Combined pcr-eia in test tube with the extended surface
SUBSTANCE: reactions are conducted in the same PCR-microtube on walls of which there is a probe immobilised. In comparison with standard, the PCR-tubes used have a feature of construction: an extended internal surface, and as consequence - a great sorption capacity. A process of selective amplificate sorption on the test tube walls occurs after each anneal stage: temperature decreasing leads to hybridisation of amplicon chain not only among themselves, and with probes preliminary immobilised on the test tube walls. Using the similar PCR-microtubes enables increasing sensitivity of an immune-enzyme assay of amplicons and decreasing a number of polymerase chain reaction cycles. The fixed analytical signal will characterise both the presence of the required DNA, and show its concentration in the sample.
EFFECT: offered test system can be adapted for any existing PCR-technique, does not require special instrumentation and is reliable, simple and efficient for all parameters.
6 cl, 1 dwg, 1 tbl, 1 ex
The technical field to which the invention relates.
The invention relates to the field of analytical biochemistry, and more specifically to DNA analysis.
The level of technology
Typically, DNA analysis techniques based on the unique ability of the chains of nucleic acids for hybridization. Under the hybridization of nucleic acids to understand the process of re-education dvuhtsepochnyj molecules by pairing nucleotides complementary circuits. This phenomenon is used in a polymerase chain reaction (PCR, polymerase chain reaction (PCR) is the main method of DNA analysis, and numerous variants of the analysis of the amplicon product of this reaction. One such method is hybridization enzyme-linked immunosorbent assay (ELISA, enzyme-linked immunosorbent assay - ELISA), which in English literature got another one, a more accurate title ELOSA (enzyme-linked oligosorbent assay) [F. Mallet, Hebrard C., Brand, D., Chapuis, E., P. Cros, Allibert P., Besnier J.M., Barin F. and Mandrand B. Enzyme-linked oligosorbent assay for detection of polymerase chain reaction-amplified human immunodeficiency virus type 1. J.Clin.Microbiol, 31, 1444-1449, 1993.].
In the future, however, when using abbreviations, we will stick to the traditional terminology English transcription. In fact, due to the extreme popularity and prevalence of enzyme immunoassay, ELISA term rather not to be regarded as the abbreviation of the names, but as the logo of the method sublattice is someway plurality of ways and a variety of components. The above equally applies to the condensed name of the polymerase reaction (PCR). That's why often in scientific publications, presented, for example, in German or French, the authors try to not use a literal translation of titles and well-known things abbreviations.
Hybridization oligofermentans analysis of the products of the polymerase reaction based on the specific interaction of amplicons with the so-called hybridization probe. The latter is odnozadachnoy oligonucleotide, complementary to the available section of one of the chains of an amplicon comprising from about 20 bases. Probe covalently attached to a solid matrix (filter paper or the wall of the wells) and after incubation with the sample specifically binds to the complementary chain of the amplicon. The use of labeled nucleotides (e.g., Biotin, digoxigenin or radioactive phosphorus), embedded in the amplicon in the process of its formation, allows not only qualitatively, but also quantitatively judge the presence of an amplicon in the reaction mixture after completion of the polymerase reaction and, therefore, the presence of the desired DNA into the source of the analyzed sample.
Generally, the immobilization of the probes is carried out in cells in 96-well plates (blades), traditionally used in immunofermentnom the analysis. The use of special equipment greatly simplifies and standardizes work with these tablets. That is why the described method of detection of amplicons is specific, reliable, and affordable method.
Sharing methods PCR and hybridization oligofermentans analysis of amplicons is a very promising direction in the development of analytical chemistry of DNA for purposes of clinical diagnosis. In the scientific and patent literature, you can find numerous examples of analysis of different DNA by PCR-ELISA [PCR-ELISA - A low technology alternative to real time PCR.http://wxvw.btc-bti.com/pcrelisa.]. This method can serve as a real and considerably cheaper alternative to the analysis of DNA by PCR in real time (real time PCR).
The main obstacle to the widespread introduction of tandem PCR-ELISA in routine clinical practice the diagnosis is its duration. Summarily, without considering the time spent on the isolation of DNA from the sample, the duration of the PCR-ELISA takes at least 6-7 hours, of which at least three hours falls on stage incubation amplificata on the blade. Thus, more than 50% of the total time is exclusively stage of analysis of the products of the polymerase reaction. Therefore, when using a blade main disadvantage of ELISA as "x is nuclear biological chemical (NBC reaction of the process of the formation of amplicons is its duration.
