RU2789387C1 - Composition for removing dna- and/or rna-containing biological material (variants) - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: group of inventions relates to liquid compositions made to remove DNA- and RNA-containing biological material from the surface of objects of inorganic and organic origin. Disclosed are compositions (variants) for removing DNA- and/or RNA-containing biological material, in the form of a mixture of the following two solutions: decontamination solution 1 (DCR-1) and decontamination solution 2 (DCR-2) at a ratio from 1:1 to 35:1, wherein DCR-1 may contain sodium dodecyl sulfate (SDS), copper (II) sulfate pentahydrate (CuSO₄×5H₂O), iron (II) sulfate heptahydrate (FeSO₄×7H₂O), tris (hydroxymethyl) aminomethane (Tris, (HOCH₂)₃CNH₂), glycerin, and deionised water; and DCR-2 contains hydrogen peroxide (H₂O₂), sodium benzoate (C₆H₅COONa), and deionised water.

EFFECT: removal of nucleic acids and destruction of cellular membranes on the surface of objects of inorganic and organic origin, including bone material, in laboratory conditions.

20 cl, 25 dwg, 11 tbl, 5 ex

Description

The field of technology to which the invention belongs

The present invention relates to liquid compositions designed to remove DNA, RNA, DNA- and RNA-containing biological material on the surface of objects of inorganic and organic origin, including samples of bone material in laboratory conditions.

State of the art

When working with biological material in modern laboratories, there is a risk of contamination (DNA/RNA contamination) of instruments, materials, objects under study, samples, etc., which can lead to unreliable results, especially in routine molecular genetic studies. The sources of contamination can be different: amplicons present in large quantities in PCR laboratories, blood, saliva, contact traces of people interacting with the test sample and/or work surface in the laboratory.

I., Hyslop Р.А., Jackson J.H. et al. Oxidant-induced DNA damage of target cells // The Journal of clinical investigation. - 1988. - V. 82. - №. 3. - P. 1040-1050; Gutteridge J. M.C. Ferrous-salt-promoted damage to deoxyribose and benzoate. The increased effectiveness of hydroxyl-radical scavengers in the presence of EDTA // Biochemical journal. - 1987. - V. 243. - №. 3. - P. 709-714).The use of decontamination tools is aimed at removing contaminants of biological origin and preventing their secondary transfer. Most of these agents are represented in the form of solutions that are easy to use and do not require special storage conditions. The most commonly used deactivating agents are metal ions, mainly iron salts, and hydrogen peroxide. Various studies have shown the use of Sodium dodecyl sulfate (SDS), sodium benzoate, and hydrogen peroxide as a deactivating agent (

I., Hyslop R.A., Jackson JH et al. Oxidant-induced DNA damage of target cells // The Journal of clinical investigation. - 1988. - V. 82. - no. 3. - P. 1040-1050; Gutteridge JMC Ferrous-salt-promoted damage to deoxyribose and benzoate. The increased effectiveness of hydroxyl-radical scavengers in the presence of EDTA // Biochemical journal. - 1987. - V. 243. - no. 3. - P. 709-714).

In laboratories where nucleic acids are studied, DNARid solutions (Biomedical Innovations, Russia), DNA-ExitusPlus (AppliChem, USA), as well as the commercial Belizna solution, an aqueous solution of 70 mM or 0.5% sodium hypochlorite, are widely used for decontamination. (NaOCl). The action of these agents is aimed at the degradation of nucleic acid molecules. However, in the environment, genetic material, as a pollutant, does not always exist in the form of free molecules. Most often, nucleic acids are part of biological material, such as cells, secretions of a physiological nature, sweat fatty substance of hands, etc.

There are patents for inventions that relate to reagents for the deactivation of nucleic acids, methods for their preparation and use. In the patent of US8765652, the use of hydrogen peroxide and metal ions, such as, for example, copper, cobalt, iron, iron as a decapivating agent, is disclosed. Compounds that were not effective in specific experiments include only 15% peroxide; peroxide + potassium, sodium or iron ions; 5 mM bromine or chlorhydantoin and KMnO ₄ . In this patent, the concentration of components: SDS from 0.005 to 1%, copper (II) sulfate (CuSO ₄ ) from 0.1 to 5 mm, hydrogen peroxide (H ₂ O ₂ ) from 0.5 to 30%, while this concentration H ₂ O ₂ can have a negative effect on the decontamination of objects of biological origin. This patent does not disclose the effect of the composition of chemical compositions on cells, but only indicates the effect on nucleic acid molecules relative to solutions of commercial whiteness.

In the application US9371556, relating to reagents for the deactivation of nucleic acids and methods for their preparation and use, the following components are indicated: the ratio of CuSO ₄ /H ₂ O ₂ (2 mm/3%), the ratio of CuSO ₄ /H ₂ O ₂ (1 mm/ 3%). However, this work does not indicate the effect of compositions on DNA-containing biological material and the effect on cells.

According to the results of research by a number of authors, a modified SDS-based DNA extraction method was disclosed to obtain high-quality DNA. The concentration of SDS 20%, Tris buffer 100 mm.

It is also known that hydrogen peroxide releases hydroxyl free radicals (•OH) when oxidizing Cu (I) to Cu (II) through a Fenton-type reaction causing DNA damage, explaining the mutual use of copper sulfate and hydrogen peroxide in the ratio CuSO ₄ /H ₂ O ₂ (5 mM / not specified) (Natarajan VP, Zhang X., Morono Y. et al. A modified SDS-based DNA extraction method for high quality environmental DNA from seafloor environments // Frontiers in microbiology. - 2016. - V. 7. - P. 986; Chattopadhyay D., Somaiah A., Raghunathan D. et al. Dichotomous effect of caffeine, curcumin, and naringenin on genomic DNA of normal and diabetic subjects // Scientifica. - 2014. - V. 2014) .

Thus, a universal decontamination method for getting rid of nucleic acids and destroying cell membranes on the surface of objects of inorganic and organic origin, including bone material, has not yet been proposed under laboratory conditions.

Disclosure of invention

The present invention proposes:

(1) The composition for the removal of DNA, RNA, DNA and RNA-containing biological material in the form of a two-component decontamination solution (DKR) No. 1, which includes component 1 (decontamination solution-1, DKR-1): 0.5-2% of predecyl sulfate sodium (Sodium dodecyl sulfate, SDS), 1-5 mM copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 1-15 mM Tris (hydroxymethyl) aminomethane (Tris, (HOCH ₂ ) ₃ CNH ₂ ), 0 ,1-1 about. % glycerol, deionized water-rest and component 2 (decontamination solution-2, DKR-2): 0.1-3% hydrogen peroxide (H ₂ O ₂ ) and 0.01-0.1% sodium benzoate (C ₆ H ₅ COONa), deionized water - the rest.

(2) Composition (1), where DCR No. 1 is a mixture of DCR-1: 100 mg Sodium dodecyl sulfate (SDS), 12.5 mg copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 3.63 mg Tris (hydroxymethyl) aminomethane (Tris, (HOCH ₂ ) ₃ CNH ₂ ), 50 µl glycerol, made up to 10 ml with sterile deionized water and DKR-2: 330 µl 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg sodium benzoate (C ₆ H ₅ COONa) adjusted to 10 ml with sterile deionized water.

(3) Composition (1), where the ratio of DCR No. 1 to the biosample is 1:1.

(4) Composition (1), where the ratio of DKR No. 1 to the biosample represented by the cell culture is 25.8:1.

(5) Composition (1) wherein the ratio of DCR-1 to DCR-2 is 8.5:1 when the biosample is a DNA or RNA preparation.

(6) Composition (1) wherein the ratio of DCR-1 to DCR-2 is 9:1 when the biosample is a DNA or RNA preparation.

(7) Composition (1) wherein the ratio of DCR-1 to DCR-2 is 3.7:1 when the biosample is an amplicon.

(8) Composition (1) wherein the ratio of DCR-1 to DCR-2 is 6:1 when the biosample is an amplicon.

(9) Composition (1), where the ratio of DCR-1 to DCR-2 is 4:1, when the biosample is a DNA and/or RNA-containing preparation/smear/trace.

