FIELD OF THE INVENTION

The present invention relates to the medical industry and molecular biology and concerns the preparation of stabilized lyophilized mixtures of reagents. The present invention can be used in veterinary medicine, medicine, the food industry, and other fields where molecular genetic methods of research are applicable.

A mixture containing Taq-polymerase modified with specific antibodies, deoxynucleoside triphosphates, buffer components, and a complex of stabilizers and preservatives is proposed.

The mixture of the present invention is characterized by high sensitivity and specificity, which makes it possible to use it for the diagnosis of infectious and hereditary diseases, nucleotide sequence analysis, gene sequencing, genotyping, DNA marking, and other molecular diagnostic methods.

BACKGROUND OF THE INVENTION

The main problem of using reagent kits is preserving the activity of enzymes and other components for a long period of storage and the dependence of the storage time on the temperature regime. Reaction mixtures are usually stored in a glycerol solution at a temperature about - 20 °C. Defrosting and heating of reagents during their long-term storage and transportation leads to a decrease and loss of their activity. Most of the existing technologies for increasing the shelf life of biologically active substances, in particular enzymes, are based on lyophilic drying of the samples.

At the same time, during the usual method of lyophilization, the components lose their properties in the further preparation of the working solution— that is, standard lyophilization does not ensure the stability of the components. To save the properties of reaction PCR mixtures for a long time, it is necessary to carry out additional technological operations with the mixture, which is the subject of the present invention.

Dry reaction mixtures obtained using existing lyophilization technologies have a high hygroscopicity and, as a result of moisture absorption during long-term storage at a positive temperature, may lose the reaction activity.

Ions Mg2+ present in the mixture may lead to the beginning of nonspecific reactions. The presence of primers in ready dry PCR mixtures limits the use of such mixtures for a wide range of studies.

One of the key advantages of the lyophilization technology used is a dry amplification mixture of PCR reagents that contains Taq-polymerase modified with specific antibodies, deoxynucleoside triphosphates, buffer components, and a complex of stabilizers and preservatives.

The method for carrying out the PCR assay involves dissolving the dry amplification mixture of the polymerase chain reaction reagents with a buffer solution containing magnesium ions, followed by the addition of specific primers and analyzed DNA.

The composition of stabilizers and preservatives in the proposed amplification mixture allows simultaneous lyophilization under milder conditions, which reduces the hygroscopicity of the dry PCR mixture. The finished mixture can be stored for a long time (for a year or more) in a wide range of temperatures (-20 °C to +30 °C), without losing reaction activity. The presence of Mg2+ ions in the solvent buffer— but not in the mixture itself— makes it possible to control the onset of the reaction and to avoid the appearance of nonspecific products and false positive results in PCR analysis using the proposed amplification mixture.

The proposed amplification mixture works with any primer systems for genomic, plasmid, mitochondria, recombinant (genetically modified) DNA and can be used for PCR analysis of the DNA of any multicellular and unicellular organisms, viruses, food products, or their components as well as for the quantitative determination of the content DNA in the test samples.

From the level of technology, a method of lyophilization for the purpose of preserving biologically active substances is known. Lyophilization is the process of removing water from the frozen material under low atmospheric pressure. In an aqueous solution, the enzymes are surrounded by a hydrated membrane formed by water molecules bound to the surface of the protein by hydrogen bonds. Such an aqueous membrane stabilizes the enzyme and ensures its activity. Stabilization of the spatial structure occurs due to the fact that the water molecules fix the mutual arrangement of individual protein groups, preventing their direct interactions and the formation of intermolecular bonds in the macromolecule. During the drying process, the three-dimensional structure of the protein can change, which leads to a violation of its activity.

A reagent US 5763157 A is known from the previous level of technology, comprising at least one biological agent and a glass-forming filler in a concentration sufficient to facilitate the formation of a glassy porous composition in which the glass-forming filler comprises a mixture of a synthetic polymer and a high molecular weight carbohydrate; wherein the reagent is soluble in water and has a Tg of at least 10 °C.

From the previous level of technology, reagent US 4451569 A is a stable enzyme composition, in lyophilized form, containing glutathione peroxidase and at least one stabilizer selected from the group consisting of pentoses, hexoses, pentatomic sugar alcohols, hexahydric sugar alcohols, and disaccharide compounds. The composition contains pentoses: - D-, L- arabinose, D-L-xylose, D-, L-lyxose, D-ribose, D-, 1 ^* xylulose, and D-, L- ligase; hexoses: D-, L-galactose, D-glucose, D-mannose, D-sorbose, and D-fructose; pentatomic sugar alcohols: D-arabitol and xylitol; hexahydric sugar alcohols: Galactite, D-sorbitol, and D-mannitol; disaccharides: xylobiose, maltose, isomaltose, cellobiose, gentibiose, lactose, and sucrose.