One of the possible options to reduce the duration of hybridization oligofermentans analysis of amplicons is preheating hybridisierung solution to the melting temperature of DNA, which exceeds 90°C. However, to implement this process in practice, the application of traditional 96-well plates and standard incubators for hybridization impossible. The fact that polystyrene is used as raw material for the manufacture of tablets, in the temperature range from 90 to 100°C thermally unstable and subject to strong mechanical strain. In addition, incubators for hybridization, as a rule, aircraft type and structurally designed in such a way that it can provide a fast and controlled a sharp temperature drop in the chamber.
From the point of view of the task is to find a simple way to reduce the time of analysis of the amplicons by hybridization method oligofermentans analysis, the use of amplifier DNA as an incubator for hybridization and standard tubes for PCR as hybridization vessels seems almost ideal. The fact that the geometric shape of such tubes is ideally suited to surface topography fuser amplifier and the desired temperature at any stage of hybridization can be set is go for seconds and maintained with great precision.
This constructive design of the hybridization process greatly simplifies the method of PCR-ELISA as a whole and makes it much more attractive for routine studies for two reasons. Firstly, you will be able to carry out all stages of tandem PCR-ELISA is not only equal, but even in the same tubes. Secondly, instead spent when using blade 3 hours or more process specific interaction of amplificata probe will last no longer than 10 minutes: for a short period of amplicon melting at 95°C actual process of hybridization will last only a few minutes required for gradual cooling the tube to room temperature.
In the scientific and patent literature describes several variants of the DNA analysis of methods PCR-ELISA in the same tube [US Patent 6844158]. One of the most well-known method is called solid-phase PCR (solid phase PCR) [US Patent 6017738; C. Adessi, Matton G., Ayala G., Turcatti G., Mermod J-J, Mayer and P. E. Kawashima Solid phase DNA amplification: characterization of primer attachment and amplification mechanisms. Nucleic Acids Res., 28, e87, 2000 A. Carmon, Vision TJ, Mitchell S.E., Thannhauser T.W., Muller U., Kresovich S. Solid-phase PCR in microwells: Effects of linker length and composition on tethering, hybridization, and extension. BioTechniques 32,410-420, 2002].
In this method, one of the primers is covalently attached to the surface of the tubes. As a consequence, the formation of some part amplicon what will happen not only in solution, but on the vessel wall. A number of subsequent operations, the traditional heterogeneous enzyme immunoassay, is relatively quick to perform quantitative analysis of immobilized amplicons. The main challenges preventing widespread this method seems to be connected with the complexity of selection conditions polymerase reaction with the immobilized primer.
In other significantly less well-known variant of the method of PCR-ELISA in the same tube without the need to conduct polymerase reaction and hybridization analysis of series - these two processes can be combined to run concurrently.
The closest in technical essence and the claimed effect is a variant of PCR-ELISA described in the patent application CN 1521269. The functioning of the proposed test was performed as follows. Hybridization probe covalently "sewn" to the inner wall of the standard PCR tubes, which subsequently conducted polymerase reaction. Since the PCR method is a cyclical process, after each stage of melting DNA following this process of hybridization has taken place not only in the solution (between newly synthesized chains of amplicons or between circuits amplicon and primers), but also on the walls of the reaction vessel (between one of the circuits of the amplicon and immob lisovanim probe). Moreover, the amount of bound peroxidase molecules amplificate was directly proportional to the concentration of applicano in the cell. Thus, the formation of the amplicon (PCR reaction) and linking them with the wall of the tube (hybridization of amplificate with probe) was held in parallel. After PCR, it was necessary to wash out the test tube and to determine the amount of bound peroxidase bulleted amplificata any appropriate standard method.
The proposed version of the PCR-ELISA completely eliminates the continuous phase hybridization amplificata on tablets, which significantly reduces the time of DNA analysis in General: without regard to time allocation DNA described variant PCR-ELISA will take no more than two hours. In addition, the development of new methods of DNA analysis this method does not require introducing any significant corrections in existing established the scheme or recipe of the reaction mixture polymerase reaction, as is the case for solid-phase PCR.