(10) Composition (1) wherein the ratio of DCR-1 to DCR-2 is 32.25:1 when the biosample is a cell culture suspension.

(11) Composition (1), where the DNA or RNA preparation, as well as the DNA and/or RNA containing preparation/smear/trace is a biosample.

(12) Composition (1), wherein the amplicon is a biosample.

(13) Composition (1), wherein the cells and/or cell culture is a biosample.

(14) Composition (1), where the bone material (bone, bone fragment, bone chips, bone powder) is a decontaminable object.

(15) Composition for removing DNA, RNA, DNA- and RNA-containing biological material in the form of a two-component decontamination solution (DKR) No. 2, including component 1 (decontamination solution-1, DKR-1): 0.5-2% Dodecyl sulfate sodium (Sodium dodecyl sulfate, SDS), 1-5 mM copper(II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 1-15 mM Tris (hydroxymethyl) aminomethane (Tris, (HOCH ₂ ) ₃ CNH ₂ ), 1 -3 mM iron(II) heptahydrate (FeSO ₄ × 7H ₂ O) and deionized water - the rest and component 2 (decontamination solution-2, DKR-2): 0.1-3% hydrogen peroxide (H ₂ O ₂ ) and 0.01-0.1% sodium benzoate (C ₆ H ₅ COONa), deionized water - the rest.

(16) Composition (15), where DKR No. 2 is a mixture of DKR-1: 100 mg Sodium dodecyl sulfate (SDS), 12.5 mg copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 3.63 mg Tris (hydroxymethyl) aminomethane (Tris, (HOCH ₂ ) ₃ CNH ₂ ), 5.6 mg iron(II) heptahydrate (FeSO ₄ × 7H ₂ O) adjusted to 10 ml with sterile deionized water and DKR-2 : 330 µl of 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg sodium benzoate (C ₆ H ₅ COONa) made up to 10 ml with sterile deionized water.

(17) Composition (15), where the ratio of DCR No. 2 to the biosample is 1:1.

(18) Composition (15) wherein the ratio of DCR-1 to DCR-2 is 8.5:1 when the biosample is a DNA or RNA preparation.

(19) Composition (15) wherein the ratio of DCR-1 to DCR-2 is 9:1 when the biosample is a DNA or RNA preparation.

(20) Composition (15) wherein the ratio of DCR-1 to DCR-2 is 3.7:1 when the biosample is an amplicon.

(21) Composition (15) wherein the ratio of DCR-1 to DCR-2 is 6:1 when the biosample is an amplicon.

(22) Composition (15), where the ratio of DCR-1 to DCR-2 is 4:1, when the biosample is represented by a DNA and/or RNA containing preparation/smear/trace.

(23) Composition (15), where the DNA or RNA preparation, as well as the DNA and/or RNA containing preparation/smear/trace is a biosample.

(24) Composition (15) wherein the amplicon is a biosample.

(25) Composition (15), where the bone material (bone, bone fragment, bone chips, bone powder) is a decontaminable object.

(26) Composition for removing DNA, RNA, DNA- and RNA-containing biological material in the form of a two-component decontamination solution (DKR) No. 3, including component 1 (decontamination solution-1, DKR-1): 0.5-2% Dodecyl sulfate sodium (Sodium dodecyl sulfate, SDS), 1-5 mM copper(II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 1-15 mM Tris (hydroxymethyl) aminomethane (Tris, (HOCH ₂ ) ₃ CNH ₂ ), 1 -3 mM iron(II) heptahydrate (FeSO ₄ × 7H ₂ O), 0.1-1 vol. % glycerin and deionized water - the rest and component 2 (decontamination solution-2, DKR-2): 0.1-3% hydrogen peroxide (H ₂ O ₂ ) and 0.01-0.1% sodium benzoate (C ₆ H ₅ COONa), deionized water - the rest.

(27) Composition (26), where DKR No. 3 is a mixture of DKR-1: 100 mg Sodium dodecyl sulfate (SDS), 12.5 mg copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 3.63 mg Tris (hydroxymethyl) aminomethane (Tris, (HOCH ₂ ) ₃ CNH ₂ ), 5.6 mg iron(II) heptahydrate (FeSO ₄ × 7H ₂ O), 50 μl glycerol, made up to 10 ml with sterile deionized water and DKR-2: 330 μl of 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg sodium benzoate (C ₆ H ₅ COONa) made up to 10 ml with sterile deionized water.

(28) Composition (26), where the ratio of DCR No. 3 to the biosample is 1:1.

(29) Composition (26) wherein the ratio of DCR-1 to DCR-2 is 8.5:1 when the biosample is a DNA or RNA preparation.

(30) Composition (26) wherein the ratio of DCR-1 to DCR-2 is 9:1 when the biosample is a DNA or RNA preparation.

(31) Composition (26) wherein the ratio of DCR-1 to DCR-2 is 3.7:1 when the biosample is an amplicon.

(32) Composition (26) wherein the ratio of DCR-1 to DCR-2 is 6:1 when the biosample is an amplicon.

(33) Composition (26) wherein the ratio of DCR-1 to DCR-2 is 4:1 when the biosample is a DNA and/or RNA containing preparation/smear/trace.

(34) Composition (26), where the DNA or RNA preparation, as well as the DNA and/or RNA containing preparation/smear/trace is a biosample.

(35) Composition (26) wherein the amplicon is a biosample.

(36) Composition (26), where the bone material (bone, bone fragment, bone chips, bone powder) is a decontaminable object.

(37) Composition for removing DNA, RNA, DNA- and RNA-containing biological material in the form of a two-component decontamination solution (DKR) No. 4, including component 1 (decontamination solution-1, DKR-1): 0.5-2% Dodecyl sulfate sodium (Sodium dodecyl sulfate, SDS), 1-5 mM copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), deionized water - the rest and component 2 (decontamination solution-2, DKR-2): 0.1- 3% hydrogen peroxide (H ₂ O ₂ ) and 0.01-0.1% sodium benzoate (C ₆ H ₅ COONa), deionized water - the rest.

(38) Composition (37), where DKR No. 4 is a mixture of DKR-1: 100 mg Sodium dodecyl sulfate (SDS), 12.5 mg copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), made up to 10 ml with sterile deionized water and DKR-2: 330 µl of 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg sodium benzoate (C ₆ H ₅ COONa), made up to 10 ml with sterile deionized water.

(39) Composition (37), where the ratio of DKR No. 4 to the biosample is 1:1.

(40) Composition (37), where the ratio of DKR No. 4 to the biosample represented by the cell culture is 25.8:1.

(41) Composition (37) wherein the ratio of DCR-1 to DCR-2 is 8.5:1 when the biosample is a DNA or RNA preparation.

(42) Composition (37) wherein the ratio of DCR-1 to DCR-2 is 9:1 when the biosample is a DNA or RNA preparation.

(43) Composition (37) wherein the ratio of DCR-1 to DCR-2 is 3.7:1 when the biosample is an amplicon.

(44) Composition (37) wherein the ratio of DCR-1 to DCR-2 is 6:1 when the biosample is an amplicon.

(45) Composition (37) wherein the ratio of DCR-1 to DCR-2 is 4:1 when the biosample is a DNA and/or RNA containing preparation/swab/trace.

(46) Composition (37) wherein the ratio of DCR-1 to DCR-2 is 32.25:1 when the biosample is a cell culture suspension.

(47) Composition (37), where the DNA or RNA preparation, as well as the DNA and/or RNA containing preparation/smear/trace is a biosample.

(48) Composition (37) wherein the amplicon is a biosample.

(49) Composition (37), wherein the cells and/or cell culture is a biosample.

(50) Composition (37), where the bone material (bone, bone fragment, bone chips, bone powder) is a decontaminable object.

In one embodiment, the invention includes a composition as described above, where the ratio of the decontamination solution to the sample of biological material is from 1:10 to 30:1, including all ratios within the specified range, for example, 1:9, 1:8, 1: 7 etc. Or, for example, 25.8:1.

In one embodiment, the invention includes a composition as defined above, where the ratio of component 1 to component 2 is from 1:1 to 35:1, including all ratios within the specified range, for example, 2:1, 3:1, 4:1 etc. Or, for example, 32.25:1.