A reagent US5565318 is known from the previous level of technology. An open emulsion of a glass-forming filler material, a biological reagent, and water, which provides a viscosity sufficient to allow the controlled distribution of droplets on an inert surface and vacuum dried to give the biological reagent a kind of hemisphere (all that is spherical form but less than sphere), which has certain advantages. The resulting hemisphere is stable at room temperature, soluble in water, and has a glass-transition temperature sufficient for storage at a temperature ranging from 22 °C and above (at room temperature). This hemisphere can be completely dissolved in < 100 μΙ of an aqueous solution for 2 minutes. The moisture content in it is < 10%, the diameter of the hemisphere is 2-6 mm (optimally about 2.5 mm). The hemisphere can contain both a single biological reagent, which itself is unstable at room temperature, and several biological reagents that can or cannot react with each other in an aqueous solution at room temperature. The biological reagent can be nucleotides, oligonucleotides, nucleic acids, DNA/RNA-modifying enzymes, restriction enzymes, proteins, and enzymes (verbatim translation— that is, all proteins and nucleic acids can be a bioreagent). The filler is a carbohydrate (mono-, di-, or trisaccharide), a polyhydroxyl compound, a collagen, a sugar polymer containing saccharide groups (sugar residues) bound by ether bridges with bifunctional groups, other than carbohydrates, a mixture of polysaccharides (mixture of sugars), or protein. Optimally, it is a polysaccharide (optimal ficoll), containing saccharide groups, linked by ether bridges with bifunctional groups, other than carbohydrates. Of all these compounds, ficoll (high molecular weight hydrophilic sucrose polymer), sucrose, or dextran (a glucose polymer with a different molecular weight) is optimal. Also, the filler may be a protein (collagen, bovine serum albumin, or gelatin), but of proteins it is optimal that it is bovine serum albumin or gelatin.

Thus, a kit is created containing at least one hemisphere described above, packed in a test tube, a sealed package into which a vial and a desiccant are packed and (optionally) a dispenser for spraying the hemispheres.

The method of manufacturing hemispheres includes dissolving a buffered biological agent in water, mixing the filler with an aqueous solution of a buffered biological agent to form a mixture in which the filler concentration is sufficient to form a glassy porous structure of a hemispherical shape, spraying the mixture in the form of fairly similar droplets, collection of droplets in an inert medium to form hemispheres, drying the drops under conditions suitable for maintaining hemispheres to form the final product— hemispheres with a biological reagent that are stable at room temperature, soluble in water, and have a glass-transition temperature above room temperature. It is optimal that the mixture was in the form of an emulsion or semiemulsion.

It is optimal that the emulsion contains 52%-62% of the dispersed phase (solids). For a semiemulsion, 10%-50% of the dispersed phase (solids) is optimal. It is optimal that the first drying takes place at a pressure of 300 Torr and 10 °C (possibly 4-20 °C) for about an hour. It is optimal that the drying will be continued until the hemisphere with the biological reagent contains < 10% of moisture. The inert medium may be an inert solid surface, a cryogenic liquid, or a cryogenically cooled inert solid surface. For semiemulsions, freeze-drying is possible. An inert solid surface can have concave indentations.

The shape of the drops formed on the inert surface can be controlled by changing the percentage of the dispersed phase in the emulsion, the composition, or the shape of the surface, on which the drying takes place, by the vacuum level during the drying process. The intensity of drying can affect the shape and activity of hemispheres with a biological reagent. A hemisphere with a biological reagent is resistant to degradation and mechanical damage and has a porous structure that facilitates its dissolution. Individual hemispheres can be sprayed if the dispenser is adapted accordingly.

Thus, it is possible to stably store those biological reagents that are unstable in an aqueous solution at room temperature. Moreover, it is possible to stably store several biological reagents that react with each other in aqueous solution at room temperature.