Since different stages of polymerase reactions proceed at different temperatures in the range from 45 to 98°C for quickly creating in solution the desired temperature, it is desirable to use small amounts. In addition, the high cost of the reagents also dictates the maximum minimize the reaction medium. Therefore, as a rule the ILO this value is 25-50 µl. Such a small amount of leads, respectively, and a small area of contact of the solution with the surface of the tubes. This fact is in contradiction with the optimal conditions for carrying out immuno(oligo)enzyme analysis reveals the presence of significant design flaws in the standard smooth PCR tubes for the purposes of PCR-ELISA in the same test tube. In addition, due to steric physically impossible to mobilitat on a small surface such amount of hybridization probes, which would provide the necessary analytical signal. The latter fact has a significant negative impact on such a fundamental parameter analytical method sensitivity. Therefore, in order to organize the "analytical" (PCR), and "indicator" (ELISA) processes in one test tube, it is necessary for each of them to ensure the most favorable conditions. For "analytical" phase, in comparison with the classical method of carrying out PCR reaction, no significant changes in the formulation or temperature is not required, while for the "indicator" process, on the contrary, it is highly desirable to radically improve the properties of the tubes as the reaction vessel for oligofermentans EN is Lisa. This requires, on the one hand, to increase the surface concentration of immobilized hybridization probes, and, on the other hand, without increasing the volume of the reaction mixture to provide the greatest possible area of contact of the solution with the walls of the reaction tubes. In other words, it is necessary, without changing the geometric dimensions PCR tubes and without affecting any other properties, to increase the reaction surface area and sorption capacity.
Disclosure of inventions
This goal is achieved by replacing the smooth inner surface of the PCR tubes on the relief with the size of microcraters commensurate with the thickness of the vessel walls (see also the drawing provides a schematic representation of the structure of the inner wall PCR tubes with well-developed inner surface).
When the smooth inner surface of the tube, the reaction medium is with the wall of the smallest area of contact. The creation of microcraters on its inner surface will significantly increase as the sorption capacity of the vessel wall (you can mobilitat per unit surface area is significantly more hybridization probes), and will provide increased area of contact of the solution with the walls without having to increase the volume of the reaction medium.
The formation of microrelief internal PCR-tubes can be achieved by casting, as well as mechanical and/or physical effects on the internal surface of the finished tubes. The heterogeneity of the irregularities formed by micro - and microcraters may correspond to 1 through 6 classes roughness and to fluctuate in a wide range from a few to hundreds of microns. The creation of microdefects the inner surface of the PCR yourock allows you to increase the reaction surface area and sorption capacity tenfold. Apart from the purely mathematical calculations, the influence of the magnitude of the reaction surface and sorption capacity can be most clearly demonstrated in experiments on oligofermentans analysis solutions amplicons of different concentration.
Thus, the first aspect of the present invention is a test system for DNA analysis, which includes:
a) a reaction vessel for carrying out PCR-ELISA, representing a PCR tube standard sizes with a developed inner surface;
b) a standard set of reagents for PCR-analysis, which includes a buffer mixture, the oligonucleotides and the enzyme Taq polymerase;
C) a standard set of reagents for ELISA, including the subject of immobilization, hybridization probe conjugate and appropriate substrate;
d) instructions for use.
The increase in the area of the inner surface of the tubes is achieved by microgeek the s wall, representing micro - and microkeratome. The heterogeneity of the roughness of the inner surface of the tube formed by these micro - and microcraters, is in the range from several microns to hundreds of microns.
The second aspect of the present invention is a method of DNA analysis, including application of the above-described test system. The method comprises the initial standard procedure for immobilization of the probe on the internal surface of the PCR tubes with subsequent conduction in this tube analytical (PCR) and indicator (ELISA) reactions.
The implementation of the invention
For experimental verification of the achievement of the set objectives, experiments were carried out in test tubes of two types: smooth and well-developed surfaces. In the manufacture reaction vessels of the first type (smooth) all operations on chemical modification of the inner walls for the purpose of immobilization, hybridization probes were conducted in the raw PCR tubes. In the manufacture reaction vessels of the second type before carrying out similar operations internal surface of the tubes was previously loosened by using a diamond cutter, a work profile which was complementary to the geometric shape of the PCR tubes. In other words, the vessels of the second type were made into the base of PCR tubes with well-developed inner surface.
In manufactured so the reaction vessels was conducted oligofermentans analysis solutions amplificate obtained by serial dilution.