I. The objects of the present invention are compositions of solutions for decontamination treatment of various surfaces of inorganic origin, for example, laboratory equipment and tools, as well as organic origin, for example, bone material, which makes it possible to get rid of exogenous DNA, RNA, DNA- and RNA-containing biological material (blood, saliva, sweat fat, etc.).

The sign "%" as used in the present invention means the percentage by weight of the specified component in the specified solution.

0.5-2% Sodium dodecyl sulfate (SDS) is one of the permanent components of liquid compositions and is the most powerful detergent, leading to the destruction of cellular structures and the release of nucleic acids.

Also, the constant component of the compositions is 1-5 mm pentagratite of copper (II) sulfate (CUSO ₄ × 5H ₂ O). It has been established that copper ions cause an inactivating effect and damage to DNA and RNA. On the other hand, the redox properties of copper can also cause cell damage, making copper an effective decontamination agent. In addition, the use of copper as a deactivating ion, instead of iron ions, increases the resistance and shelf life of solutions (De Bendictis P., Beato MS, Capua I. Inactivation of Avian Influenza Viruses by Chemical Agens and Physical Conditions: A Reviews/ zzoones Health. - 2007. - V. 54. - No. 2. - P. 51-68; Steuer P., Tejeda C., Martinez O. et al. Effectivence of Copper Ins Against Mycobacterium Subsp. Paratuberculosis And Bactiriat naturally raw cow's milk // Journal of applied microbiology - 2021. - V. 131. - No. contaminated 1. - P. 146-154).

The addition of 1-3 mM iron (II) heptahydrate (FeSO ₄ × 7H ₂ O) to the solution accelerates the chemical reaction when interacting with hydrogen peroxide (H ₂ O ₂ + Fe ²⁺ \u003d HO • + HO ^- + Fe ²⁺ - Fenton reaction ) and leads to an increase in the rate of chemical degradation of nucleic acid molecules under the action of emerging reactive oxygen species (ROS).

To create and maintain an alkaline pH value, as well as to destabilize nucleic acids, 1-15 mM Tris (hydroxymethyl) aminomethane (Tris, (HOCH ₂ ) ₃ CNH ₂ ) is added to the solution.

0.1-1 vol. % glycerol is used in solution to prevent precipitation of copper(II) hydroxide. To do this, add 50 μl of glycerol and shake the contents. The precipitate dissolves and the solution acquires a dark blue color due to the formation of copper glycerate.

Hydrogen peroxide (H ₂ O ₂ ) 0.1-3%, which is part of the second component of the decontamination solution, is a powerful deactivating agent for DNA and RNA, and also contributes to the destruction of cell membranes (Schraufstatter I., Hyslop R.A., Jackson JH et al., Oxidant-induced DNA damage of target cells, The Journal of Clinical Investigation, 1988, V. 82, No. 3, P. 1040-1050.

Sodium benzoate (C ₆ H ₅ COONa), which is a preservative, allows you to increase the shelf life of a hydrogen peroxide solution. In the second component of the decontamination solution, its concentration is 0.01-0.1%.

The present invention is a combination of solutions of the above components:

1. Liquid composition (1) is a mixture of solutions (DKR-1: 0.5-2% Sodium dodecyl sulfate (SDS), preferably 1%, 1-5 mm copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), preferably 5 mm, 1-15 mm Tris (hydroxymethyl) aminomethane (Tris, (HOCH ₂ ) ₃ CNH ₂ ), preferably 3 mm, 0.1-1 vol.% glycerol, preferably 0.5 vol. % DKR-2: 0.1-3% hydrogen peroxide (H ₂ O ₂ ), preferably 1%, 0.01-0.1% sodium benzoate (C ₆ H ₅ COONa), preferably 0.05%). For example, a mixture of DCR-1: 100 mg sodium dodecyl sulfate, 12.5 mg copper (II) sulfate pentahydrate, 3.63 mg Tris (hydroxymethyl) aminomethane, 50 μl of glycerol, made up to 10 ml with sterile deionized water, and DCR-2: 330 µl of 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg of sodium benzoate (C ₆ H ₅ COONa) made up to 10 ml with sterile deionized water.

2. Liquid composition (2) is a mixture of solutions (DKR-1: 0.5-2% of sodium predecylsulfate (Sodium Dodecyl Sulfate, SDS), preferably 1%, 1-5 mm pentagratite of copper (II) (CUSO ₄ × 5H ₂ O), preferably 5 mM, 1-15 mM Tris (hydroxymethyl) aminomethane (Tris, (HOCH ₂ ) ₃ CNH ₂ ), preferably 3 mM, 1-3 mM iron(II) heptahydrate (FeSO ₄ × 7H ₂ O ), preferably 2 mm DKR-2: 0.1-3% hydrogen peroxide (H ₂ O ₂ ), preferably 1%, 0.01-0.1% sodium benzoate (C ₆ H ₅ COONa), preferably 0, 05%). For example, a mixture of DKR-1: 100 mg sodium dodecyl sulfate, 12.5 mg copper (II) sulfate pentahydrate, 3.63 mg Tris (hydroxymethyl) aminomethane, 5.6 mg iron (II) heptahydrate (FeSO ₄ × 7H ₂ O) , brought to 10 ml with sterile deionized water, and DKR-2: 330 μl of a 30% aqueous solution of hydrogen peroxide (H ₂ O ₂ ) and 5 mg of sodium benzoate (C ₆ H ₅ COONa), brought to 10 ml with sterile deionized water.

3. Liquid composition (3) is a mixture of solutions (DKR-1: 0.5-2% Sodium dodecyl sulfate (SDS), preferably 1%, 1-5 mm copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), preferably 5 mM, 1-15 mM Tris (hydroxymethyl) aminomethane (Tris, (HOCH ₂ ) ₃ CNH ₂ ), preferably 3 mM, 1-3 mM iron(II) heptahydrate (FeSO ₄ × 7H ₂ O ), preferably 2 mM, 0.1-1 vol.% glycerol, preferably 0.5 vol.% DKR-2: 0.1-3% hydrogen peroxide (H ₂ O ₂ ), preferably 1%, 0.01 -0.1% sodium benzoate (C ₆ H ₅ COONa), preferably 0.05%). For example, a mixture of DKR-1: 100 mg sodium dodecyl sulfate, 12.5 mg copper (II) sulfate pentahydrate, 3.63 mg Tris (hydroxymethyl) aminomethane, 5.6 mg iron (II) heptahydrate (FeSO ₄ × 7H ₂ O) , 50 µl of glycerol, made up to 10 ml with sterile deionized water, and DKR-2: 330 µl of 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg of sodium benzoate (C ₆ H ₅ COONa), made up to 10 ml with sterile deionized water.

4. Liquid composition (4) is a mixture of solutions (DKR-1: 0.5-2% Sodium dodecyl sulfate (SDS), preferably 1%, 1-5 mm copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), preferably 5 mm DKR-2: 0.1-3% hydrogen peroxide (H ₂ O ₂ ), preferably 1%, 0.01-0.1% sodium benzoate (C ₆ H ₅ COONa), preferably 0.05%). For example, a mixture of DCR-1: 100 mg sodium dodecyl sulfate, 12.5 mg copper(II) sulfate pentahydrate, adjusted to 10 ml with sterile deionized water, and DCR-2: 330 μl of 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg of sodium benzoate (C ₆ H ₅ Coon), brought to 10 ml with sterile deionized water.

The proposed method of purification from DNA, RNA, DNA- and RNA-containing biological material is carried out by its mechanical and chemical removal from the surface under study. To clean surfaces of inorganic origin, for example, laboratory equipment and instruments, and organic, for example, bone, bone fragment, it is necessary to successively apply components (component 1 (DKR-1), then component 2 (DKR-2)) of decontamination solutions to the treated surface . Before processing, closed containers with the components of the solution should be slightly mixed in the hands. In cases where a bone or a bone fragment is presented as an object of processing, it is first necessary to mechanically get rid of surface deposits and contamination (soft tissue remnants, soil, etc.). The surface to be treated is wiped with a napkin (cotton cloth) impregnated with DKR-1. The exposure is 15 minutes. Next, the napkin is washed in running water, squeezed out, and then impregnated with DKR-2. The surface treated with DKR-1 is wiped with a cloth impregnated with DKR-2. Exposure at least 15 minutes. The ratio of DKR-1 and DKR-2 volumes should be 1:1. Then the treated surface is wiped with a dry sterile cloth. The exposure time of solutions can be changed depending on the object and the degree of its contamination.