A reagent US8835146/WO2010144683 is known from the previous level of technology. The dry storage of molecular biology reagents used in the analysis of nucleic acids, especially Taq-polymerase, at room temperature on microfluidic maps (cartridges) is rather difficult. Reagents are usually printed in liquid form in a matrix containing lyoprotectants on the microfluidic channels of the map and then dried without freezing or lyophilization, which can disrupt the structure of the map. We hypothesized that an enzyme adapted for activity in a high-temperature environment is likely to have a high decomposition temperature, which was noticed at the maximum reaction rate for many Taq-polymerases around 75 °C. For better storage, the enzyme must be in a vitrified state together with a glassy consistency reagent (filler) with a sufficiently high glass-transition temperature. We also noticed that other fillers, such as surfactants, need the high-folded Taq-polymerase structure to be stabilized during dry storage, especially on microfluidic devices, since the reagent material is optimally printed on a low-activity polyethylene terephthalate (PET) surface, and it undergoes interfacial adsorption and denaturation during drying and rehydration. Passivation can also be used. After the reagents are printed, the microfluidic devices are further laminated or ultrasonically welded to form a map. The use of automatic printers for dispensing reagents and the subsequent vitrification of the gel make lyophilization difficult or impossible due to the technical difficulties of freezing and creating a vacuum when processing a sealed plastic package containing reagents.

After a period of drying at controlled room temperature, the method is based on the use of a gel dryer in hermetically sealed moisture-proof packages to complete the vitrification of the enzyme within the microfluidic map. The inlet and the hole for waste allow the dehydration process to continue slowly (Dh <0.2). The enzyme passes through a partially hydrated state for several weeks while dehydration is taking place. We believe that an extended progressive "time-dehydration" curve is necessary for the stabilization of the enzyme in the natural state during the gradual replacement of a polysaccharide or another polyol with water as a hydrogen bond donor. We found that the activity of Taq-polymerase rises sharply during this drying compared to the initial activity in the wet state. We explain this by restoring the hidden activity of conformers in a frozen sample as a result of refolding in a partially hydrated state, which more closely resembles a cytosol in a state of osmolarity. In this process, the material changes from gel to composite gel-like glass, and its glass- transition temperature becomes higher than room temperature due to the high glass-transition temperature of melezitose, the optimal polysaccharide-lyoprotectant. It has been found that co-lyoprotectants, such as high molecular weight polyethylene glycol (PEG), cellulose gums, bovine serum albumin, gelatin, enhancers, amino acids, and optionally selectable fluorosurfactants also help in this process and contribute to an increase in the composite glass-transition temperature. It is believed that the co-lyoprotectants are selectively associated with the protein membrane, without destroying the vitreous consistency. The method is optimized for Taq-polymerase but can be used for stabilization for other biological reagents (dNTP, DNA, and RNA polymerase, reverse transcriptase, proteinase K, RNase H, primers, buffers, template nucleic acid, and probes). Melesitosis proved itself in this respect better than trehalose.

The microfluidic map (cartridge) containing the necessary molecular biological reagents consists of a plastic corpus and microfluidic contents and contains at least one chamber or channel. There is one flow through the inlet and another through the opening for the waste. There may be several chambers or channels connected in parallel for different gel spots (with reagents). It is possible to form a layer with different microfluidic maps. Thus, it is possible to place in the card/maps at least all reagents for a particular analysis of nucleic acids.

The method of printing and stabilizing Taq-polymerase in the form of a gellike glass above the freezing point of water without lyophilization for storage in a chamber or channel of a microfluidic device includes:

a) Combining of Taq-polymerase with a stabilizing aqueous solution (in which the presence of a PCR enhancer such as betaine, N- formylmorpholine, δ-valerolactam (2-piperidone), ε-caprolactam, 1 ,2- cyclopentanediol, polyvinylpyrrolidones 0 or -40 or its mixture, also the presence of inulin, cellulose, derivatized cellulose, polyvinylpyrrolidone, lysine, arginine, or Mayyar inhibitor is optimal. In an aqueous solution, the following is combined: 1 %-10% (weight/volume) disaccharide (optimally trehalose), trisaccharide, or trisaccharide hydrate (optimally melezitose or raffinose); optionally 0.001 %-0.1 % (weight/volume) of high molecular weight PEG (optimally PEG-90M, molecular weight PEG 0.1-5 mDa, linear or branched polyoxyethylene glycol) or polyhydroxyl compound; optional 0.001 %-0.3% fluorosurfactant (optimally nonionic fluoroalkylsulfuractant, of which is fluorosurfactant FC-4430, with a short alkyl side chain); optionally 0.001 %-1 % of a nonprotein compound (amino acid) with an amino or amide functional group; 0.1 %— 10% (0.1-10 mg/ml? in the formula and text in different ways) of the carrier protein (optimally bovine serum albumin or fish gelatin); compatible buffer, thereby forming a composite printable solution of Taq-polymerase; possible passivation of the chamber or channel to the next stage.)