As a test object used the DNA of the microorganisms Chlamydia trachomatis, which causes one of the most common infectious diseases. Therefore, PCR-diagnosis of chlamydia is carried out in virtually every clinical diagnostic laboratory, and konosioni the composition of the reaction mixture, the reaction scheme, the structure of the primers or hybidization probes, as a rule, are well known. In our case, to obtain amplificata standard way used one of the first PCR methods proposed for routine diagnosis [Claas H.C., Melchers WJ, de Bruijn I.H., de Graaf, M., W.C. van Dijk, J. Lindeman, Quint W.G. Detection of Chlamydia trachomatis in clinical specimens by the polymerase chain reaction. Eur.J.Clin.Microbiol.Infect.Dis., 9, 864-868,1990].
Immobilization hybridization probe (aminirovanie 5'- or 3'-oligonucleotide) on the walls of the tubes was performed by using techniques borrowed from the works on creation of DNA biochips (microarray technology) [Beier M., J.D. Hoheisel Versatile derivation of solid support media for covalent bonding on DNA-by microchips. Nucleic Acids Res., 27, 1970-1977, 1999; M.C. Pirrung How to make a DNA Chip.Angewandte Chemie 41, 1276-1289, 2002]. The sequence of processes may be reflected in a simple two-step scheme: silanization surface+chemisorption [S.L. Beaucage Strategies in the preparation of DNA Oligonucleotide Arrays or Diagnostic Application. Curr.Med.Chem., 8, 1213-1244, 2001].
In the first stage of the test tube was treated with alcoholic solution of gamma-propyltriethoxysilane. As a result of this processing on the surface of the polypropylene was the formation of a silicon film containing a primary amino group. These groups were further involved in the process of covalent immobilization of the oligonucleotide. To do this just before proshivkoi" hybridization probes amino group of the walls of blood vessels has been modified succinic anhydride, and the process of immobilization was performed using water-soluble carbodiimide [Jiang X-S., Chai, S., Zhang Y., Zhuo R-X., Mao H-q, Leong K.W. Surface-immobilization of adhesion peptides on substrate for ex vivo expansion of cryopreserved umbilical cord blood CD34+cells. Biomaterials 27, 2723-2732, 2006].
The measurement of fluorescence after carrying out the immunoassay was carried out on an FL-800 (Biotech company), intended in particular for the signal directly from the PCR tube.
The results of the analysis of the amplicons by hybridization to PCR tubes with different inner surface presented in the table. As follows from the presented data, in comparison with the smooth, in test tubes with a developed inner surface, the value of the minimum detectable concentration of the amplicon was reduced by almost two orders of magnitude.
|The values of fluorescence depending on the concentration of the amplicon, hybridisierung in PCR tubes with different reaction surface. [Conditions: 50 μl of 20 mm Tris-Hcl buffer solution, 10 minutes hybridization: 95°C - 3 min, 45°C - 5 min; 15 minutes of interaction with the conjugate streptavidin-alkaline phosphatase; 10 minutes of incubation with the substrate (4-methylumbelliferyl) at 45°C]|
|The number of analyzed amplificate (ál)||The values of fluorescence|
|Standard PCR tube||PCR tube with a developed surface|
|Horizotally control (1 μl)||986||1147|
In addition, as can be seen in the table, already at low concentrations of analyte in a PCR tube with a developed surface is logged quite a high level signal. This suggested that when conducting PCR-ELISA in the same test tube, after a certain number of cycles of amplification, had no sense continuing to accumulate amplificat. The experiments confirmed the assumption. Indeed, to achieve the same signal magnitude when separated using PCR and ELISA, when conducting PCR-ELISA in smooth tubes or tubes with a developed surface, the number of cycles of amplification of the same original DNA concentration was 40, 33 and 28, respectively.
The presented results clearly indicate organic interaction and mutual influence of the three main components of the inventive test system: PCR, ELISA and reactor. Using PCR-tubes with large surface allows to improve the simultaneous occurrence of these processes, which shows the I in a significant reduction in the number of cycles of amplification and a significant improvement of the analytical characteristics of the ELISA as a method of quantitative analysis of the amplicons.
1. Test-system for DNA analysis, including:
a) a reaction vessel for carrying out PCR-ELISA, which is a tube of standard size, with well-developed inner surface;
b) a standard set of reagents for PCR analysis;
C) a standard set of reagents for ELISA;
d) instructions for use.
2. The test system according to claim 1, where the increase in the area of the inner surface of the tube is achieved at the expense of microdefects its walls, representing the micro - and microcrater.