Thus, the present invention relates to compositions capable of getting rid of contamination by causing lysis of cell membranes and degradation of nucleic acids.

II. Another object of the present invention is a method for decontamination of foreign DNA and cells in bone chips and bone powder (hereinafter referred to as "sample"):

(i) adding liquid compositions of the present invention as a decontaminating agent during sample preparation after homogenization of bone material and before decalcification and extraction of nucleic acids.

(ii) mixing DKR and a sample of the present invention in a ratio of 25.8:1.

(iii) the ratio of DCR-1 to DCR-2 is 32.25:1.

In one embodiment, the objects of the present invention are liquid compositions for the removal of exogenous DNA and DNA-containing biological material in the composition of the sample with the sample.

The present invention is a combination of the above components in solution:

Liquid composition (4) is a mixture of solutions (DKR-1: 0.5-2% Sodium dodecyl sulfate (SDS), preferably 1%, 1-5 mm copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), preferably 5 mM DKR-2: 0.1-3% hydrogen peroxide (H ₂ O ₂ ), preferably 1%, 0.01-0.1% sodium benzoate (C ₆ H ₅ COONa), preferably 0 .05%). For example, a mixture of DCR-1: 100 mg sodium dodecyl sulfate, 12.5 mg copper(II) sulfate pentahydrate, adjusted to 10 ml with sterile deionized water, and DCR-2: 330 μl of 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg sodium benzoate (C ₆ H ₅ COONa) adjusted to 10 ml with sterile deionized water.

Decontamination treatment of samples is carried out during their sample preparation before decalcification and extraction of nucleic acids. To do this, first obtain a sample in the form of chips or powder (difference in particle size). The sample is added to a sterile 50 ml tube. Based on 1 g of the sample, 800 μl of DKR-2 are added to the tube, and then 25 ml of DKR-1. Mix for 10 seconds on a vortex. Incubate on a horizontal shaker at 200 rpm. within 10 min. Centrifuge at 1500 g for 5 minutes. Drain off the excess. 800 μl of DCR-2 is re-added to the pellet, then 25 ml of DCR-1 is added. Mix for 10 seconds on a vortex until the sediment is completely broken. Incubate on a horizontal shaker at 200 rpm. within 10 min. Centrifuge at 1500 g for 5 minutes. Drain off the excess. Add 20-25 ml of DKR-1 to the supernatant. Mix for 30 seconds on a vortex until the precipitate is completely broken. Centrifuge at 1500 g for 5 minutes. Drain off the excess. Further, decalcification and extraction of nucleic acids are carried out according to standard protocols.

Thus, the present invention relates to liquid compositions that make it possible to get rid of DNA and DNA-containing biological material contamination of surfaces of organic origin.

Additional objects and advantages of the invention are set forth in the following section of the description and, in part, become apparent from the description, or may be learned by practice of the invention. The objects and advantages of the invention are understood and realized with the help of the features and combinations detailed in the appended claims.

It is to be understood that, within the scope of the foregoing, the foregoing general description and the following detailed description are illustrative and explanatory only and do not limit the scope of the invention. Complementing the included and forming part of this description, the Figures illustrate some of the advantages of the invention and together with the description help to explain the principles of the invention.

Brief description of the drawings

On FIG. Figure 1 shows the results of electrophoresis in 0.9% agarose gel of preparations of high molecular weight DNA K562 (Promega, USA) after its treatment with DCR nos. 1-4 (samples 1-7), commercial solutions of 0.1X Belizna (samples 8), DNARId (Biomedical Innovations, Russia) (samples 9), DNA-ExitusPlus (AppliChem, USA) (samples 10), and also without decontamination effects (control, samples 11) in two parallels; L - denotes a molecular weight marker.

On FIG. 2A-D show electropherograms of genotyping of high-molecular-weight DNA K562 (Promega, USA) after its treatment with DCR nos. 1-4 (A), commercial solutions 0.1X Belizna (B), DNARid (Biomedical Innovations, Russia) (C), DNA-ExitusPlus (AppliChem, USA) (D) and without decontamination treatment (control) (E) (test system COrDIS "EXPERT" (GORDIZ, Russia)).

On FIG. Figure 3 shows the results of electrophoresis in 2% agarose gel of PCR products of DNA 9947A after their treatment with DCR nos. 1-4 (samples 1-7), commercial solutions 0.1X Belizna (samples 8), DNARid (Biomedical Innovations, Russia) (samples 9), DNA-ExitusPlus (AppliChem, USA) (samples 10), and also without decontamination effect (control, samples 11) in two parallels; L - denotes a molecular weight marker.

On FIG. 4 Micrographs of Goryaev's chamber with applied HeLa cell suspension (10k) after treatment and incubation in DCR No. 4 in three parallels (A-B).

On FIG. Fig. 5 Micrographs of Goryaev's chamber with applied suspension of HeLa cells (10k) after treatment and incubation in DBPS in three parallels (A-B).

On FIG. Fig. 6 Micrographs of Goryaev's chamber with applied suspension of HeLa cells (10k) after treatment and incubation in deionized water in three parallels (A-B).

On FIG. Figure 7 shows electrophoregrams for testing DCR in samples of bone powder contaminated with HeLa cell culture: the profile of a bone sample contaminated with HeLa cell culture without treatment with DCR No. 4 (A), the profile of a bone sample contaminated with HeLa cell culture after treatment with DCR No. 4 (B), the profile is not of a contaminated bone sample (C), a HeLa cell culture profile (D) and a blank negative control profile (E) (COrDIS Plus and COrDIS EXPERT test systems (GORDIZ, Russia)).

On FIG. Figure 8A shows the results of RT and RT-PCR on the ROX channel of swabs from the nasal cavity and oropharynx of patients with a new coronavirus infection (causative agent SARS-CoV-2) after their treatment with DCR and its effect on the genetic material (RNA) of the causative agent of coronavirus infection (SARS-CoV- 2, gene E) (test system RU No. RZN 2020/9948 (DNA-Technology, Russia)).

On FIG. Figure 8B shows the results of RT and RT-PCR using the FAM channel of swabs from the nasal cavity and oropharynx of patients with a new coronavirus infection (causative agent SARS-CoV-2) after their treatment with DCR and its effect on the genetic material (RNA) of the causative agent of coronavirus infection (test system RU No. RZN 2020/9948 (DNA-Technology, Russia)).

On FIG. Figure 8C shows the results of RT and RT-PCR by the Cy5 channel of swabs from the nasal cavity and oropharynx of patients with a new coronavirus infection (causative agent SARS-CoV-2) after their treatment with DCR and its effect on the genetic material (RNA) of the causative agent of coronavirus infection (SARS-CoV-2). 2, gene N) (test system RU no. RZN 2020/9948 (DNA-Technology, Russia)).

On FIG. Figure 9 shows the results of RT and RT-PCR using the ROX channel (registration of the SARS-CoV-2 cDNA signal) of swabs from the nasal cavity and oropharynx of patients with a new coronavirus infection (pathogen SARS-CoV-2) after their treatment with DCR, as well as with a commercial disinfectant " Forispot" (Alfa, Russia) (test system "RealBest RNA SARS-CoV-2" (Vector Best, Russia)).

Implementation of the invention

Example 1

A comparative study of the decontamination effect on high molecular weight DNA of liquid compositions disclosed in the present invention DKR Nos. 1-4, commercial solutions: Belizna, DNARid (Biomedical Innovations, Russia) and DNA-ExitusPlus (AppliChem, USA) was carried out. The experimental study was carried out in eight repetitions.