b) Depositing on the plastic surface of said microfluidic drop map a printable Taq-polymerase solution containing an amount of Taq- polymerase sufficient to polymerize the nucleic acid

c) A short (about 10 minutes) drying of the drop at controlled room temperature (about 20 °C) to form a gel spot (with a reagent) on the surface

d) Closing and sealing of the gel spot (with the reagent) on the surface (the conclusion of the microfluidic chamber or channel in the plastic casing) by lamination, gluing, ultrasonic welding, or by any other suitable means e) The conclusion of a microfluidic map with one or more gel spots (with reagent) in a gas-tight bag in a dry atmosphere with a desiccant, which also vitrifies the spot or spots of the gel during storage

Thus, lyophilization or storage in a frozen form is not required. In a microfluidic map or maps, several reagents can be stored for the analysis of nucleic acids in the form of several gel spots. Reagents can be stored for at least six months. The method can find application primarily in the production of microfluidic devices and kits for diagnostic assays of nucleic acids.

From the previous level of technology, the reagent US5861251 / US6153412 is known. The instant reagent for PCR (the "PCR-reagent") is prepared by freeze-drying a standard aqueous reaction mixture consisting of a reaction buffer, MgCI2, dNTP, and DNA-polymerase. This PCR- reagent is stable at room temperature. Depending on the purpose of use, the lyophilized reagent can be used in practice along with other combinations of components (dH20, primers and template DNA, dH20 and template DNA, only dH20). For example, for the diagnosis of HIV infection, hepatitis B, and tuberculosis, the PCR-reagent can be mixed with the genomic DNA/RNA of the pathogen and complementary primers; for DNA sequencing it can be mixed with universal or individual primers.

It is well known that materials such as gelatin, bovine serum albumin, ammonium sulfate, or polydocanol (Thesit) stabilize DNA polymerase, dNTP, and nonionic surfactants (NP40, Tween 20), improving the reactivity of the PCR mixture (Saiki, R. K. et al., Science, 239:487-491(1988)). However, ammonium sulfate can significantly influence the process of PCR in the case of its use in a PCR-reagent. Therefore, it is optimal that the PCR-reagent contains a stabilizer such as gelatin, bovine serum albumin, polydocanol (Thesit, polyoxyethylene-9-lauryl ether), PEG-8000 (polyethylene glycol-8000), or polyol (ficoll, sucrose, glycerol, glucose, mannitol, galactitol, glucitol, and sorbitol). It is perfectly optimal that it will be a polyol since it is also a sedimentation agent.

The method of preparing the instant reagent for PCR (PCR-reagent) involves adding a stabilizer to the PCR mixture containing the reaction buffer, MgCI2, dNTP, and DNA polymerase followed by lyophilization of the whole mixture.

This PCR reagent may also contain a sedimentation agent or a water- soluble colorant in the presence or absence of a stabilizer. As mentioned above, the polyol fulfills the dual function of the stabilizer and sedimentation agent, and it is most optimal to use glucitol, glucose, ficoll, or sucrose. As a water-soluble colorant, bromophenol blue, xylenocyanol, bromocresol, or cresol red can be used. The water-soluble colorant facilitates the determination of the complete mixing of the PCR-reagent with the test sample and allows for not adding the loading buffer, usually necessary for the PCR product analysis that can prevent possible contamination.

In general, this PCR-reagent is prepared by lyophilizing an aqueous reaction mixture containing a reaction buffer, MgCI2, dNTP, DNA polymerase, stabilizing and sedimentation agent (optimally polyol, from polyols-glucitol), a water-soluble colorant (bromophenol blue, xylenocyanol, bromocresol or cresol red), and a primer.

This PCR reagent has the following advantages: 1 ) it facilitates multistage manipulations with PCR, in which each component of the reaction mixture is added to each of the test samples; 2) it increases the thermostability of the reaction mixture; 3) it prevents possible contamination since it allows for not adding a boot buffer; 4) it improves the reliability of PCR-diagnostics of diseases and experiments related to PCR since it reduces the likelihood of erroneous pipetting.

This PCR reagent can be designed as a kit for analyzing nucleotide sequences or diagnosing diseases as well as for amplifying a specific region of the genome. In addition, dNTPs can also be replaced with ddNTPs.