3. The test system according to claim 1 or 2, where the heterogeneity of the roughness of the inner surface of the tube formed by micro - and microcraters, is in the range from several to hundreds of microns.
4. The test system according to any one of claims 1 to 3, in which a standard set of reagents for PCR-analysis includes: buffer mixture, the oligonucleotides and the enzyme Taq polymerase.
5. The test system according to any one of claims 1 to 4, in which a standard set of reagents for ELISA includes subject immobilization hybridization probe and the appropriate substrate.
6. The method of DNA analysis, including the use of a test system according to any one of claims 1 to 5, which provides a standard procedure for immobilization of the probe on the internal surface of the PCR test tubes, followed by successive conduction in this tube analytical (PCR) and the indicator reaction.
SUBSTANCE: faeces sample is examined in patients with acute enteric infections by bacteriological and microscopical methods. One faeces sample of the patient is concentrated by acetate sedimentation, and the produced sediment is used to prepare simultaneously 3 smears: the first one is stained with Ziehl-Nielsen carbolic fuchsin for acid-fast intestinal protozoa indication, the second one is stained with 1% Lugol's solution for lamblia detection, the third one is stained with polyvalent adsorbed serums in an indirect immunofluorescence reaction for salmonella indication, and is observing specific luminescence, the salmonellosis-protozoal acute enteric infection is diagnosed. 1-4 cells in a field of view enable to diagnose a mild form, and 5-12 cells - a severe infection.
EFFECT: use of the declared method allows producing results already in 1-1,5 h after sampling the material and providing higher diagnostic accuracy ensured by quantitative analysis of adhesive activity of simultaneously examined faeces samples, both for salmonellas, and for intestinal protozoa.
2 ex, 1 dwg
SUBSTANCE: urine is sampled, centrifuged that is followed by solid-phase extraction with Oasis HLB sorbent with using 100% acetonitrile as an extraction fluid for dimethyl terephthalate extraction. Said solid-phase extraction is conducted by consequent passing 100% acetonitrile, distilled water, the urine sample after centrifugation, distilled water, 20% aqueous acetonitrile and 100% acetonitrile as the extraction fluid through the sorbent; then the prepared extract is analysed by liquid chromatography with using as a mobile phase mixed acetonitrile and water in the at the varying ratio 25:75 vol. % to 90:10 vol. % respectively in a gradient mode which is enabled with combining chromatography by supplying at first the mobile phase containing mixed acetonitrile and water in the ratio 25:75 vol. % for 10 minutes. Then increasing the acetonitrile concentration in the mobile phase to 90 vol. % for 5 minutes and passing such mobile phase for another 5 minutes is followed by decreasing a volume amount of acetonitrile to 25 vol. % for 5 minutes and passing such mobile phase through a column for 10 minutes, while an amount of dimethyl terephthalate is determined by a calibration chart.
EFFECT: high sensitivity of the method combined with selectivity and availability for routine analyses.
5 cl, 6 tbl
SUBSTANCE: amniotic fluid of a pregnant woman on her 16-19 weeks of pregnancy is analysed for the concentration of arginine and praline by capillary electrophoresis. An aspect ratio is calculated, and if its value is equal to 3.2 and higher, foetus growth inhibition is diagnosed.
EFFECT: higher diagnostic accuracy of foetus growth inhibition.
SUBSTANCE: substance of the method for prediction of a CNS pathology in newborns consists in the fact that that blood of a pregnant woman on her 3rd trimester is examined for a nitrogen oxide. If the blood NO concentration is 6.12 mcmol/l and more, the developing CNS pathology is predicted in a newborn.
EFFECT: use of the declared method enables effective screening assays of the pregnant women for identification of the CNS pathology in newborns.
SUBSTANCE: peripheral blood lactate level is measured. If the postischemic lactate level exceeds 10 mmol/l, reperfusion syndrome is diagnosed.
EFFECT: improved diagnostic accuracy.
5 dwg, 1 ex
SUBSTANCE: substance of the laboratory diagnostic technique for sepsis consists in a quantitative analysis of blood serum for nonprotein microorganism metabolism products, particularly n-hydroxyphenyllactic (HPLA), phenyllactic (PLA), n-hydroxyphenylacetic (HPAA) and homovanillic (HVA) acids. If the contents of said four acids listed above synchronously excess the a healthy level by 5 times or more, sepsis is diagnosed.
