Diagnostic technique for inflammatory process in early rheumatoid arthritis

FIELD: medicine.

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

 

The technical field to which the invention relates.

The proposed method relates to the field of medicine, biology and biotechnology and can be used as an additional method of screening patients for early diagnosis of inflammatory diseases based on the polymerase chain reaction with the accumulation of reaction products, "real-time" (PCR "real-time").

Polymerase chain reaction (PCR) is widely used for nucleic acid amplification and allows, in particular, to identify the presence and to determine the amount of nucleic acid with a specific sequence of nucleotides in the RNA sample.

The results of polymerase chain reaction to analyze directly during the reaction. For registration of the results of the PCR process is performed detecting the amplifier in the presence of either fluorescent intercalating dyes or fluorescently labeled oligonucleotide primers or probes.

Rheumatoid arthritis (PA) is an autoimmune disease of unknown etiology, characterized by symmetrical erosive arthritis (synovitis) and a wide range of extra-articular (system) manifestations. RA represents a vivid model of chronic inflammation, which allows to study the fundamental problems of modern medicine, associated with pathoge Tom and therapy of various human diseases. PA is an extremely common disease, affecting approximately 1-2% of the population of the globe. The cardinal signs of RA is steadily progressing defeat of the joints, leading to disability and reduced life expectancy of patients. Traditionally RA is considered as a model antigen-specific activation of CD4+ T-lymphocytes TX1 should be the type characterized by the predominance of the synthesis of interleukin-2 (IL-2), interferon-γ (IFN-γ) on the production of interleukin-4 (IL-4) [1-3]. Imbalance TX1 should be/TX2 response enhances the activation of macrophages, dendritic cells, b-lymphocytes, accompanied by the overproduction of proinflammatory cytokines (tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-18 (IL-18) and others) and decreased expression of anti-inflammatory cytokines (IL-4, soluble antagonist receptor IL-1, soluble TNF-α receptors) [4-7].

There are several subpopulations of T-lymphocytes, including T-helper 1 (TX1 should be) and 2 (TX2) of the order. Each of them allocates certain cytokines: Tx1 - IFN-γ, IL-2, TNF-α; TX2 - IL-4, IL-5, IL-6. The body is supported by a certain ratio of Tx1 and TX2-cells, respectively, and secreted them of cytokines. In pathological conditions, this balance may be disturbed.

The characteristics of the different C is Takenov

TNF-α. The main source of TNF-α are activated macrophages, in vitro lysis of lymphoma cells, necrosis sarcoma in vivo activate polymorphonuclear leukocytes exhibit antiviral activity. Under the influence of TNF-α dramatically increases the formation of macrophages and neutrophils hydrogen peroxide and other free radicals.

IL-1β. The main source of production of IL-1β are monocytes and macrophages of different tissue localization, as well as endothelial cells, T-lymphocytes and b-lymphocytes, fibroblasts, NK-cells, keratinocytes and neutrophils. Is a mediator of acute and chronic inflammation. Performs many important functions, including the stimulation and release of neutrophils from the bone marrow; the activation of lymphocytes and neutrophils.

IL-2. IL-2 is an important proinflammatory cytokine that stimulates the proliferation and differentiation of activated T lymphocytes into effector T-lymphocytes or cytotoxic T cells. IL-2 can stimulate large granular lymphocytes, macrophages and b cells. IL-2 is secreted by T-lymphocytes CD4+and T-cells of some other lymphocyte subpopulations. IL-2 belongs to the group of hematopoietic cells, it was opened one of the first. In the mid 60-ies have shown that culture stimulated with mitogen or antigen lymphocytes accumulate in the supernatant facto is, which greatly enhances proliferation of freshly isolated peripheral blood lymphocytes in vitro. Of particular interest was the fact that adosados from cultures stimulated lymphocytes capable of a long time to support the proliferation of intact T cells and to ensure the functional activity of clones of these cells. The active compound in the culture fluid of stimulated lymphocytes were cytokine T-cell nature, originally called T-cell growth factor. The main result of the action of IL-2 in resting or stimulated by antigen or mitogen cells is to ensure their proliferation. This biological activity of IL-2 defines it as a typical growth factor cell lympho-myeloid complex.

IL-4 belongs to the group of hematopoietic cells. It used to be called the growth factor In cells. The source of IL-4 are T-helper cells stimulated by mitogen, fat cells, unidentified stromal cells of the bone marrow. It is known that IL-4 increases the expression of antigens of the major histocompatibility class II (MHC II) in resting b cells, and synthesis of immunoglobulins IgG and IgE after stimulation by lipopolysaccharide (LPS), supports the viability and growth of intact T cells, increases the activity of cytotoxic T-lymphocytes, enhances proliferation before the of estevanico haematopoiesis in response to growth factors. In relation to b-cells IL-4 acts as costimulatory proliferation. So, he does not have any effect on resting b cells, but enough to act on them one of the specific data cells inductor activation that showed biological activity of IL-4: a sharp increase in the proliferation of this cell type. Initially, IL-4 has been described as b-cell growth factor I. In retrospect, that he is able to enhance the proliferation of T-cells belonging to different subpopulations. Its effect on T-cells and b-cells occurs only after prior activation by antigen or mitogen. By its nature, the IL-4 acts as a pleiotropic regulator, as it interacts with a variety of cell types. So, its effect on macrophages is manifested in the increased expression of class II molecules MHC and Fc receptor for IgG, acquisition antitumor activity against fibrosarcoma, enhancing antigen presenting function.

IL-6. Interleukin-6 is produced by activated monocytes or macrophages, endothelial cells, fibroblasts, activated T-cells, as well as a number of cells, non-immune cells. Some of the effects of IL-6, similar to the observed under the action of IL-1 and TNF. However, the main effect of IL - due to his involvement as a cofactor in the differentiation of b-lymphocytes, Mature and transform into plasma cells secreting immunoglobulins. In addition, IL-6 promotes the expression of the receptor for IL-2 by activated immune cells, and also induces the production of IL-2 by T-cells. This cytokine stimulates the proliferation of T-lymphocytes and reactions of hemopoiesis.

