Combined pcr-eia in test tube with the extended surface

FIELD: medicine.

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.

Table
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 tubePCR tube with a developed surface
552139696
128968896
0,520608719
0,29606974
0,18216743
0,05-5293
0,01-3740
Horizotally control (1 μl)9861147
Background657689

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.



 

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9 cl, 3 dwg, 7 tbl, 7 ex

FIELD: agriculture.

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 39TG in SEQ ID NO:1, a marker of single-nucleotide polymorphism 29GA in SEQ ID NO:2, a marker of single-nucleotide polymorphism 193CT 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

FIELD: medicine.

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

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

FIELD: mechanics.

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.

2 dwg

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