1. The composition of the liquid composition of DKR No. 1 (DKR-1: 1% sodium predecyfate (Sodium Dodecyl Sulfate, SDS), 5 mm pentagitrate of copper (II) (CUSO ₄ × 5H ₂ O), 3 mm Trisa (hydroxymethyl) aminomethan Tris, (Nosn ₂ ) ₃ CNH ₂ ), 0.5 vol.% Glycerin; DKR-2: 1% hydrogen peroxide (h ₂ O ₂ ), 0.05% sodium benzoate (C ₆ H ₅ COONA)): mixture) DKR-1 solutions: 100 mg of sodium dodecyl sulfate, 12.5 mg of pentagrath of copper sulfate (II), 3.63 mg of tris (hydroxymethyl) aminomethan, 50 μl glycerol, brought to 10 ml of sterile deionized water, and DKR-2: 330 μl 30% of the aqueous solution of hydrogen peroxide (H ₂ O ₂ ) and 5 mg of sodium benzoate (C ₆ H ₅ COONA), brought to 10 ml with sterile deionized water.

2. Composition of the liquid composition of DKR No. 1 (DKR-1: 1% Sodium dodecyl sulfate, SDS), 5 mM copper(II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 6 mM Tris (hydroxymethyl) aminomethane ( Tris, (HOCH ₂ ) ₃ CNH ₂ , 0.5 vol% glycerol, DKR-2: 1% hydrogen peroxide (H ₂ O ₂ ), 0.05% sodium benzoate (C ₆ H ₅ COONa)): mixture solutions of DCR-1: 100 mg sodium dodecyl sulfate, 12.5 mg copper (II) sulfate pentahydrate, 7.26 mg Tris (hydroxymethyl) aminomethane, 50 μl of glycerol, adjusted to 10 ml with sterile deionized water, and DCR-2: 330 μl 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg sodium benzoate (C ₆ H ₅ COONa) adjusted to 10 ml with sterile deionized water.

3. Composition of the liquid composition DKR No. 1 (DKR-1: 1% Sodium dodecyl sulfate, SDS), 5 mM copper(II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 12 mM Tris (hydroxymethyl) aminomethane ( Tris, (HOCH ₂ ) ₃ CNH ₂ , 0.5 vol% glycerol, DKR-2: 1% hydrogen peroxide (H ₂ O ₂ ), 0.05% sodium benzoate (C ₆ H ₅ COONa)): mixture solutions of DCR-1: 100 mg sodium dodecyl sulfate, 12.5 mg copper (II) sulfate pentahydrate, 14.52 mg Tris (hydroxymethyl) aminomethane, 50 μl of glycerol, made up to 10 ml with sterile deionized water, and DCR-2: 330 μl 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg sodium benzoate (C ₆ H ₅ COONa) adjusted to 10 ml with sterile deionized water.

4. Composition of the liquid composition of DKR No. 2 (DKR-1: 1% Sodium dodecyl sulfate, SDS), 5 mM copper(II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 3 mM Tris (hydroxymethyl) aminomethane ( Tris, (HOCH ₂ ) ₃ CNH ₂ ), 2 mM iron(II) heptahydrate (FeSO ₄ × 7H ₂ O), DKR-2: 1% hydrogen peroxide (H ₂ O ₂ ), 0.05% sodium benzoate (C ₆ H ₅ COONa)): a mixture of DKR-1 solutions: 100 mg sodium dodecyl sulfate, 12.5 mg copper (II) sulfate pentahydrate, 3.63 mg Tris (hydroxymethyl) aminomethane, 5.6 mg iron (II) heptahydrate (FeSO ₄ ×7H ₂ O) adjusted to 10 ml with sterile deionized water, and DKR-2: 330 μl of 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg sodium benzoate (C ₆ H ₅ COONa) adjusted to 10 ml with sterile deionized water.

5. Composition of the liquid composition of DKR No. 3 (DKR-1: 1% Sodium dodecyl sulfate, SDS), 5 mM copper(II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 3 mM Tris (hydroxymethyl) aminomethane ( Tris, (HOCH ₂ ) ₃ CNH ₂ ), 2 mM iron(II) heptahydrate (FeSO ₄ × 7H ₂ O), 0.5 vol% glycerol, DKR-2: 1% hydrogen peroxide (H ₂ O ₂ ), 0.05% sodium benzoate (C ₆ H ₅ COONa)): mixture of DKR-1 solutions: 100 mg sodium dodecyl sulfate, 12.5 mg copper (II) sulfate pentahydrate, 3.63 mg Tris (hydroxymethyl) aminomethane, 5.6 mg iron(II) heptahydrate (FeSO ₄ × 7H ₂ O), 50 µl glycerol, made up to 10 ml with sterile deionized water, and DKR-2: 330 µl 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg benzoate sodium (C ₆ H ₅ COONa) adjusted to 10 ml with sterile deionized water.

Fig. 6. Composition of the liquid formulation DKR No. 4 (DKR-1: 1% Sodium dodecyl sulfate (SDS), 5 mM copper(II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O); DKR-2: 1% hydrogen peroxide (H ₂ O ₂ ), 0.05% sodium benzoate (C ₆ H ₅ COONa)): a mixture of DKR-1 solutions: 100 mg sodium dodecyl sulfate, 12.5 mg copper (II) sulfate pentahydrate, brought to 10 ml with sterile deionized water, and DKR-2: 330 μl of 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg sodium benzoate (C ₆ H ₅ COONa), adjusted to 10 ml with sterile deionized water.

7. Composition of the liquid formulation DKR No. 4 (DKR-1: 2% Sodium dodecyl sulfate (SDS), 5 mM copper(II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O); DKR-2: 1% hydrogen peroxide (H ₂ O ₂ ), 0.05% sodium benzoate (C ₆ H ₅ COONa)): a mixture of DKR-1 solutions: 200 mg sodium dodecyl sulfate, 12.5 mg copper (II) sulfate pentahydrate, brought to 10 ml with sterile deionized water, and DKR-2: 330 μl of 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg sodium benzoate (C ₆ H ₅ COONa), adjusted to 10 ml with sterile deionized water.

8. Commercial solution "Belizna" is an aqueous solution of 70 mM or 0.5% sodium hypochlorite (NaOCl) diluted 1:10 with deionized water to obtain 7 mM or 0.05% sodium hypochlorite (NaOCl).

9. DNARId (Biomedical Innovations, Russia) is a two-component set of solutions and includes a DNA decontamination reagent 1 and a DNA decontamination reagent 2, the composition of which is not disclosed by the manufacturer.

10. The composition of the DNA-ExitusPlus reagent (AppliChem, USA) is not disclosed by the manufacturer.

11. Control sample with DNA and deionized water (diH ₂ O).

To study the stability of high-molecular-weight DNA under the decontamination effect of the studied reagents, we used standard K562 DNA (Promega, USA) at a concentration of 20 ng/μL. DNA concentration was measured with a QuantiFluor-P fluorometer (Green/Blue, Promega Corporation) in triplicate.

Before starting the experiment, K562 DNA was mixed and briefly centrifuged. 20 μl of DNA were added to 0.2 ml PCR tubes for PCR. The test solutions were added to DNA in the following ratio:

1. 20 µl DNA + 20 µl composition (1): 18 µl DCR-1 + 2 µl DCR-2

2. 20 µl DNA + 20 µl composition (2): 18 µl DCR-1 + 2 µl DCR-2

3. 20 µl DNA + 20 µl composition (3): 18 µl DCR-1 + 2 µl DCR-2

4. 20 µl DNA + 20 µl composition (4): 18 µl DCR-1 + 2 µl DCR-2

5. 20 µl DNA + 20 µl composition (5): 18 µl DCR-1 + 2 µl DCR-2

6. 20 µl DNA + 20 µl composition (6): 18 µl DCR-1 + 2 µl DCR-2

7. 20 µl DNA + 20 µl composition (1): 18 µl DCR-1 + 2 µl DCR-2

8. 20 µl DNA + 20 µl commercial 0.1X Whiteness solution

9. 20 µl DNA + 10 µl DNA decontamination reagent 1 and 10 µl DNA decontamination reagent 2 DNARid (Biomedical Innovations, Russia)

10. 20 µl DNA + 20 µl DNA-ExitusPlus (AppliChem, USA)

11. 20 µl DNA + 20 µl diH ₂ O

The test samples were mixed and briefly centrifuged. Then, they were incubated with a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, USA) for 15 min at 25°C. After incubation, they were briefly centrifuged and electrophoresis of the studied samples was carried out in a 0.9% agarose gel. To do this, 6 μl of each DNA preparation was mixed with 1.5 μl of a six-fold loading buffer (5:1, v/v) containing bromophenol blue and xylene cyanol in a 30% glycerol solution. A molecular weight marker of phage λ DNA treated with HindIII restriction enzyme was added to separate wells (marked as "L" in the electrophoresis photo of Fig. 1). Electrophoresis was carried out for 5 min at 120 V, then 15 min at 100 V. The gel was placed in the UV transilluminator of a ChemiDoc XRS+System (Bio-Rad, USA) and photographed. The luminescence intensity of the experimental DNA preparations and the PCR product was assessed relative to the control samples (Kornienko I.V., Kharlamov S.G. Methods for the study of human DNA. DNA isolation and its quantitative assessment in the aspect of forensic examination of material evidence of biological origin: educational and methodological allowance Rostov n/D: SFedU, 2012. - 216 p.).