IL-8. Interleukin-8 belongs to the group of chemokines, the main property of which is to provide chemotaxis in the area of inflammation of different types of cells: neutrophils, monocytes, eosinophils, T cells. IL-8 has a pronounced anti-inflammatory properties, causing the expression of molecules intercellular adhesion and reinforcing the adhesion of neutrophils to endothelial cells and subendothelial matrix proteins, suggesting its primary role in mediating the inflammatory process. Cells producing IL-8 are macrophages, lymphocytes, epithelial cells, fibroblasts, epidermal cells. The main function of IL-8 to act as chemoattractant for neutrophils, macrophages, lymphocytes, and eosinophils. In addition, the biological actions of IL-8 enhances the adhesive properties of neutrophils, changing the expression of integrins and other compounds with adhesive properties. Properties of IL-8 to induce the migration of cells and promote their adhesion define it as an active participant in the acute inflammatory reactions is.

IL-10. Is a key regulator of immunolo response with immunosuppressive properties. It is synthesized mainly by T-lymphocytes, mast cells and macrophages.

IL-17 is characterized as a proinflammatory cytokine, is produced by a subpopulation of lymphocytes Th functions as a factor of protection of the organism against pathogens. Stimulates the synthesis of IL-6, IL-8, TNF-α, IL-1β, are involved in maintaining the number of neutrophils (hypothesis M. Stark, 2005).

IFN-γ has antiviral activity. The main property - regulation of immunity. Is produced mainly by activated T-lymphocytes and NK-cells. Main functions: activation of macrophages; stimulation of the expression of molecules of class II MHC; activation of NK cells; regulation of synthesis of isotypes of immunoglobulines B-lymphocytes; direct antiviral activity.

The progression of RA is an evolving process that can be divided into several stages: early (asymptomatic) stage, which is characterized by vascular and cellular activation; expanded stage (fast chronic inflammation), manifested violation of angiogenesis, activation of endothelial cell migration, infiltration of activated SV+T-lymphocytes in the synovial tissue, the formation of rheumatoid factors and immune complexes, synthesis "PR is inflammatory" cytokines, prostaglandins, collagenase, matrix metalloproteinases; late stage, which is characterized by induced chronic inflammation offline "tumor-like processes"due to somatic mutations and defects in apoptosis of synovial cells.

One of the major problems of modern rheumatology is getting the correct diagnosis of RA in its early stages and differentiation of this disease from other rheumatic diseases. The diagnosis of RA in the first place is made on the basis of the clinical picture. Due to lack of specificity of clinical criteria for setting the objective of the diagnosis it is necessary to conduct additional laboratory tests (detection of antibodies to rheumatoid factor (IgM RF) and antibodies to cyclic citrullinaemia peptide (ACCP)), as well as research, the results of which allow us to more accurately assess the patient's condition and adjust therapy. One of such studies can serve as an evaluation of cytokine profile in peripheral blood cells (hereinafter "PC") and synovial fluid (hereinafter "SG") using polymerase chain reaction in real-time with pre-production reaction of reverse transcription ("RT-PCR").

The method of RT-PCR "real-time" is widely used in scientific research, including Chesley for quantitative evaluation in the study of mRNA expression of cytokine genes man. The level of mRNA expression is evaluated relative to the mRNA expression of genes that ensure the viability of the cells (referred to as "genes of the household", the control genes, normalization genes reference genes), ΔCt methods (method 1) or ΔΔCt (method 2) [8]. The Ct value is automatically determined by the software of the device is the cycle at the point of intersection with the baseline.

The disadvantages of these methods include the absence of "perfect control gene", satisfying all the requirements (especially the constant expression in all organs and tissues). Therefore, in practice, the normalization is carried out by several control genes, determine the normalization factor, and comparing the expression levels in different samples (method 2). The disadvantages 2 method include the need to conduct a full investigation with all the samples, which is very inconvenient in terms of clinically meaningful application in routine practice.

The task proposed by the invention is to remedy these disadvantages, in achieving faster, more reliable and easier to diagnose the presence of inflammatory process in early rheumatoid arthritis by analyzing only one biological material.

The proposed method for the diagnosis of vos is Alitalia diseases (model of rheumatoid arthritis at an early stage) on the basis of the quantification of the transcripts of cytokine genes by PCR "real-time" includes:

- the use of the control gene (gipoksantin-guanine phosphoribosyltransferase 1 - HPRT1) to a greater extent for assessing the quality of execution of all stages of the reaction than for normalization when determining the level of expression of mRNA (messenger RNA) genes of cytokines,

- determination of the spectrum of cytokines, characterized by an increased expression of mRNA in PC and SG,

- determination of the spectrum of cytokines that are characterised by decreased expression of mRNA in PC and SG (i.e. those cytokines that are most informative for determining the condition of the patient),

- search functional linkage and causing molecular processes of mutual influence in the network cytokine cascade, reflecting the development and course of inflammatory process in early rheumatoid arthritis (hereinafter "PPA"),

- calculation of the markers (i.e. the most informative cytokines)that takes into account the dependence of the intensity of the inflammatory process in the PPA, according to the formula

[IL1]/[IL2]=Eil2Ct2/Ei11Ct1(formula 1),

where

[IL1] is the concentration of mRNA cytokine gene with increased expression in PC or LF;

[IL2] is the concentration of mRNA of the gene of cytokine characterized by low (or constant) expression in PC or LF;

E - the efficiency of amplification is a quantity that characterizes the growth of the matrix on each cycle of amplification (the form is and 2);

Ct1 and Ct2 - value threshold cycles that are automatically determined by the instrument, the sample for the respective cytokine.

In practice, the efficiency of amplification is determined experimentally by the formula

E=10-(1/slope)(formula 2),

where

slope is the difference in Ct values with a 10 fold dilution of the sample (angular coefficient of the linear equation according to Ct from the logarithm (lg) concentration matrix) (Fig.1).

Thus, during the NDP "real-time" get standardized according to the intensity of fluorescent radiation from non cycle polymerase chain reaction (Fig.2). The software automatically computes the threshold cycle. These values can be substituted into the formula 1.

Technical result achieved in the implementation of the proposed invention consists, first, in assessing the severity of the inflammatory process in the PPA immediately after the reaction; secondly, if the calculations are not taken into account the value of Ct for normalization gene that reduces the probability of error of the method associated with individual differences in the expression of the control gene in a given tissue type; third, to more accurately assess the levels of expression of the relevance of multiple normalization is ENES questioned.

Brief description of drawings

Figure 1 shows a plot of Ct against log (lg) the concentration matrix. The slope coefficient is equal to -3,3919. Accordingly, the efficiency of amplification was 1.97. The efficiency of amplification is determined in a series of experiments with breeding matrix in a concentration range linear range of test systems as an average value.