A reagent WO2005103277 is known from the previous level of technology. The purpose of the invention is a multipurpose dry amplification mixture of reagents for PCR, in which its reactivity will be preserved under long-term storage conditions at temperatures above 0 °C and in which it will be allowed to perform both conventional and specific PCR. These goals can be achieved as follows: the dry amplification mixture of reagents for PCR contains DNA polymerase, dNTP, buffers, water-soluble colorant for electrophoresis, stabilizers (composition of D-glucose, disaccharide (inulin, sucrose, trehalose, maltose), and polysaccharide (D-mannitol, dextrans, ficoll, polyvinylpyrrolidone). The PCR-analysis technique involves dissolving the dry mixture of amplification reagents in a buffer solution containing Mg ions, followed by the addition of specific primers and an analyzed DNA sample. The carbohydrate mixture in the proposed amplification mixture not only serves as a stabilizer in the lyophilization process but also allows the mixture to be active under mild conditions by reducing the hygroscopicity of the dry PCR mixture. The prepared mixture can be stored for a long time (a year or more) in a wide temperature range (-20 °C to +30 °C) without loss of activity. The presence of Mg ²⁺ ions in a buffer solvent and not in the mixture itself makes it possible to control the moment of the onset of the reaction to avoid the appearance of nonspecific products and false positive results. The proposed amplification mixture is compatible with any primer system for genomic DNA, plasmid DNA, mitochondrial DNA, recombinant DNA and can be used for PCR analysis of the DNA of any multicellular or unicellular organism, food, or their components as well as for quantifying DNA in the sample. The proposed mixture contains DNA polymerase (0.1 U/μΙ), a mixture of dNTPs (500 mM each dNTP), 80 mM Tris-HCI (pH = 8.0), 0.1 % Triton X-100, 24 mM (NH ₄ ) ₂ S0 ₄ , 0.5 mM EDTA, D-glucose (0.16%), disaccharide, for example inulin (1.6%), polysaccharide, for example D-mannitol (8%), and water- soluble colorant for electrophoresis, for example xylenethanol (0.04%). The proposed PCR mixture is prepared by mixing the aqueous solutions of the constituent components and then placed in amplification tubes (10 μΙ each) and lyophilized at -20 °C for 4 hours. The lyophilized mixture is used for PCR analysis: the mixture is dissolved in 10 μΙ buffer solution (buffer- solvent) containing 5 mM MgCI ₂₎ 10 mM Tris-HCI (pH = 8.0), 0.1 % of Triton X-100, 5% glycerol, then oligonucleotide primers (5 μΙ) and solution of the DNA sample (5 μΙ) are added. The following reagents are used to prepare the proposed dry amplification PCR mixture: OligoTaq as a DNA polymerase; Pfu; Vent (Promega, USA) dNTP, Tris-HCI, Triton X-100, (NH) ₂ S0 ₄ , EDTA, carbohydrates, dye for electrophoresis (Sigma, USA). The water-soluble dye for electrophoresis is selected from bromophenol blue, xylenocyanol, bromocresol, or cresol purple. The present invention can also be used with similar reagents from other companies.

A reagent WO2008155529 / WO2008155524 / WO2010001162 is known from the previous level of technology. A mixture for chemical or biochemical reaction is invented, which is prepared by lyophilization and consists of a set of reagents necessary for carrying out a particular reaction (possibly a fluorescent or bioluminescent reagent) forming a vitreous consistency of the reagent (a nonreducing polysaccharide but not a polyol, optimal raffinose pentahydrate, 2.5%-10% (w/v)) and threonine (2-10 mM) and fish gelatin (0.0001 %-0.02 wt%). Also, the mixture may contain a stabilizer of the vitreous-forming reagent-PEG, polyvinylpyrrolidone or polysaccharide. The kit of these reagents can be a set of reagents that are specifically adapted for PCR, PCR with a fluorescent reagent, or RT-PCR (reverse transcriptase). In addition, it can include a polymerase (RNA/DNA- dependent polymerase of the Tsec type) capable of interacting with a primer when attached to a template nucleic acid during the PCR process. It can include all buffers, salts (magnesium or manganese), primers, and nucleotides suitable for PCR to amplify the DNA sequence. A fluorescently tagged reagent (fluorescent colorant, DNA-binding agent, or fluorescently tagged oligonucleotide) contains a tagged oligonucleotide to monitor realtime PCR. This tagged oligonucleotide is a probe that carries two tags- one as an energy donor and the other that can act as an acceptor of this energy. The labeled oligonucleotide acts as a molecular signal. It is also possible to use the fluorescently tagged reagent as a pair of labeled probes, one of which carries a fluorescent energy donor tag, and the other, a fluorescent energy acceptor tag from this donor. The probes are hybridized in the immediate vicinity of the PCR product helix. The mixture may also contain a DNA-binding agent capable of exchanging energy with the abovementioned fluorescently tagged oligonucleotide. The mixture may include one or more reagents capable of controlling the initiation of PCR (for example, anti-Taq antibody). The mixture may contain an RNase inhibitor (in an amount at least the same as that of the polymerase). The mixture may contain an antioxidant (optimally threonine), a Maillard reaction inhibitor (optimally threonine), and a blocking compound. The mixture is prepared by mixing its components in water and then by lyophilization. Also, the kit with the mixture must contain a buffer for rehydration (it can contain salts necessary for conducting PCR if the mixture does not contain them). To carry out the reaction, the lyophilized mixture is hydrated and used to conduct a chemical or biochemical reaction under appropriate conditions with monitoring of the fluorescent signal. As a stabilizer of a lyophilic-dried mixture containing fluorescent reagents, L- threonine and an RNase inhibitor can be used.