EFFECT: use of said technique allows higher reliability of diagnosing sepsis in the patients with hyperthermia of an uncertain aetiology, in impaired state of the patients in hopital, in the intensive care patients, and in surgical postoperative complications.
4 tbl, 3 ex
SUBSTANCE: invention describes a method of quantitative evaluation of blood acetic, propionic, isobutyric, butyric, valeric, isocapronic and capronic acids by gas chromatography analysis wherein a blood sample is acidified with 1 % sulphuric acid to pH 2-3, evaluated acids are extracted with isobutyl alcohol volume of which is related to the blood sample volume as 1:1. The protein separation is enabled by centrifugation. 2-3 drops of 0.4 % alkali is added, and the extract is evaporated dry, further the solid residue is added consistently with 1 % sulphuric acid and isobutyl alcohol that is followed with gas chromatography separation of the mixed acids in a capillary column with a flame ionisation detector, and the amount of each acid is evaluated by a calibration diagram.
EFFECT: higher sensitivity and accuracy of the method of quantitative evaluation of acetic, propionic, isobutyric, butyric, valeric, isocapronic and capronic acids if found in blood together.
5 cl, 1 ex, 4 tbl
SUBSTANCE: invention relates to field of medicine, namely, to gastroenterology. For non-invasive diagnostics of fibrosis and cirrhosis in case of HBV and HCV infections complex ultrasonic examination of liver and spleen tissue is carried out. Additionally duplex scanning with colour Doppler mapping of porto-hepatic region vessels is performed and quantitative indices of hemodynamics of rate of blood flow in vessels, including splenic vein, are determined. Blood test is analysed and used to determine number of platelets, biochemical blood tests are performed, most significant for determination of disease degree indices of coagulogram are taken. After that, discriminant analysis of obtained characteristics and indices is carried out, and taking into account age and experimentally obtained coefficients, total value of two canonical discriminant functions F1 and F2 for HCV and HBV is calculated. Further, by obtained in empiric way territory map position of point for calculated by patient's concrete indices values F1 and F2 for cases of HCV-infection and HBV-infection is determined. Depending on point location on territory map case of mild fibrosis, severe fibrosis or liver cirrhosis is diagnosed.
EFFECT: method increases reliability of fibrosis and cirrhosis diagnostics in case of HBV and HCV infections.
10 dwg, 2 ex
SUBSTANCE: peripheral blood thrombocytes of women suffering gestosis of various severity levels on their 32-38 weeks of pregnancy are analysed for the activity of glutathione reductase (GY), NADF-dependent glutamate dehydrogenase (NADFGDG) and NADF-dependent isocitrate dehydrogenase (NADFICDG). A nicotine amide adenine dinucleotide phosphate transfer coefficient (NTC) represented by the relation of the GY activity to a product of the NADFGDG and NADFICDG activities is calculated. At the NTC value is equal to 1.3 and lower, the newborn's Apgar score is predicted to be equal to 6 and less, and the NTC value exceeding 1.3 provides the Apgar score being 7-10 points.
EFFECT: more accurate prediction of the newborn's state.
2 tbl, 5 ex
SUBSTANCE: areas highly exposed to harmful chemical agents are chosen. A random group of children without clinical signs of living in this territory is tested using chemical laboratory tests of blood to identify the content of chemical compounds, which are priority chemical environmental factors on the selected area of residence and clinical and laboratory studies are conducted to determine a set of laboratory indicators of adaptation system. Then using the results of the study the average values of chemical compounds in the blood are fixed and then they are compared to the background and average values for each of the above laboratory parameters are fixed and compared to the physiological norm; deviation of this value from the normal rate reveals children bodies response to chemical exposure. Next, a causal relationship is established between the level of content of chemical compound in the blood and the response of the child body through deviation of laboratory parametres from the norm using a logistic regression model. Using method based on analysis of odds ratios the maximally inactive level of marker of exposure and corresponding response marker based on the conditions are determined under which the odds ratio that characterises the degree of the connection between exposure to a chemical compound and the body's response will be greater than or equal to one; for this a model of dependence between the level of a marker of exposure and the specified index odds ratio is designed, the parametres of the model are determined and they reflect the change in the probability using which the value of the maximally inactive level of marker of exposure is calculated, i.e. maximum ineffective concentrations of chemical compounds. From the entire spectrum of defined concentrations of certain chemical compounds for each laboratory parametre of adaptation systems choose the smallest value that is accepted as the maximally inactive concentrations on a child adaptation system for a given chemical compound i. In future diagnosis of violation of adaptation of children living in the selected area is performed by comparing the content Ci of certain chemicals in their blood with previously established value of inactive concentration for this chemical compound; and if a ratio is a violation of adaptation is diagnosed.