Figure 2 shows the standardized according to the intensity of fluorescent radiation from non cycle polymerase chain reaction for the whole investigated range of cytokines. The threshold cycle is determined automatically by the instrument, as the point of intersection of the graph data with the baseline (Threshold).

Table 1 presents the values of the efficiency of amplification for all test systems.

Table 2 shows the indicators of the inflammatory process.

In table 3 shows the volume of the conducted research: examined 116, were studied 101 sample PC and 91 sample SG.

Figure 3 shows the ratio of expression levels of cytokines in peripheral blood identified in the study PC, where the left scale is the ratio of the expression level of IL-10/IL-4 and IL-10/IL-2 in early rheumatoid arthritis patients.

Fig.4 shows the ratio of expression levels of cytokines in synovial fluid, to detect the Noe research results OOH, left scale is the ratio of the expression level of Pro-inflammatory cytokines (TNF-α, IL-6, IL-8, IL-17, IFN-γ, IL-2 and IL-4 in rheumatoid and early rheumatoid arthritis patients.

In table 4 above correlation statistical indicators of the levels of expression of cytokines in synovial fluid from patients of the comparison group with osteoarthritis, where M and σ - respectively the average values and the average quadratic deviation.

In table 5 shows the coefficients of correlation of C-reactive protein (CRP) and rheumatoid factor (RF) with a ratio of cytokines (Spearman).

In table. 6 presents the performance of cytokine profile for example, patients with weak (patient No. P11/12) and pronounced (patient No. 31/32) inflammatory process.

The implementation of the invention

stage 1. Material capture

Material research is peripheral blood (PC) or synovial fluid (LF) of patients with suspicion on the PPA.

The severity of the inflammatory process in the PPA can be identified by any of these liquids. The most informative is SG.

The sampling is carried out in tubes with EDTA (anticoagulant that prevents blood clotting) at a final concentration of 2 mg/ml

PC taken from a vein in the usual way, OOH taken from the affected joint with a syringe.

stage 2. The selection of cells

option 1: from your PC you who elaut mononuclear cells.

The separation of the cellular elements of blood is carried out by centrifugation in a density gradient ficoll-urografin (density of 1.077 g/ml). The selection of lymphocytes spend from 1 ml of whole blood. The obtained cells resuspended in 100 μl of physiologic saline.

The methodology of extracting blood lymphocytes on ficelle: Boyum A. Separation of leukocytes from blood and bone marrow //Scand.J.Clin.Lab.Investig. - 1968. - Vol.21. - Suppl.97. p.1-9

2 variant: SG precipitated all cells.

to 1 ml of LF add 1 ml of saline;

- besiege cells by centrifugation at 6000 rpm for 10 minutes.

- remove supernatant,

- derived cells to resuspendable 100 ál fishriver.

stage 3. The allocation of RNA.

For RNA extraction using sets "Sample NC" (manufactured by JSC "APF of DNA technology") with modification (the phenol deproteinization). The method is based on the lysis of samples in a 4M solution guanidinoacetate, phenol deproteinization, the precipitation of nucleic acids with isopropanol in the presence of shoesadidas, followed by cleaning with ethanol and acetone.

The volume of sample after extraction of 50 µl.

Differences with respect to PC and SG no.

Methods RNA extraction:

- to prepare one tube for negative control sample - "K-";

add to each tube of cells PC and SG, as well as "To-" 300 ál lyse solution;

- tightly closed kr is the loud and tubes, shake on vortex for 3-5 s;

to thermostatically tubes 15 min at 65°C, precipitated condensate by centrifugation at 13,000 rpm for 30 s;

add 400 µl of saturated phenol, shake the test tube on the vortex for 30 s;

- precipitate drops from walls of tubes by centrifugation at 13,000 rpm for 30 s;

- add 160 μl of a solution of chloroform:isoamyl alcohol in the ratio of 24:1, shake the test tube on the vortex for 30 s;

- centrifuge the tube at 13,000 rpm for 10 min;

- to select the supernatant to a new tube;

add 400 µl of saturated phenol, shake the test tube on the vortex for 30 s;

- precipitate drops from walls of tubes by centrifugation at 13,000 rpm for 30 s;

- add 160 μl of a solution of chloroform:isoamyl alcohol in the ratio of 24:1, shake the test tube on the vortex for 30 s;

- centrifuge the tube at 13,000 rpm for 10 min;

add 400 ál of reagent for precipitation (isopropanol) and shake the test tube on the vortex for 3-5 s;

- centrifuge the tube at 13,000 rpm for 15 min;

- not touching the sediment, remove supernatant (separate tip for each tube);

to add to the precipitate 500 ál of wash solution №1 (70%ethanol) and 3-5 times gently invert each tube;

<> - centrifuge the tube at 13,000 rpm for 5 min;

- not touching the sediment, remove supernatant (separate tip for each tube);

to add to the precipitate 300 ál of wash solution # 2 (acetone) and 3-5 times gently invert each tube;

- centrifuge the tube at 13,000 rpm for 5 min;

- not touching the sediment, remove supernatant (separate tip for each tube);

- open the lid of the tube and dry the precipitate at 65°C for 5 min;

to add to the precipitate in 50 μl of buffer for dissolving;

- to warm up the tubes at 65°C for 10 min;

- shake the samples for the vortex to dissolve the material, to besiege drops by centrifugation of the tubes at 13,000 rpm for 30 sec.

Preparation of RNA is ready for arming the reaction of reverse transcription.

The resulting preparation RNA it is recommended to use for the given reaction reverse transcription. Preparation of RNA can be stored at a temperature of minus 20°C for 1 month or at minus 70°C for 1 year.

stage 4. Reverse transcription

The reaction of reverse transcription is carried out by the enzyme reverse transcriptase in a particular buffer in the presence of messenger RNA (mRNA), deoxynucleotides and primers at 40°C for 60 min Inactivate the reverse transcriptase inhibitor is carried out at 95°C for 15 minutes.

During the reaction the enzyme reverse transcriptase with matrix RNA (mRNA) synthesis of she complementary DNA (cDNA) by the polymerization of deoxynucleotides, using as seed crystal a complementary chain primers.

As primers for reverse transcription using specific oligonucleotides.

The composition of the reaction mixture in the test tube when conducting a reverse transcription reaction:

- trichitillomania water treated with DEPC - 0,7 µl;

- the reaction buffer* - 4 ál

- a mixture of deoxyribonucleotides four types at a concentration of 25 μm each deoxyribonucleotide - 0.2 µl;

primers** (11 specific oligonucleotides) at a concentration of 100 μm each 0.1 µl;

- reverse transcriptase at a concentration of 500 international units of activity - 1 mm;

- analyzed sample mRNA - 33 μl.