After decontamination treatment, the concentration of the remaining DNA was measured using a QuantiFluor-P fluorimeter (Green/Blue, Promega Corporation, USA) in 3 replicates. The results are presented in table. 1.

Genotyping of the studied samples was carried out using a set of reagents for multiplex analysis of 19 STR markers (D3S1358, TH01, D12S391, D1S1656, D10S1248, D22S1045, D2S441, D7S820, D13S317, FGA, TROX, D18S51, D16S517, D8S119, D8S119, D8S119, vWA, D21S11, SE33) and a human amelogenin gene fragment (Amelogenin) according to the COrDIS EXPERT test system (GORDIZ, Russia) in accordance with the user manual.

To carry out PCR, a reaction mixture consisting of components included in the kit was pre-prepared: 10 μl of the reaction mixture (containing TAQ polymerase enzyme, fluorescent primers, DNTPS), 5 μl of activator solution (containing MGCL were added to each PCR. ₂ ), 5 µl of DNA and diluted with sterile deionized water to a final volume of 25 µl. To check the purity of the reagents and the experiment, 5 µl of deionized water was added to the negative control tube. DNA amplification was carried out by PCR using a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, USA) in the 9600 emulation mode; the reaction parameters are presented in Table 1. 2.

Amplicon electrophoresis was performed using an ABI PRISM 3130xl genetic analyzer (Applied Biosystems, USA) in accordance with the user manual. Alleles were identified according to the S550 size standard relative to the allelic ladder supplied with the EXPERT COrDIS kit.

Fluorimetry data show that after treatment with DCR nos. 1-4 of high-molecular-weight DNA, the concentration of the latter does not exceed 0.21 ng/μl on average (Me = 0.18, Q ₁ = 0.09, Q ₃ = 0.23). Samples treated with a commercial 0.1-fold (0.1X) Belizna solution did not contain DNA (0 ng/µl). At the same time, the values of DNA concentrations after decontamination treatment with commercial reagents DNARid (Biomedical Innovations, Russia) (Me = 1.98, min = 1.95, max = 1.99) and DNA-ExitusPlus (AppliChem, USA) (Me = 2, 18, min = 2.17, max = 2.21) were close to the control (without decontamination treatment) (Me = 2.32, min = 2.32, max = 2.35).

On FIG. 1 shows the results of electrophoresis of samples in 0.9% agarose gel. The absence of white bands in wells 1-8 and traces of "shmer" of low molecular weight DNA samples treated with DCR No. 1-4 and commercial 0.1-fold (0.1X) solution "Belizna" indicates complete DNA degradation. In the wells of 9 samples treated with DNARid (Biomedical Innovations, Russia), high molecular weight DNA is not visualized, however, a “shmer” is clearly visible, which indicates the presence of low molecular weight DNA in the samples. In the wells of 10 samples treated with DNA-ExitusPlus (AppliChem, USA), high molecular weight DNA is clearly visible, as well as a “shmer” of low molecular weight DNA.

When genotyping high-molecular-weight DNA samples treated with DCR No. 1-4 (Fig. 2A), 0.1-fold (0.1X) solution "Belizna" (Fig. 2B) and DNARid (Biomedical Innovations, Russia) (Fig. 2C ) the amplification product was not obtained, which is reflected in the absence of allelic peaks on electrophoregrams. In samples of high molecular weight DNA after exposure to DNA-ExitusPlus (AppliChem, USA), a PCR product was obtained in the form of a complete DNA profile of K562 (Promega, USA) on electrophoregrams (Fig. 2D), which is confirmed by the results of positive control genotyping (Fig. 2E).

Thus, the data obtained indicate the presence of a stable effect exerted by DKR Nos. 1-4 and 0.1-fold (0.1X) solution of "Belizna" on high-molecular DNA, which manifests itself in the form of its degradation. The use of DNARId commercial reagents (Biomedical Innovations, Russia) did not allow us to obtain stable results regarding the degradation of high-molecular-weight DNA. DNA-ExitusPlus (AppliChem, USA) did not have a significant effect on high molecular weight DNA, which is reflected in the absence of its degradation.

Example 2

Conducted a comparative study of the decontamination effect on the amplicons of liquid compositions disclosed in the present invention DKR No. 1-4, commercial solutions: Belizna, DNARid (Biomedical Innovations, Russia) and DNA-ExitusPlus (AppliChem, USA). The experimental study was carried out in eight repetitions.

1. The composition of the liquid composition DKR No. 1 (DKR-1: 1% Sodium dodecyl sulfate (SDS), 5 mm copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 3 mm Tris (hydroxymethyl) aminomethane ( Tris, (HOCH ₂ ) ₃ CNH ₂ , 0.5 vol% glycerol, DKR-2: 1% hydrogen peroxide (H ₂ O ₂ ), 0.05% sodium benzoate (C ₆ H ₅ COONa)): mixture solutions of DCR-1: 100 mg sodium dodecyl sulfate, 12.5 mg copper (II) sulfate pentahydrate, 3.63 mg Tris (hydroxymethyl) aminomethane, 50 μl of glycerol, made up to 10 ml with sterile deionized water, and DCR-2: 330 μl 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg sodium benzoate (C ₆ H ₅ COONa) adjusted to 10 ml with sterile deionized water.

2. Composition of the liquid composition of DKR No. 1 (DKR-1: 1% Sodium dodecyl sulfate, SDS), 5 mM copper(II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 6 mM Tris (hydroxymethyl) aminomethane ( Tris, (HOCH ₂ ) ₃ CNH ₂ , 0.5 vol% glycerol, DKR-2: 1% hydrogen peroxide (H ₂ O ₂ ), 0.05% sodium benzoate (C ₆ H ₅ COONa)): mixture solutions of DCR-1: 100 mg sodium dodecyl sulfate, 12.5 mg copper (II) sulfate pentahydrate, 7.26 mg Tris (hydroxymethyl) aminomethane, 50 μl of glycerol, adjusted to 10 ml with sterile deionized water, and DCR-2: 330 μl 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg sodium benzoate (C ₆ H ₅ COONa) adjusted to 10 ml with sterile deionized water.

3. Composition of the liquid composition DKR No. 1 (DKR-1: 1% Sodium dodecyl sulfate, SDS), 5 mM copper(II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 12 mM Tris (hydroxymethyl) aminomethane ( Tris, (HOCH ₂ ) ₃ CNH ₂ , 0.5 vol% glycerol, DKR-2: 1% hydrogen peroxide (H ₂ O ₂ ), 0.05% sodium benzoate (C ₆ H ₅ COONa)): mixture solutions of DCR-1: 100 mg sodium dodecyl sulfate, 12.5 mg copper (II) sulfate pentahydrate, 14.52 mg Tris (hydroxymethyl) aminomethane, 50 μl of glycerol, made up to 10 ml with sterile deionized water, and DCR-2: 330 μl 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg sodium benzoate (C ₆ H ₅ COONa) adjusted to 10 ml with sterile deionized water.