EFFECT: method allows diagnosing violation of children adaptation under chemical hazards of environmental factors with high precision at an early preclinical stage, with simultaneous simplicity and accessibility for a wide practical application.
5 tbl, 2 dwg
SUBSTANCE: during selection by markers, a set of alleles related to loci of quantity tokens (QTL) that contribute to expression of certain phenes of economic value is introduced into a corn idioplasm. The criteria are selected among grain crop capacity, grain moisture when picked, early and late root fit, stem drowning, frequency of common rust, frequency of ear rotting caused by Fusarium (ear wilt), resistance to Sulcotrione and panicle structure. Invention also relates to the method of such plants production, and also to methods of analysis and screening to identify plants with a required profile of alleles.
EFFECT: improved method of new corn plants production.
33 cl, 1 dwg, 19 tbl
SUBSTANCE: synthetic oligonucleotides for indentifying DNA of Torque teno virus of all known genotypes are disclosed. Primers are combined in a set for DNA identification in blood and other biomaterials of the infectious agent of the latent viral infection Torque teno virus of Circoviridae family by polymerase chain reaction.
EFFECT: invention allows reliable identification of said virus in a biological material.
SUBSTANCE: analysed sample is studied simultaneously by two methods: the first one is a indirect immunofluorescence (IIMF) reaction, while the second one involves a polymerase chain reaction (PCR); the IIMF provides using monoclonal antibodies "BCKK" (P-384D and 434D). The antibodies interact with the capsular antigen F1 specific for Y.pestis species, or the plasmid-temperature-independent surface protein PFV which is found in all Y.pestis strains and rare R-form Y.pseudotuberculosis strains. The bacteria luminescence shows the presence of native or fraction-less Y.pestis bacteria, or typical and PFV-atypical Y.pseudotuberculosis strains in the sample. The PCR is conducted by two pairs of primers vlm33for/ISrevl754 - specific for Y.pestis species, and JS - specific for Y. pseudotuberculosis species. The values derived with the first pair of the primers are estimated as positive if observing the amplicons in 400 bps, and with the second pair if observing the amplicons in 223 bps; the analysed sample is identified by the matching the IIMF and PCR values with the reference strains.
EFFECT: method provides quick identification and high-reliability differentiation of all strains.
7 tbl, 6 ex
SUBSTANCE: method for bioindication of water bodies involves collecting samples of planktons inhabiting in a water body, determining the contamination level by analysing said samples and assessing the analysis results. The contamination level is determined via phylogenetic analysis of ribosomal RNA genes (18S rRNA) of planktons in the sample. Phylogenetic trees built from the conservative 18S rRNA gene are determined and evolutionary relationships of the analysed object with other saprobionts are identified. Analysis results are assessed as follows: at high (over 85%) value of bootstrap support of clusters containing the analysed planktons and resistant saprobionts, the following conclusions are made: resistant indicator organisms xeno- or oligosaprobic (or exclusively xenosaprobic) of water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in a safe ecological state and there is no threat of negative anthropogenic action, if resistant indicator organisms oligo- and mesosaprobic (or exclusively oligosaprobic) of the water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in an unstable (transition from safe to unsafe state) ecological state, is under insignificant anthropologic load, is capable of self-recovery and does not need additional environmental protection measures, if resistant indicator organisms meso- and polysaprobic (or exclusively mesosaprobic) of water bodies and the analysed plankton merge into one cluster, it is concluded that the water body is in an unsafe state and is under considerable anthropologic load, natural capability of self-recovery is insufficient and the water body needs environmental protection measures, if resistant indicator organisms of polysaprobic water bodies and the analysed plankton merge into one cluster, it is concluded that there is a local ecological disaster and there is need for urgent recovery measures.
EFFECT: high reliability of the biomonitoring result for use without territorial limit, independent of the geographical location of the investigated water body.
SUBSTANCE: bacteria freely immobilised on a slide are processed with a lysis solution containing an ionogenic protein-denaturing detergent and EDTA. Further, DNA-nucleoid is dry heating and microwave incubation stabilised. DNA is stained by high-sensitivity DNA-specific fluorochrome, in conclusion DNA integrity is evaluated. Offered is a kit for evaluating the DNA bacteria integrity which comprises all the components required.