*the composition of the reaction buffer: 0.5 M Tris-HCl pH 8.3, 0.75 M KCl, 30 mm MgCl2, 50 mm DTT, with 0.1% DEPC.

** primers:

TNF-α 5'-ggg TTC gAg AAg - 3';

IL-1β 5'- gTC CAT ggC CAC - 3';

IL-2 5'- gTT gTT TCA gAT C - 3';

IL-4 5' - CCT TCA CAg gAC - 3';

IL-6 5' - CAC CAg gCA AgT C - 3';

IL-8 5' - CTC TSC, TCC ATC - 3';

IL-10 5' - CAC Agg gAA gAA - 3';

IL-17 5' - Cgg CAC TTT gCC - 3';

IFN-γ 5' - Tgg ACA TTC AAg - 3';

HPRT1 5' - TCC ACA ATC Aag - 3'.

To increase the samples after reverse transcription of cDNA diluted 5-fold with deionized water.

stage 5. P is R.

Primers and probes for PCR were selected taking into account the structures of exons and introns in such a way as to prevent annealing of the matrix genomic DNA of cytokines and HPRT1 (control gene). This allows not to use an additional stage of processing nucleic acids DNA asoi.

In standard previously proposed protocols this step was performed to remove from the DNA sample, because the reaction may not go on cDNA obtained in the course of the reaction the reverse transcripti, and genomic DNA. It was necessary to eliminate the response on the genomic DNA, for example by removing her DNA asoi. The proposed method due to the specificity of the primers for mRNA prevents annealing of the matrix on the genomic DNA, so this step is not necessary.

DNA probes used for detection of amplification products of cytokine genes and HPRT1 marked FAM. Amplification reaction of cytokines and HPRT1 are used in different test tubes. To increase the sensitivity and specificity of PCR was applied "hot start", which is ensured by the use of paraffin. The optimum temperature for annealing of the primers and probes were selected experimentally using the "temperature gradient", for all test systems, the temperature was uniform and was 62°C.

Amplification is carried out in "real time" in a volume of 35 ál according to the following program: 1 cycle - 80°C 30 sec, 94°C 1 min; 50 cycles of 94°C 10 sec, 62°C for 20 sec, used instruments DT-322" and "DT-964" production company "NPO DNA Technology". Measuring the level of fluorescence is performed on each cycle at a temperature of 62°C.

The composition of the reaction mixture in vitro by PCR:

- bidistilled water - 25,37 ál

- the reaction buffer* and 3.5 ál

- a mixture of deoxyribonucleotides four types at a concentration of 25 μm each deoxyribonucleotide - 0,24 ál

primer No. 1** at a concentration of 100 μm - 0,125 ál

primer No. 2** at a concentration of 100 μm - 0,125 ál

- detection test** at a concentration of 50 μm (reagent labeled with a fluorophore, to determine the desired target) of 0.07 ál

- Taq polymerase at a concentration of 5 international units of activity of 0.5 ál

- analyzed sample cDNA obtained by reverse transcription reaction (see step 4 above) and 5 µl.

* the composition of the reaction buffer: 100 mm Tris-HCl (pH 8.8 at 25°C), 500 mm KCl, 0.8% R40, 20 mm MgCl2

** primers and detecting the sample for TNF-α primer No. 1 -5'- gAT Cgg CCT CCA gAg ggA Ag - 3'; primer No. 2 - 5'- ggg TTC gAg AAg ATg ATC - 3'; the detecting sample- (FAM)-5'- gCC CTC Tgg CCC AggCAg-3'-(BHQ1);

IL-1β primer No. 1 5'- gCC HUNDRED AAC AgA TgA AgT - 3';

primer No. 2 -5'-gTC CAT ggC CAC AAC AAC - 3'; the detecting sample - (FAM) -5'- gCg gCC TgC CTg AAg CCC - 3'-(BHQ1);

IL-2 primer No. 1 -5'-CCC AAA CTC ACC Agg ATg C - 3';

primer No. 2 -5'- gTT gTT TCA gAT CCC TTT Ag-3'; the detecting sample - (FAM)- 5'- CAg TTC TgT ggC CTT CTT gg - 3'-(BHQ1);

IL-4 primer is 1 -5'-gAC ATC TTT gCT gCC TCC AAg - 3';

primer No. 2 -5'-CCT TCA CAg gAC Agg AAT T-3'; the detecting sample - (FAM)-5'- TTT GCC GGA GCA CAG TCG CAG - 3'-(BHQ1);

IL-6 primer No. 1 -5'- gAA CTC CTT CTC CAC AA - 3';

primer No. 2 -5'-CAC CAg gCA AgT CTC CTC - 3'; the detecting probe (FAM)-5'- GCC TGC TGC CTT CCC TGC C - 3'- (BHQ1);

IL-8 primer No. 1 -5' - TgC AgC TCT gTg TgA Ag - 3'; primer No. 2 - 5' - CTC TCT TCC ATC AgA AAg CTT - 3'; the detecting sample - (FAM)- 5'- gCg Cag TgT ggT CCA CTC TCA - 3'- (BHQ1);

IL-10 primer No. 1 -5'- CTT gCT ggA ggA CTT TAA gg-3'; primer No. 2 -5'- CAC Agg gAA gAA ATC gAT gA - 3'; the detecting sample -(FAM)5'-CCT ggA ggA ggT gAT gCC C - 3'-(BHQ1);

IL-17 primer No. 1 -5'- Tgg gAA gAC CTC ATT ggT - 3'; primer No. 2 5'- Cgg CAC TTT gCC TCC CAg A-3'; the detecting probe(FAM)- 5'- CCA CgA AAT CCA ggA TgC CC - 3'- (BHQ1);

IFN-γ primer No. 1 -5'- gAA gAA TTg gAA AgA ggA gAg - 3'; primer No. 2 5'- Tgg ACA TTC AAg TCA gTT AC - 3'; the detecting sample - (FAM)-5'- gTC TCC ACA CTC TTT Tgg AT -3'-(BHQ1);

HPRT1 primer No. 1 5'- ggA HUNDRED ATT ATg gAC Agg ACT g - 3'; primer No. 2 -5'- TCC ACA ATC AAg ACA TTC - 3'; the detecting sample - (FAM)-5'- CAG CCC CCC TTG AGC ACA CAG - 3'-(BHQ1).