4. Composition of the liquid composition of DKR No. 2 (DKR-1: 1% Sodium dodecyl sulfate, SDS), 5 mM copper(II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 3 mM Tris (hydroxymethyl) aminomethane ( Tris, (HOCH ₂ ) ₃ CNH ₂ ), 2 mM iron(II) heptahydrate (FeSO ₄ × 7H ₂ O), DKR-2: 1% hydrogen peroxide (H ₂ O ₂ ), 0.05% sodium benzoate (C ₆ H ₅ COONa)): a mixture of DKR-1 solutions: 100 mg sodium dodecyl sulfate, 12.5 mg copper (II) sulfate pentahydrate, 3.63 mg Tris (hydroxymethyl) aminomethane, 5.6 mg iron (II) heptahydrate (FeSO ₄ ×7H ₂ O) adjusted to 10 ml with sterile deionized water, and DKR-2: 330 μl of 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg sodium benzoate (C ₆ H ₅ COONa) adjusted to 10 ml with sterile deionized water.

5. Composition of the liquid composition of DKR No. 3 (DKR-1: 1% Sodium dodecyl sulfate, SDS), 5 mM copper(II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 3 mM Tris (hydroxymethyl) aminomethane ( Tris, (HOCH ₂ ) ₃ CNH ₂ ), 2 mM iron(II) heptahydrate (FeSO ₄ × 7H ₂ O), 0.5 vol% glycerol, DKR-2: 1% hydrogen peroxide (H ₂ O ₂ ), 0.05% sodium benzoate (C ₆ H ₅ COONa)): mixture of DKR-1 solutions: 100 mg sodium dodecyl sulfate, 12.5 mg copper (II) sulfate pentahydrate, 3.63 mg Tris (hydroxymethyl) aminomethane, 5.6 mg iron(II) heptahydrate (FeSO ₄ × 7H ₂ O), 50 µl glycerol, made up to 10 ml with sterile deionized water, and DKR-2: 330 µl 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg benzoate sodium (C ₆ H ₅ COONa) adjusted to 10 ml with sterile deionized water.

Fig. 6. Composition of the liquid formulation DKR No. 4 (DKR-1: 1% Sodium dodecyl sulfate (SDS), 5 mM copper(II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O); DKR-2: 1% hydrogen peroxide (H ₂ O ₂ ), 0.05% sodium benzoate (C ₆ H ₅ COONa)): a mixture of DKR-1 solutions: 100 mg sodium dodecyl sulfate, 12.5 mg copper (II) sulfate pentahydrate, brought to 10 ml with sterile deionized water, and DKR-2: 330 μl of 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg sodium benzoate (C ₆ H ₅ COONa), adjusted to 10 ml with sterile deionized water.

7. Composition of the liquid formulation DKR No. 4 (DKR-1: 2% Sodium dodecyl sulfate (SDS), 5 mM copper(II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O); DKR-2: 1% hydrogen peroxide (H ₂ O ₂ ), 0.05% sodium benzoate (C ₆ H ₅ COONa)): a mixture of DKR-1 solutions: 200 mg sodium dodecyl sulfate, 12.5 mg copper (II) sulfate pentahydrate, brought to 10 ml with sterile deionized water, and DKR-2: 330 μl of 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg sodium benzoate (C ₆ H ₅ COONa), adjusted to 10 ml with sterile deionized water.

8. Commercial solution "Belizna" is an aqueous solution of 70 mM or 0.5% sodium hypochlorite (NaOCl) diluted 1:10 with deionized water to obtain 7 mM or 0.05% sodium hypochlorite (NaOCl).

9. DNARId (Biomedical Innovations, Russia) is a two-component set of solutions and includes a DNA decontamination reagent 1 and a DNA decontamination reagent 2, the composition of which is not disclosed by the manufacturer.

10. The composition of DNA-ExitusPlus liquid compositions (AppliChem, USA) is not disclosed by the manufacturer.

11. Control sample with DNA and deionized water (diH ₂ O).

To study the stability of amplicons under the decontamination effect of the studied reagents, PCR products of standard DNA 9947A were used. Amplicons were obtained using a Real-Time PCR iQ5 PCR analyzer (Bio-Rad, USA). For enzymatic amplification of the HBG (Human beta globin) gene locus, 120 nucleotide pairs long, the following pair of primers was used (Faleeva T.G., Ivanov I.N., Mishin E.S. et al. human fingers // Forensic medical examination. - 2016. - V. 59, No. 2. - P. 14-18):

HBG1: 5'-GCTTCTGACACAACTGTGTTCACTAGC-3'

HBG2: 5'-CACCAACTTCATCCACGTTCACC-3'

Amplification was performed using the EVA Green reagent kit (Sintol, Russia). 20 μl of the reaction mixture and 5 μl of 9947A DNA were added to each tube. The volumes of the components of the PCR mixture are presented in table. 3.

Amplification was carried out in the following mode:

The real-time PCR efficiency calculated from the standard curve was at least 0.92. Data analysis was carried out using the iQ5 Optical System Software (version 2.0). The melting temperature of the PCR product was 84.5±0.5°C.

The concentration of the resulting HBG 9947A PCR products was also measured using a QuantiFluor-P fluorimeter (Green/Blue, Promega Corporation, USA) in three agility. The concentration was: Me = 7.363, min = 7.327, max = 8.112.

Before starting the experiment, the HBG 9947A PCR products were mixed and briefly centrifuged. 14 μl of amplicons were added to 0.2 ml PCR tubes for PCR. The test solutions were added to DNA in the following ratio:

1. 14 µl PCR product + 14 µl composition (1): 12 µl DCR-1 + 2 µl DCR-2

2. 14 µl PCR product + 14 µl composition (2): 12 µl DCR-1 + 2 µl DCR-2

3. 14 µl PCR product + 14 µl composition (3): 12 µl DCR-1 + 2 µl DCR-2

4. 14 µl PCR product + 14 µl composition (4): 12 µl DCR-1 + 2 µl DCR-2

5. 14 µl PCR product + 14 µl composition (5): 12 µl DCR-1 + 2 µl DCR-2

6. 14 µl PCR product + 14 µl composition (6): 12 µl DCR-1 + 2 µl DCR-2

7. 14 µl PCR product + 14 µl composition (7): 11 µl DCR-1 + 3 µl DCR-2

8. 14 µl of PCR product + 14 µl of commercial 0.1X Whiteness solution

9. 14 µl PCR product + 7 µl DNA decontamination reagent 1 and 7 µl DNA decontamination reagent 2 DNARid (Biomedical Innovations, Russia)

10. 14 μl of PCR Product + 14 μl DNA-ExitusPlus (Applichem, USA)

11. 14 µl PCR product + 14 µl diH ₂ O

The test samples were mixed and briefly centrifuged. Then it was incubated with a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems) for 15 min at 25°C. After incubation, they were briefly centrifuged and electrophoresis of the studied samples was carried out in a 2% agarose gel. To do this, 6 μl of each amplicon was mixed with 1.5 μl of a six-fold loading buffer (5:1, v/v) containing bromophenol blue and xylene cyanol in a 30% glycerol solution. The Bench Top 100 bp DNA Ladder marker (Promega, USA) was used as a size standard (marked as “L” in the electrophoresis photo of Fig. 3). Electrophoresis was carried out for 5 min at 120 V, then 15 min at 100 V. The gel was placed in the UV transilluminator of a ChemiDoc XRS+ System (Bio-Rad, USA) and photographed. The luminescence intensity of the experimental DNA preparations and the PCR product was assessed relative to the control samples (Kornienko I.V., Kharlamov S.G. Methods for the study of human DNA. DNA isolation and its quantitative assessment in the aspect of forensic examination of material evidence of biological origin: educational and methodological allowance Rostov n/D: SFedU, 2012. - 216 p.).

Next, the concentration of PCR products was measured after exposure to various test reagents using a QuantiFluor-P fluorimeter (Green/Blue, Promega Corporation, USA) in 3 replicates. The results are presented in table. 4.

Fluorimetry data show that the concentration of PCR products after treatment with DCR nos. 1-4 does not exceed 0.12 ng/μl on average (Me = 0.10, Q ₁ = 0.09, Q ₃ = 0.18). The content of PCR products in samples after decontamination processing with a commercial 0.1 multiple (0.1x) solution “Whiteness” (Me = 2.92, Min = 2.89, Max = 2.94) and DNA-ExitusPlus (Applichem, USA ) (Me = 3.17, min = 3.14, max = 3.19) were close to the control (without decontamination treatment) (Me = 3.45, min = 3.44, max = 3.49). The concentrations of PCR products treated with DNARid commercial reagents (Biomedical Innovations, Russia) occupied an intermediate position (Me = 0.80, min = 0.79, max = 0.83).