A reagent WO2007137291 is known from the previous level of technology. The invention is a method for DNA polymerase and dNTP processing for use in amplification as well as a formula for a reaction mixture comprising a DNA polymerase and/or dNTP, a buffer solution, and at least one stabilizing agent in combination with 50%-100% dehydration of the reaction mixture (heating at 55 °C, lyophilization, vacuum, spray drying, fluidized bed drying, or drum drying). The reaction mixture is dehydrated at a temperature of 0-100 °C. The invention also provides a kit for amplifying a nucleic acid comprising an initially dehydrated mixture comprising a thermophilic DNA polymerase, dNTP, a buffer solution, at least one stabilizing agent (at least one nonreducing polysaccharide, optimally sucrose in a concentration of 1 %-20% and at least one protein, optimally bovine serum albumin in a concentration of 0.5-3 mg/ml), MgCI2, a pair of oligonucleotide primers, a water-soluble colorant, an oligonucleotide probe, or a fluorescent colorant, and a template DNA. MgCI2 may not be included in the original mixture and may be added during rehydration of the mixture. The kit can be delivered in a microtube for PCR, stripped microtubes, or in the wells of the PCR plate where the mixture is added prior to dehydration. Using different pairs of primers and different probes, it is possible to quantify different nucleic acids. The kit is capable of successful amplification of the nucleic acid after storage at room temperature for up to two years, and before use it must be rehydrated. SUMMARY OF THE INVENTION

A technical problem solved in the present invention is providing a method for producing a stabilized dry mixture intended for the amplification of nucleic acids, namely a mixture containing at least a thermostable DNA polymerase.

The technical result is achieved by lyophilizing an aqueous solution to amplify the nucleic acid using at least two stabilizers.

The claimed method makes it possible to obtain a homogeneous, vitreous, and at the same time readily soluble mixture spaced in individual test tubes. The mixtures obtained by the claimed method have a long shelf life at room temperature and are highly stable throughout the shelf life. The present invention also simplifies the analysis.

The aim of the present invention is to provide a dry mixture producing method for amplifying a nucleic acid comprising the following steps:

- Preparation of an aqueous solution for the amplification of a nucleic acid containing a thermostable Taq-polymerase modified with specific antibodies, deoxynucleoside triphosphates, buffer components, and stabilizers

- Lyophilization of the abovementioned aqueous solution

The other aim of the present invention is to provide a method in which an aqueous solution of a reaction mixture for amplifying a nucleic acid comprises a thermostable DNA-polymerase.

The other aim of the present invention is to provide a method in which the thermostable DNA polymerase is a Taq-polymerase from Thermus aquaticus.

The other aim of the present invention is to provide a method in which a thermostable DNA polymerase is a hot-start DNA polymerase.

The other aim of the present invention is to provide a method in which an aqueous solution contains primers and an oligonucleotide probe for detection, containing a fluorescent label.

The other aim of the present invention is to provide a method in which the aqueous solution contains buffer components and nucleotide triphosphates.

The other aim of the present invention is to provide a method in which compounds selected from the group consisting of carbohydrates, polyalcohols, polymers, proteins, amino acids, or various combinations of them are used as stabilizers.

The other aim of the present invention is to provide a method in which trehalose in a concentration of 3%-8% is used as the stabilizer.

The other aim of the present invention is to provide a process in which raffinose is used as a stabilizer in a concentration of 2%-6%.

The other aim of the present invention is to provide a method in which hydroxyectoin is used as a stabilizer in a concentration of 1 %- %.

The other aim of the present invention is to provide a process in which polyvinylpyrrolidone is used as the thickener in a concentration of 0.4%- 0.8%.

The other aim of the present invention is to provide a method in which xanthine gum is used as the thickener in a concentration of 0.1 %— 1 %, and/or locust bean gum, in a concentration of 0.05%-0.5%.

The other aim of the present invention is to provide a method in which lyophilization of the abovementioned aqueous solution is carried out in vials, strips, or plates.

