibodies to a particular antigen
(immunological methods), the other on the identification of specific nucleotide sequences by hybridization.
Advantages of molecular diagnostics are its universality, the ability to use for analysis any DNA-containing cells or tissues, and the analysis can be performed at any stage of ontogenesis, beginning with the zygote stage
Status andprospects of usingPCRin veterinary practice.
The PCR is based on repeated repetition of DNA replication cycles. Each cycle consists of three stages with different temperature regimes. In the first stage, at 93-95°C, the complementary chains of the double-stranded DNA molecule are separated (denaturation). The second stage consists in introducing into the sample containing the DNA of the detected microorganism a pair of primers, one of which is complementary to one chain and the other to the opposite one.
Primers are artificially synthesized single-stranded deoxyoligonucleotides consisting, as a rule, of 20-27 base pairs, representing the terminal sequences of the DNA fragment of interest. At a temperature of 50-65 C, polynucleotide chains are synthesized by elongation of primers with deoxyribonucleotide triphosphates (dNTP) in the presence of a thermostable DNA polymerase. During the reaction primers are annealed on these chains and initiate enzymatic DNA synthesis towards each other. At the end of the synthesis, new strands of double-stranded DNA are melted, the same primers are annealed, and DNA synthesis is again performed. Repeating 3 stages 30-40 times in 1.5-3 hours, millions copies of a specific region of DNA or RNA of a particular microorganism are obtained, i. E. nucleic acid is produced in an amount sufficient for its visualization by means of agarose gel electrophoresis without the use of radioisotopes.
PCR has two main disadvantages: false positive reactions caused by contamination with DNA fragments from previously obtained products (amplicons), and false-negative reactions resulting from the action on Taq-polymerase inhibitors present in biological fluids and tissues. In addition, the shortcomings of the test should include and the high cost of reagents, as well as equipment used in its formulation.
In recent years, molecular biological methods, in particular polymerase chain reaction (PCR), have been increasingly used to identify pathogens for certain infectious diseases.
In 1983 K. Müllis discovered the principle of selective multiplication of nucleotide sequences, and in 1984 developed a PCR method that allows several hours to obtain millions of copies of a selected DNA fragment, which results in the accumulation of DNA in the reaction mixture.
The PCR method is based on the identification of the causative agent of the species-specific region of the DNA, which allows obtaining a positive result when there are single cell cells in the test material.
The advantages of PCR in laboratory diagnostics are associated with rapidity, high sensitivity and specificity, which allows to detect microorganisms available in the material under study in a very small amount (D. Cousins et al., 1991; G. Buck et al., 1999).
Currently, studies are under way to study the possibility of using a polymerase chain reaction in the diagnosis of tuberculosis in humans and animals. Nevertheless, there is no consensus on the effectiveness of PCR in diagnosing tuberculosis (D. Thierry et al., 1990, 1992; VV Punga, 1998).
Thus, the methods of molecular diagnostics as highly sensitive and specific tests can find their practical application in veterinary medicine for the timely detection of infected animals and the detection of pathogens in biomaterial or objects of the external
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