EFFECT: invention can be used for determining the bacterial DNA fragmentation levels by a precise express technique.
16 cl, 11 dwg, 5 tbl, 9 ex
SUBSTANCE: offered invention can be used for the quantitative determination of alive bacteria. The offered method involves PCR-based product amplification with using the primers able to specific hybridisation with rRNA of a bacterium of interest. It is followed with the analysis of cell number of the alive bacterium of interest taking into account PCR cycle number.
EFFECT: invention allows precise determination of cell number of the alive bacterium of interest in a test sample.
8 cl, 7 dwg, 4 tbl, 10 ex
SUBSTANCE: it is described that immunogenicity of hemagglutinin (HA) molecule of influenza virus can be increased by substituting amino acids in the HA sequence. Substituting specific residues in HA such as asparagine introduction in position 223 in HA H5 allows ensuring more sensitive hemagglutination inhibition (HI) test provided by changing receptor specificity and/or ability to antibody-antigen linkage. The HA molecules having such substitutions can find application in creating diagnostic prototype viruses.
EFFECT: improved influenza virus vaccines.
9 cl, 3 dwg, 7 tbl, 7 ex
SUBSTANCE: specified plant is a plant of Cucumis sativus type and includes at least one area at one chromosome, which gives resistance to closterovirus, and at least one area, which gives resistance to powdery mildew. The area of the chromosome, which gives resistance to closterovirus, is linked to at least one marker selected from the group, made of markers E16/M50-244, E16/M50-188, and E11/M48-251. The area of the chromosome, which gives resistance to powdery mildew, is linked to at least one marker selected from the group made of the following components: a marker of single-nucleotide polymorphism 39T→G in SEQ ID NO:1, a marker of single-nucleotide polymorphism 29G→A in SEQ ID NO:2, a marker of single-nucleotide polymorphism 193C→T in SEQ ID NO:3, mutation of an insert 5'-AATTT-3' in position 221 in SEQ ID NO:4, and markers E16/M50-F-194, E11/M48-F-251, E23/M38-M001, E23/M40-M003, E24/M46-M002, E24/M46-M003, E12/M91-M003, E26/M43-M003, E14/M59-F-134 and E14/M59-F-200.
EFFECT: production of the plant resistant to closterovirus and to powdery mildew of cucumbers.
25 cl, 8 dwg, 4 tbl
SUBSTANCE: elaborated is method of obtaining factor, which takes part in process of control of appetite and/or body weight. Also described are genes, obtained by said method, polypeptides, coded by said genes, intended for treatment, control or diagnostics of diseases, associated with eating disorders and/or control of body weight. Invention also relates to substances, which inhibit activity of said genes or said polypeptides, intended for treatment, control or diagnostics of diseases, associated with process of appetite and/or body weight control.
EFFECT: using tiasolidindions, possessing PPARγ-agonistic activity, it is possible to obtain genes and polypeptides, involved into regulation of appetite and/or body weight reduction.
27 cl, 41 dwg, 35 ex
SUBSTANCE: RNA is recovered from peripheral blood or synovial liquid. Further, cytokine balance is evaluated by quantitative analysis of interleukin-2 (IL-2), interleukin-4 (IL-4) and interleukin-10 (IL-10) or interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-17 (IL-17), interleukin-1p (IL-1p) cytokines mRNA genes expression, as well as by quantitative analysis of interferon-gamma (IFNG) and a tumour necrosis factor (TNF) by reverse transcription and polymerase chain reaction with recording the accumulation of reaction products by direct fluorescence. The direct fluorescence is used to evaluate the cytokine balance. Further, a pair balance of expression of various cytokines is calculated on the basis a functional interrelation.
EFFECT: use of the invention reduces an evaluation error related to specific properties of the control gene expression.
4 dwg, 6 tbl, 2 ex
SUBSTANCE: container (1) consists of tube (2) closed at one end and pierced with point of sample lap of needle, of membrane disk (7), of integral choking cup (3) designed to close another open end of tube (2); also membrane disk (7) adjoins internal surface of external flat even head wall (5) of choking cup (3); notably, choking cup (3) has internal collar (4) retaining and propping membrane disk (7) inserted from beneath.
EFFECT: increased reliability of sampling and keeping sample inside.