The tubes are placed in the detection amplifier (a device that allows simultaneous PCR and record the fluorescent radiation in vitro), such as the detection amplifier DT-96 (manufactured by JSC "APF of DNA-Technology, Russia), and performed PCR with the check results in real-time."

stage 6: analysis of results

As shown in figure 2, for each sample PC or SG will receive a set of values Ct (automatically defined software both the biscuits device is the cycle at the point of intersection with the baseline) for each cytokine. These Ct values substituted into the formula 1:

[IL1]/[IL2]=Eil2Ct2/Eil1Ct1

The efficiency of amplification was determined experimentally (table 1). For different test systems it was 1,96-1,98, except for IL-4 (1,92).

In the course of the study it was found that when the PPA may be increased mRNA expression of proinflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8 and IL-17), and immunosuppressive IL-10 and reduced the mRNA expression of anti-inflammatory IL-4. The most informative for assessing the inflammatory process in the PPA is LF (option 2). The following indicators (table 2) in PC and SG can be predictor inflammatory process:

option 1 for PC: interleukin - 10/interleukin - 2>1,5

interleukin - 10/interleukin - 4>1,0

option 2 for SG: interleukin - 2/interleukin - 4>10

interferon-γ/interleukin - 4>300

the tumor necrosis factor-α/Il - 4>2000

interleukin - 1β/Il - 4>5 000

interleukin - 6/interleukin - 4>10

interleukin - 8/interleukin - 4>10000

interleukin - 17/interleukin - 4>5

The principal difference of the proposed method is that there is no need to use the normalization gene.

Examples of the implementation of the proposed method

Were surveyed 116 people, studied 101 sample PC and 91 sample SG (table 3). Formed 4 is a group of the study:

- 1 group consisted of conditionally healthy patients (control). In the current study is infectious, respiratory viral, autoimmune, allergic and other diseases were excluded. The size of the group amounted to 25 people. Due to the difficulty of taking this group SG in the study was taken only PC.

- in the 2nd group included patients with a diagnosis of rheumatoid arthritis (RA). The size of the group amounted to 49 people. The study was taken SG and PC.

- 3 group included patients with a diagnosis of early rheumatoid arthritis (the PPA). Group size was 27. The study was taken SG and PC.

- 4 group included patients with a diagnosis of osteoarthritis (OA). The size of the group amounted to 15 people. The study was taken only LF. This group was chosen as the comparison group because the capture material in this case was possible, and also due to the fact that this disease is a chronic non-inflammatory joint disease in contrast to rheumatoid arthritis.

The results of the study is represented in figure 3. The ratio of the expression level of IL-10/IL-4 and IL-10/IL-2 in early rheumatoid arthritis was significantly higher (respectively p=0,0063, p=0,00009)than in the control group. Fig.3 shows the medians of the groups, as well as 25% and 75%quartiles for the control group. Media is its value (M) in the control group IL-10/IL-4 was 0.4, standard deviation (σ) of 0.2. General formula (2) for all correlations was calculated as

Interleukin-1/interleukin-2≥M+3σ (formula 3).

The average value (M) in the control group IL-10/IL-2 was 0.4, standard deviation (σ) of 0.3.

Thus, the calculations for the formula 3 in the control group IL-10/IL-4=1, the IL-10/IL-2=1,3. In the presence of an inflammatory process, these figures will be higher.

The results of the study OOH presented on Fig.4. The ratio of the expression level of Pro-inflammatory cytokines (TNF-α, IL-6, IL-8, IL-17, IFN-γ, IL-2 and IL-4 in rheumatoid and early rheumatoid arthritis was significantly higher than in the comparison group with osteoarthritis. Figure 4 presents the median in groups, as well as 25% and 75%quartiles for the control group and the group of rheumatoid arthritis. The average value (M) and the average quadratic deviation (σ) in the control group are presented in table 4. On the basis of them was the formula of the invention.

The comparison of the received cytokine profile and activity of inflammatory process in the PPA.

To evaluate the activity of the inflammatory process in the PPA was chosen such laboratory marker of inflammation like C-reactive protein (CRP). As an immunological marker of RA was used IgM rheumatoid factor (RF). The level of CRP and RF were measured by a highly sensitive the method for laser turbidity meter "BN-100" (Dade Behring, USA), units of measure mg/l

Received moderate and a good positive correlation of these indicators with indicators of cytokine ratios (table 5).

Consider two example

Patient No. P11/12. Male, age 54 years. The number of affected joints - 14. At the time of the survey the CRP in the blood of 0.67, SG 0.26 mg/l; RF in the blood and SG 0.1 mg/l, indicating low activity of the inflammatory process. Ratios of cytokines (table 6) also indicate low activity of the inflammatory process at the moment.

Patient No. P31/32. Female, age 59 years. The number of affected joints 1. At the time of the survey the CRP in the blood to 20.88, SG 19,28 mg/l; RF in the blood of 247.2; SG 324,7 mg/l, indicating high activity of the inflammatory process. Ratios of cytokines (table 6) also indicate a high activity of the inflammatory process at the moment.

Sources of information

1. Dolhain R.J.E.M., van der Heiden A.N., ter Haar N.T. et al. Shift toward T lymphocytes with a T helper 1 cytokine-secretion profile in the joints of patient with rheumatoid arthritis // Arthritis Rheum. - 1996. - Vol.39. - P.1961-1969.

2. Morita Y., Yamamura M., Kawashima M. et al. Flow cytometric singl-cell analisis of cytokine production by CD4+ T cell in synovial tissue and periferal blood from patients with rheumatoid arthritis // Arthritis Rheum. - 1998. - Vol.41. - P.1669-1676.

3. Simon A.K., Seipelt E., Sieper J. Divergent T-cell cytokine patterns in iflammatory arthritis // Proc Nati Acad Sci USA. - 1994. - Vol.91. - P.8562-8566 series.

4. Abbas A.K. Muphy C.M., Sher A. Functional diversity of T helper lymphocytes. //Nature. - 1996. - Vol.383. - P.787-793.

5. Miosses P., van den Berg W. Th1/Th2 cytokine balance inarthritis // Arthritis Rheum. - 1997. - Vol.40. - P.2105-2115.

6. Van Roon J.A., van Roy, J.L., A. Duits et al. Proinflammatory cytokine production and cartilage damage due to rheumatoid synovial T-helper-1 activition is inhibited by interleukin-4.// Ann Rheum Dis. - 1995. - Vol.54. - P.836-840.