The other aim of the present invention is to provide a process in which, in order to increase the stability, a mixture of reagents is subject to vacuum drying at a temperature of 15-25 °C and a pressure of 0.6-6.6 Pa.

The other aim of the present invention is to provide a process in which the mixture is not preliminary frozen prior to vacuum drying.

The other aim of the present invention is to provide a process in which the reaction mixtures are dried in separate individual tubes, and the volume of the mixture is from 20-50 μΙ.

The other aim of the present invention is to provide a method in which the mixture is diluted with a dilution solution containing a buffer pH = 0.1-9.0 and reaction enhancers: betaine in a concentration of 0.2-2 mol, sodium azide in a concentration of 0.05%-0.1 %, and prokline 300 in concentrations of 0.01 %-0.1 %.

The other aim of the present invention is to provide a method in which the dilution solution contains Mg ^{2 +} in a concentration of 4-6 mmol.

The proposed stabilized mixture of reagents is prepared by mixing the aqueous solution components. The finished mixture is poured in amplification tubes and dried at a temperature of 15-25 °C and a pressure of 0.6-6.6 Pa for 4-5 hours. The dried mixture is used for PCR analysis. For this, the mixture is dissolved with 10 μΙ of buffer solution (buffer- solvent).

THE INVENTION IS ILLUSTRATED BY THE FOLLOWING EXAMPLES.

The given examples are not intended to limit the invention but are offered solely as an illustration.

Comparison of the results of PCR amplification of Mycobacterium tuberculosis (Mtu) DNA in the presence of internal control showed a slight difference in the values of threshold cycles, within 0.5-1.0 cycles, with the use of Mastermixes stored at -20 ^° C compared with Mastermixes stored at room temperature for one year (Tab. 1 , Figs. 1-2).

Table 1 Evaluation of the amplification efficiency of Mycobacterium tuberculosis (Mtu) DNA using lyophilized PCR Mastermixes within one year after storage 1 ) at room temperature 2) at a temperature of -20 C

Mastermix Threshold cycle values

storage 1 2 3 4 5 6 7 8 average conditions

At room temp. 27.6 26.1 26.5 27.5 26.1 27.5 27.3 26.9 26.9 (Fig. 1 ) 26.9 27.4 26.6 27.7 26.3 26.6 27.4 26.3 26.9

26.8 26.1 26.6 26.3 26.1 27.5 27.3 26.9 26.7

During the storage of lyophilized PCR mastermixes for 2 years, there is a slight loss of activity of the mastermixes (less than one threshold cycle), which does not affect the sensitivity of detection of single copies of the DNA matrix (Tables 2-5; Figs. 3-6).

Table 2

The results of the amplification of Mycobacterium tuberculosis (Mtu) DNA using lyophilized PCR Mastermixes, depending on the storage time at room temperature after manufacture 1 ) original freshly prepared PCR mastermixes

Table 3

The results of the amplification of Mycobacterium tuberculosis (Mtu) DNA using lyophilized PCR Mastermixes, depending on the storage time at room temperature after manufacture 1 ) PCR mastermixes after 6 months of storage

Table 4

The results of the amplification of Mycobacterium tuberculosis (Mtu) DNA using lyophilized PCR Mastermixes, depending on the storage time at room temperature after manufacture 1 ) PCR mastermixes after 1 year of storage

Table 5 The results of the amplification of Mycobacterium tuberculosis (Mtu) DNA using lyophilized PCR Mastermixes, depending on the storage time at room temperature after manufacture 1 ) PCR mastermixes after 2 years of storage

During the storage of lyophilized PCR mastermixes for 1 month at (4, 25, 35, and 45) °C, a slight decrease in PCR mastermix activity occurs, in particular at a temperature of 45 °C (Tables 6-9; Figs. 7-10).

Table 6 Results of the amplification of Chlamydia trachomatis (Ctr) DNA using lyophilized PCR Mastermixes, depending on the different storage temperatures after manufacturing during the month 1 ) PCR mastermixes during the month storage at 4 ^° C

Table 7

Results of the amplification of Chlamydia trachomatis (Ctr) DNA using lyophilized PCR Mastermixes, depending on the different storage temperatures after manufacturing during the month 1 ) PCR mastermixes during the month storage at 25 C

Table 8

Results of the amplification of Chlamydia trachomatis (Ctr) DNA using lyophilized PCR Mastermixes, depending on the different storage temperatures after manufacturing during the month 1 ) PCR mastermixes during the month storage at 35 ^° C

Table 9

Results of the amplification of Chlamydia trachomatis (Ctr) DNA using lyophilized PCR Mastermixes, depending on the different storage temperatures after manufacturing during the month 1 ) PCR mastermixes during the month storage at 45 ^° C

Thus, the ready lyophilized PCR mastermixes proposed in the composition patent retain their original activity for a long shelf life of at least two years at room temperature.