7. Nasonov EL, Ghukasyan D.A., Nasonova MB Immunopathology of rheumatoid arthritis and osteoporosis: new data. // Osteoporosis and osteopathy. - 2000. No. 2. - P.4-7.

8. Vandesompele j, De Preter K, Pattyn F. et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.// Genome Biology. - 2002. - Vol.3 (7).

1. A method for diagnosing inflammatory process in early rheumatoid arthritis, namely, that of peripheral blood mononuclear cells secrete, of which there are RNA-based quantification of mRNA expression of cytokine genes interleukin-2 (IL-2), interleukin-4 (IL-4) and interleukin-10 (IL-10) by the method of reverse transcription and polymerase chain reaction with the accumulation of reaction products on flourescence directly during the reaction appreciate the balance of cytokines by the formula
[IL']/[ILA]=E IL"^t"/E IL'^t',
where [IL'] is the concentration of mRNA cytokine gene with increased expression;
[IL"] is the concentration of mRNA cytokine gene, characterized by low expression;
E - the efficiency of amplification is a quantity that characterizes the nature of the t matrix for each cycle of amplification;
Ct' and Ct is the threshold cycles for the respective cytokine, and ascertain the presence of inflammatory process in obtaining the following balances:
IL-10/IL-2>1,5;
IL-10/IL-4>and 1.0.

2. A method for diagnosing inflammatory process in early rheumatoid arthritis, namely, that of the cells of the synovial fluid precipitated all cells, of which there are RNA-based quantification of mRNA expression of genes of cytokines interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-17 (IL-17), interleukin-1β (IL-1β), and also on the basis of quantitative determination of interferon-gamma (IFNG) and tumor necrosis factor (TNF) by the method of reverse transcription and polymerase chain reaction with the accumulation of reaction products on flourescence directly during the reaction appreciate the balance of cytokines by the formula
[IL']/[ILA]=E IL"^t"/E IL'^t',
where [IL'] is the concentration of mRNA cytokine gene with increased expression;
[IL"] is the concentration of mRNA cytokine gene, characterized by low expression;
E - the efficiency of amplification is a quantity that characterizes the growth of the matrix on each cycle of amplification;
Ct' and Ct is the threshold cycles for the respective cytokine, and ascertain the presence of inflammatory process in obtaining the trace of the operating balance:
IL - 2/IL - 4>10;
IFNG/IL - 4>300;
TNF/ IL - 4>2000;
IL - 1β/IL - 4>5000;
IL - 6/IL - 4>10;
IL - 8/IL - 4>10000;
IL - 17/IL - 4>5.



 

Same patents:

FIELD: medicine.

SUBSTANCE: offered is a method and a set of primers for determining a haplotype of a DNA-target containing two heterozygous polymorphic sites. The method according to the invention provides a) taking a genome DNA sample and amplifying the area on the 3'-side of which there is a first polymorphic site, and on the 5'-side of which the second polymorphic site is found; b) conducting a PCR of said specific area with using one of two primers allele-specific relatively to the first polymorphic site and a downstream primer which is located so that to include the second polymorphic site in an amplicon; c) hybridising the prepared PCR product with two probes one of which is specific to a mutant type sequence, and another one - to a wild type sequence within the second polymorphism and d) determining a haplotype where the first polymorphism is described by a type of an extended upstream primer, and the second polymorphism - by a type of the probe hybridised with the PCR product.

EFFECT: use of the invention provides relatively simple and high precise diagnostic system.

4 cl, 5 dwg, 5 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to synthetic oligonucleotide primers, complementary to high conservative VP60 gene region of a genome of rabbit viral hemorrhagic disease virus, to a method for identifying rabbit viral hemorrhagic disease virus and to a test system for identifying RNA of rabbit viral hemorrhagic disease virus. The offered invention can be used in veterinary virology. The method for identifying rabbit viral hemorrhagic disease virus involves sample preparation, RNA recovery from the biological material. It is followed with conducting a polymerase chain reaction with using primers 5'-caa cgt get cca gtt ttg gta cg-3', 5'-att ctg tct ggt tgg ggc gtg t-3'. Further, viral RNA is amplified. Then, the reaction is assessed by agarose gel electrophoresis, with a reaction result considered as positive if the PCR product corresponds to the size of 398 base pairs.

EFFECT: invention allows higher sensitivity of the method, as well as reduced time of diagnostic manipulations with organ and blood samples of the infected animals.

3 cl, 4 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, namely to synthetic oligonucleotide primers complementary to a conservative S-segment region of a genome of sheep Nairobi disease virus, to a method for identifying sheep Nairobi disease virus and to a test system for identifying DNA of sheep Nairobi disease virus. The offered invention can be used in veterinary virology. The method for identifying sheep Nairobi disease virus involves RNA recovery from the biological material. It is followed with conducting a polymerase chain reaction with using primers NSD F1 5'TATGCTTCTGCCTTGGTTG-3' NSD R1 5'-ATCCGATTGGC AGTGAAG-3', NSD F2 5'-AGAGCACATTGACTGGGC-3', NSD R2 5'-GCCTTCCAAAGCCAGTAG-3' Thereafter, virus RNA is amplified. Then, the reaction is assessed by agarose gel electrophoresis, with a reaction result considered as positive if the PCR product corresponds to the size of 360 base pairs.

EFFECT: offered invention allows identifying genome RNA of sheep Nairobi disease virus by means of a nidicolous version of RT-PCR with using two synthesized pairs of oligonucleotide primers complementary to conservative gene region of core protein N.

3 cl, 4 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: sequence typing is used to differentiate Yersinia pestis strains. The technique provides recovery of chromosomal DNA of the investigated strain, polymerase chain reaction (PCR) with amplification of rhaS, araC, metB, asp A and thiH gene fragments to be analysed for nucleotide sequences. A genotype of the investigated strain is stated by nucleotides being in the positions 482, 494, 671 reHarhaS, in the position 773 of the gene rhaS, in the positions 988 and 989 of the gene metB, in the positions 1087-1089 of the gene aspA and in the position 552 of the gene thiH. A sequence type (ST) of the Y. pestis strain is specified by rhaS, araC, metB, aspA and thiH gene alleles, while the subspecies differentiation is enabled by comparing to the sequence types of the major and minor subspecies. The sequence type (ST) is specified for each subspecies.

EFFECT: use of the method provides fast, reliable and effective differentiation of Yersinia pestis subspecies.