BRIEF DESCRIPTION OF THE FIGURES

Fig.1 Amplification in real time of Mycobacterium tuberculosis (Mtu) DNA using lyophilized PCR Mastermixes one year after storage at room temperature. A comparison of the amplification of 8 DNA samples in 8 parallel duplicates was made. The red color indicates the exponents of amplification of the internal control of the sample, and the blue color indicates the specific DNA under study.

Fig. 2 Amplification in real time of Mycobacterium tuberculosis (Mtu) DNA using lyophilized PCR Mastermixes one year after storage at -20 °C. A comparison was made of the amplification of 8 DNA samples in 8 parallel duplicates. The red color indicates the exponents of amplification of the internal control of the sample, and the blue color indicates the specific DNA under study.

Fig. 3 Amplification in real time of Mycobacterium tuberculosis (Mtu) DNA using freshly prepared (without storage) lyophilized PCR Mastermix at room temperature. A series of DNA dilutions of 10 ¹ , 10 ² , and 10 ³ copies of the template were amplified in the reaction in three parallel duplicates.

The red color indicates the exponents of internal control of the sample amplification, according to which the inhibition of the reaction and its reproducibility were evaluated with an increase in the storage time of PCR mastermixes.

Amplification in real time of Mycobacterium tuberculosis (Mtu) DNA using lyophilized PCR Mastermixes stored for 6 months after manufacturing at room temperature. A series of DNA dilutions of 10 ¹ , 10 ² , and 10 ³ copies of the template were amplified in a reaction in three parallel duplicates.

Fig. 5 Amplification in real time of Mycobacterium tuberculosis (Mtu) DNA using lyophilized PCR Mastermixes stored at room temperature for 1 year. A series of DNA dilutions of 10 ¹ , 10 ² , and 10 ³ copies of the template were amplified in the reaction in three parallel duplicates.

The red color indicates the exponents of internal control of the sample amplification, according to which the inhibition of the reaction and its reproducibility were evaluated with an increase in the storage time of PCR mastermixes.

Fig. 6 Amplification in a real time of Mycobacterium tuberculosis (Mtu) DNA using lyophilized PCR Mastermixes stored at room temperature for 2 years. A series of DNA dilutions of 10 ¹ , 10 ² , and 10 ³ copies of the template were amplified in the reaction in three parallel duplicates.

The red color indicates the exponents of internal control of the sample amplification, according to which the inhibition of the reaction and its reproducibility were evaluated with an increase in the storage time of PCR mastermixes.

Fig. 7 Real-time amplification of Chlamydia trachomatis (Ctr) DNA using lyophilized PCR Mastermixes stored for 1 month at 4 °C. A series of DNA dilutions of 10 ¹ , 10 ² , and 10 ³ copies of the template were amplified in the reaction in three parallel duplicates.

The red color indicates the exponents of internal control of the sample amplification, according to which the inhibition of the reaction and its reproducibility were evaluated with an increase in the storage temperature of PCR mastermixes.

Fig. 8 Real-time amplification of Chlamydia trachomatis DNA (Ctr) using lyophilized PCR Mastermixes stored for 1 month at 25 °C. A series of DNA dilutions of 10 ¹ , 10 ² , and 10 ³ copies of the template were amplified in the reaction in three parallel duplicates.

The red color indicates the exponents of internal control of the sample amplification, according to which the inhibition of the reaction and its reproducibility were evaluated with an increase in the storage temperature of PCR mastermixes.

Fig. 8 Real-time amplification of Chlamydia trachomatis DNA (Ctr) using lyophilized PCR Mastermixes stored for 1 month at 35 °C. A series of DNA dilutions of 10\ 10 ² , and 10 ³ copies of the template were amplified in the reaction in three parallel duplicates.

The red color indicates the exponents of internal control of the sample amplification, according to which the inhibition of the reaction and its reproducibility were evaluated with an increase in the storage temperature of PCR mastermixes.

Fig. 10 Real-time amplification of Chlamydia trachomatis DNA (Ctr) using lyophilized PCR Mastermixes stored for 1 month at 45 °C. A series of DNA dilutions of 10 ¹ , 10 ² , and 10 ³ copies of the template were amplified in the reaction in three parallel duplicates.

The red color indicates the exponents of internal control of the sample amplification, according to which the inhibition of the reaction and its reproducibility were evaluated with an increase in the storage temperature of PCR mastermixes.