2 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention refers to a synthetic oligonucleotide kit for identifying DNA of human monocytic ehrlichiosis (HME) agent - pathogenic representatives of Ehrlichia genus by a polymerase chain reaction. The offered invention can be used for the diagnostic purposes for detecting monocytic ehrlichiosis Ehrlichia spp. by the real-time polymerase chain reaction. Said kit includes primers as follows: 5'- GGG GAA AGA TTT ATC GCT ATT AG -3', 5'- CGG CAT AGC TGG ATC AGG CT -3' and a sample: (BHQl)-5'- CCC ACT GCT GCC (FdT)CC CGT AGG AGT CTG G - 3'P, where BHQ1 means a dark fluorescence killer attached to 5'-terminal nucleotide, while FdT is a fluorescent dye FAM attached to nucleotide T.

EFFECT: invention allows reliable identification of Ehrlichia representatives in the biological material.

1 ex

FIELD: medicine.

SUBSTANCE: live attenuated Pasteurella multocida bacterium is modified by introduing a mutation into the gene Orf-15. The mutation represents insertion and/or deletion in the gene Orf-15. As a result of modification, the bacterium is not able to express the functional protein Orf-15 that leads to its attenuation. Also, disclosed is the use of such bacterium for preparing a related vaccine for human or animal protection against Pasteurella multocida bacteria infection or pathogenic effects of the infection.

EFFECT: increased efficacy of applying the composition.

16 cl, 1 dwg, 3 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: genetic predisposition is determined by analysing polymorphism of genes ACTN3 (R577X), CNB (5I/5D), AMPD1 (C34T) through a multiplex polymerase chain reaction. For this purpose, a set of primers is used, wherein for the gene ACTN3 (R577X) the primes used are ACTGCTGCCCTTTCTGTTGCCT-3' and S'-CTGCAGGTGGCACTGACCATA3', for the gene CNB (5I/5D) the primers used are 5'-GGAGTTTAAAAGCCAGCCAGTCATACTA-3' and 5'-TGGAAGATCACACCATTTGATTAGCAGT-3', for the geneAMPD1 (C34T) the primers used are S'-GCAATCTACATGTGTCTACCCCAAAG-3' and 5'-CACTGCTGAAAAATAGCCATGTTTCTG-3'. Further, 3 pairs of primers with average melting point of 58-59°C are merged in a single test tube at standard conditions. The reaction product is analysed through analysis of polymorphism of the length of restriction fragments.

EFFECT: invention provides a simple, cheap, specific method which enables to identify functionally significant polymorphous gene loci, grow the number of analysed genes for further introduction into any standard clinical laboratory.

2 cl, 2 dwg, 3 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: present invention can be used during DNA diagnostics in medicine, veterinary, sanitary and epidemiological analysis and in criminalistics to identify criminals. The method of amplifying specific nucleic acid fragments employs thermally stable DNA polymerase having chain-displacement activity. Also, the present method employs direct and reverse primers in form of head-to-tail arranged tandem recurrent sequences of the main primer. The present invention enables to amplify specific DNA or RNA fragments with increasing multiplication factor for each cycle.

EFFECT: shorter reaction time and high sensitivity of the reaction.

4 cl, 8 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: to determine the type of a potato plant, DNA is extracted from fresh plant material and analysed through PCR analysis. PCR analysis is carried out using a set comprising a biological marker, a reaction mixture consisting of 160 mcM of each dinucleotide triphosphate dNTP, 1.6 mM MgCl2, 0.3 mcM of each primer from the set, 0.3 units of a Taq-polymerase enzyme, 1x buffer for the Taq-polymerase and standard DNA of known potato types in amount of 50 ng. Polymorphous DNA markers are obtained and viewed using gel electrophoresis. In order to carry out titration of alleles extracted in the examined group, allele spectra of the analysed and control groups of samples are compared. The biological marker, which contains polymorphous DNA, is a specific nucleotide sequence obtained using a diagnostic set of three pairs of primers.

EFFECT: marker characterises the type of widespread and essential potato types, and samples of related species Solarium, most frequency used in crossing when creating new potato types.

5 cl, 3 dwg, 4 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: claimed is method of determining hypermethylated CpG is the region of suppressor genes of tumour growth in human DNA, which includes obtaining of samples of highly purified DNA; fragmentation of isolated DNA with restriction endonuclease which does not have a recognition site in amplified region, in particular TaqI; hydrolysis of fragmented DNA with methyl-dependent site-specific endonucleases GlaL and Blsl, carried out in two separate samples; further amplification of hydrolysis products with application of a pair of primers, flanking gene area which contains CpG islands and making a conclusion on the basis of changes in comparison with control, determined in at least one of the two test samples during their electrophoretic separation.

EFFECT: invention method ensures possibility of efficient diagnostics of cancer diseases.

2 cl, 3 dwg, 3 ex

FIELD: medicine, psychiatry.

SUBSTANCE: one should isolate DNA out of lymphocytes of peripheral venous blood, then due to the method of polymerase chain reaction of DNA synthesis one should amplify the fragments of hSERT locus of serotonin carrier gene and at detecting genotype 12/10 one should predict the risk for the development of hallucino-delirious forms of psychoses of cerebro-atherosclerotic genesis.

EFFECT: more objective prediction of disease development.

3 ex

FIELD: biotechnology, medicine, proteins.

SUBSTANCE: invention describes new polypeptide in isolated form relating to subfamily of superfamily human immunoglobulins (Ig-Sf). This polypeptide shows at least 70% of homology level with amino acid sequence of murine molecules CRAM-1 or CRAM-2 regulated by the confluence of adhesive (figures 3, 6 are represented in the claim). Also, invention relates to antibodies showing specificity with respect to the polypeptide. Antibodies and soluble polypeptide can be used for treatment of inflammation and tumors. Invention describes polynucleotide or oligonucleotide encoding the full-size polypeptide or its moiety and represents primer, probe, anti-sense RNA and shows the nucleotide sequence that is identical conceptually with human CRAM-1. Invention provides preparing new adhesive proteins from superfamily Ig-Sf that are regulated at the transcription level in endothelium by effect of tumors. Invention can be used for treatment of different diseases, in particular, inflammatory responses.

EFFECT: valuable medicinal properties of polypeptide.

19 cl, 33 dwg, 1 ex

FIELD: biotechnology, genetics.

SUBSTANCE: invention relates to methods used for detecting low frequency mutations occurrence in gene encoding cytochrome b. Method involves isolation of DNA from known fungi for constructing oligonucleotide probe or primer. Then polymerase chain reaction (PCR) is carried out for assay of binding the nucleotide probe with amplicon generated by this reaction, or the presence of amplicon is detected that is generated as result of PCR using indicated primers. Invention provides rapid and precise detection of mutations conferring resistance of fungus against fungicide.

EFFECT: improved diagnostic methods for detecting mutations.

24 cl, 18 dwg, 14 tbl, 18 ex

FIELD: genetic engineering, molecular biology, pharmacy.

SUBSTANCE: invention relates to methods of genome screening and can be used for identification of pharmacological agent in vegetable extract. Method is realized by treatment of cells with a vegetable extract, isolation of protein or RNA from these cells, identification of isolated protein or RNA wherein their concentration differs from that in untreated cells and detection of compound(s) in indicated vegetable extracts. Then cells are treated with the found compound(s), protein or RNA are isolated from cells treated with this compound(s) and compound(s) are identified that cause the stimulation or inhibition of expression of protein or RNA wherein their concentration differs from that in untreated cells. Invention provides carrying out the characterization of biological properties of vegetable extract and to detect the individual compound(s) that elicit unknown or disclosed biological property of this extract.

EFFECT: improved identifying method.

6 cl

Thrombopoietin // 2245365

FIELD: medicine, molecular biology, polypeptides.

SUBSTANCE: invention describes homogenous polypeptide ligand mpI representing polypeptide fragment of the formula: X-hTPO-Y wherein hTPO has amino acid sequence of human fragments TPO (hML); X means a amino-terminal amino-group or amino acid(s) residue(s); Y means carboxy-terminal carboxy-group or amino acid(s) residue(s), or chimeric polypeptide, or polypeptide fragment comprising N-terminal residues of amino acid sequence hML. Also, invention relates to nucleic acid encoding polypeptide and expressing vector comprising nucleic acid. Invention describes methods for preparing the polypeptide using cell-host transformed with vector, and antibodies raised against to polypeptide. Invention describes methods and agents using active agents of this invention. The polypeptide ligand mpI effects on replication, differentiation or maturation of blood cells being especially on megacaryocytes and progenitor megacaryocyte cells that allows using polypeptides for treatment of thrombocytopenia.

EFFECT: valuable medicinal properties of polypeptide.

21 cl, 92 dwg, 14 tbl, 24 ex

FIELD: medicine, biology, molecular biology.

SUBSTANCE: invention proposes a new method for differential diagnosis of representatives of family Chlamydiaceae. Method involves isolation of DNA of pathogen, amplification using real-time polymerase chain reaction (PCR) and primers CM1 and CM2 exhibiting specificity to 5'-terminal fragment of gene omp1 followed by post-amplification analysis of curves of PCR-products melting in the presence of nonspecific fluorescent dye SYBR Green I for separation of Chlamydia species and electrophoretic separation of PCR-products. Identification of species is carried out on the basis of differences in PCR-products melting point wherein melting point curves of all fragments of omp1 are characterized by the presence of two peaks reflecting two-stage dissociation of DNA chains in sites with different A/T-saturation degree. Proposed method provides carrying out the differentiation of all species of Chlamydia that are pathogenic for humans. Except for, method provides carrying out the differentiation of Chlamydiaceae causing diseases in animals and also method is simple, rapid and can be used for direct diagnosis of clinical material samples. Invention can be used in medicine, veterinary science and virology for differential diagnosis of representatives of family Chlamydia.

EFFECT: improved method for diagnosis.

2 ex

FIELD: medicine, biology, molecular biology.

SUBSTANCE: invention proposes a new method for differential diagnosis of representatives of family Chlamydiaceae. Method involves isolation of DNA from pathogen, amplification of target using primers CM1 and CM2 showing specificity to 5'-terminal fragment of gene omp1 and electrophoretic separation of polymerase chain reaction (PCR) products. Electrophoresis is carried out in agarose gel with addition of sequence-specific DNA-ligand - bis-benzimide-PEG. The species belonging of PCR-products is determined by comparison of the migration rate of PCR-products in gel with electrophoretic mobility of control. Proposed method provides carrying out the differentiation of all species of family Chlamydiaceae and method is simple and rapid and can be used for direct diagnosis of clinical material samples also. Invention can be used in medicine and virology for differential diagnosis of representatives of family Chlamydiaceae.

EFFECT: improved method for diagnosis.

2 dwg, 2 ex

FIELD: molecular biology, medicine, biochemistry.

SUBSTANCE: invention proposes a method for assay of mononucleotide changes in the known sequences of nucleic acids. Method involves hybridization with PCR-amplified matrix DNA and the following ligation a tandem on its consisting of tetranucleotide that comprises the diagnosed change and two oligonucleotides of the size 8-10 nucleotides being one of that is immobilized on surface of a solid-phase carrier through a 5'-phosphate linker, and the second oligonucleotide is labeled by 3'-end with biotin label. Then tetranucleotide is hybridized with the matrix DNA chain between two oligonucleotides directly that can be ligated with immobilized oligonucleotide through the 5'-end and with non-immobilized oligonucleotide through the 3'-end. The ligation product is detected by its transformation to enzyme label through the complex with high-affinity enzymatic catalyst followed by development of enzyme label in the presence of chromogenic, luminogenic or fluorogenic substrates. Applying a method provides preparing the simple and highly selective agent used for detection of known changes in gene structure.

EFFECT: improved assay method.

8 cl, 3 dwg, 30 ex

FIELD: genetic engineering, biotechnology, biochemistry, agriculture, food industry, medicine.

SUBSTANCE: invention relates to the transformation of plant with nucleic acid encoding enzyme Δ6-desaturase in C. elegans that results to preparing a plant with enhanced content of gamma-linolenic acid and resistance to cold. Desaturase extracted from the plant can be used for preparing a drug used for treatment of disorder in body associated with deficiency of gamma-linolenic acid in it.

EFFECT: valuable biological properties of genes and desaturases.

36 cl, 9 dwg, 2 ex

FIELD: medicine, hematology.

SUBSTANCE: one should isolate DNA out of peripheral blood lymphocytes due to polymerase chain reaction (PCR) technique of DNA synthesis, carry out genotyping for polymorphism of promoter area of TNF-alpha and TNF-beta gene. While detecting genotype LT*22 being characterized by availability of gene TNF-beta mutation in homozygous state, detect persons predisposed to the development of chronic lympholeukosis, and at certain combinations of genotypes TNF*22/LT*22 or TNF*12/LT*11 in patients with chronic lympholeukosis on should predict an aggressive flow of this disease.

EFFECT: higher accuracy of prediction.

3 ex, 3 tbl

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