On FIG. 3 presents the results of electrophoresis of samples in 2% agarose gel. In samples 1-7 and 9, the absence of bands and traces of white "schmer" is noted, which indicates the degradation of the amplicon in the samples treated with DCR no. 1-4 and the DNARid reagent (Biomedical Innovations, Russia). In 8 samples treated with a commercial 0.1-fold (0.1X) Belizna solution and 10 samples treated with the DNA-ExitusPlus reagent (AppliChem, USA), a PCR product is clearly visible, similar in intensity to the control, which reflects the absence of decontamination exposure to these reagents in relation to amplings.

The data obtained reflect the presence of a stable effect exerted by DKR No. 1-4 on the PCR product, which manifests itself in the form of its significant degradation. The use of the commercial DNARid reagent (Biomedical Innovations, Russia) did not allow us to obtain stable results with respect to the degradation of amplicons. The commercial solution Belizna and the DNA-ExitusPlus reagent (AppliChem, USA) did not have a significant decontamination effect on the PCR product.

Example 3

Conducted a study of the stability of cell membranes under the influence of the proposed in the present invention liquid compositions. Commercial analogues in this example were not considered, since their manufacturers did not declare the ability of these solutions to destroy cell membranes.

The composition of the liquid composition DKR No. 4 (DKR-1: 1% Sodium dodecyl sulfate (SDS), 5 mm copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O); DKR-2: 1% hydrogen peroxide (H ₂ O ₂ ), 0.05% sodium benzoate (C ₆ H ₅ COONa)): a mixture of DKR-1 solutions: 100 mg sodium dodecyl sulfate, 12.5 mg copper (II) sulfate pentahydrate, brought to 10 ml with sterile deionized water, and DKR-2: 330 μl of 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg sodium benzoate (C ₆ H ₅ COONa) made up to 10 ml with sterile deionized water.

To study the stability of cells when exposed to various decontamination agents, cells of the HeLa cell line were studied. The cells were cultured on Eagle's nutrient medium MEM (Biolot, Russia) in a Sanyo 18M incubator at 37°C in an atmosphere with 5% CO ₂ . For treatment of cells, they were separated from the substrate by treatment with trypsin in Versene's solution (2:1) for 5 min. After adding culture medium to stop trypsinization, the cells were suspended and then pelleted in an ELMI CM-6M centrifuge at 1 kr. (~188 G) for 5 min. After separation of the supernatant, the cells were resuspended in DPBS solution without Ca ²⁺ and Mg ²⁺ and re-sedimented on a centrifuge. After separation of the wash solution, Dulbecco's phosphate-buffered saline (DPBS) without Ca ²⁺ and Mg ²⁺ was added to the precipitated cells to obtain a suspension with a cell density of 0.5-1 million per ml. 0.1 ml of the resulting cell suspension was added to 1.5 ml conical tubes with 0.9 ml of test solutions previously added. The test tubes with samples were incubated for 30 min on a solid state thermostat at room temperature with the table shaking at a frequency of 500 rpm. Half of the treated samples were additionally incubated for 9 min on a solid-state thermostat at a temperature of 99°C with the table shaking at a frequency of 500 rpm. To stabilize the state of the samples, they were cooled to 4°C.

The samples were then microscopically examined. The number of cells in the suspension of developed solutions, water and DBPS (without Ca ²⁺ and Mg ²⁺ ) was determined. After intensive mixing of the contents of the tubes for 5 seconds, two samples (9 μl each) of the contents were taken from the tubes: one at the surface of the liquid, the second at the bottom of the tube. Each of the samples was placed in a chamber for counting blood cells (Camera Goryaeva, TU 9443-007-29508133-2007, 2 sets) (MiniMed, Russia), where cells were counted on three lanes of marked camera surface. The length of each of the strips was 3 mm with a width of 0.2 mm, which, with a chamber height of 0.1 mm, corresponds to a liquid volume of 0.06 mm. For each of the bands, the cell density in 1 μl was obtained by multiplying the counted amount by a factor of 1/0.06. From the sample scores obtained, an arithmetic mean was calculated and its 95% confidence interval was calculated. The results are shown in table. 5 and also in FIG. 4-6.

The results presented in table. 5, reflect the high ability of decontamination solutions proposed in the present invention to destroy cell membranes in the studied regimes. The data obtained confirms microphotography in the Goryaeva camera of cell suspensions of the HELA line (10K) after processing and incubation in DKR No. 4: whole cells and cellular structures are not visualized (Fig. 4A-B). At the same time, the treatment and incubation of HeLa cells (10k) in DBPS (Fig. 5A-B) and deionized water (control) (Fig. 6A-B) did not lead to the destruction of cell membranes and cell structures, which is reflected in the abundant presence of cells in micrographs .

Example 4

We investigated the possibility of removing DNA-containing biological material (cell line) from samples with biological material. For this, the following liquid composition according to the present invention was used:

The composition of the liquid composition DKR No. 4 (DKR-1: 1% Sodium dodecyl sulfate (SDS), 5 mm copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O); DKR-2: 1% hydrogen peroxide (H ₂ O ₂ ), 0.05% sodium benzoate (C ₆ H ₅ COONa)): a mixture of DKR-1 solutions: 100 mg sodium dodecyl sulfate, 12.5 mg copper (II) sulfate pentahydrate, brought to 10 ml with sterile deionized water, and DKR-2: 330 μl of 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg sodium benzoate (C ₆ H ₅ COONa) made up to 10 ml with sterile deionized water.

The cell line HeLa was used as a contaminating material, and bone powder was used as a contaminated biological material. The studies were carried out in 5 repetitions. Powder was obtained from a compact layer of long tubular bones (humerus, femur) of two human skeletons using a Retsch MM 200 vibration mill for 2.5-3 min at a speed of 1500 rpm. or with the Hammer Flex MD135A mini drill. 1 g of bone powder was placed in 50 ml tubes, and 1 ml of an aqueous solution of HeLa DNA was added (total 60 ng, 60 ng/ml). For efficient mixing of the mixture and breaking up the sediment after centrifugation, a sterile glass ball 16 mm in diameter was placed separately in each test tube with the mixture. Vortexed for several seconds until all of the bone powder was mixed with the HeLa DNA solution.

Then, 800 μl of the DKR-2 composition were added to the test tubes with the contaminated powder, and then 25 ml of DKR-1 were added. Mixed for 10 seconds on a vortex. Incubated on a horizontal shaker at 200 rpm. within 10 min. Centrifuged at 1500 g for 5 min. Carefully pour off the supernatant so that the glass ball remains in the test tube. 800 µl of the DKR-2 composition was added to the sediment again. Then, 25 ml of DKR-1 were added again. Mixed for 10 seconds on a vortex until the precipitate was completely broken. Incubated on a horizontal shaker at 200 rpm. within 10 min. Centrifuged at 1500 g for 5 min. Carefully pour off the supernatant so that the glass ball remains in the test tube. 20-25 ml of DKR-1 composition was added to the supernatant. Mixed for 30 seconds on a vortex until the precipitate was completely broken. Centrifuged at 1500 g for 5 min. Carefully pour off the supernatant so that the glass ball remains in the test tube. To the supernatant was added 20-25 ml of a solution of 0.5% laurylsarcosyl in 0.5 M EDTA. Mixed for 40 seconds on a vortex until the sediment was completely broken. Centrifuged at 1500 g for 5 min. Carefully pour off the supernatant so that the glass ball remains in the test tube. 20-25 ml of a solution of 0.5% laurylsarcosyl in 0.5 M EDTA was added to the supernatant again. Mixed for 40 seconds on a vortex until the sediment was completely broken. Centrifuged at 1500 g for 5 min. Carefully pour off the supernatant so that the glass ball remains in the test tube. To the supernatant for the third time was added 20-25 ml of a solution of 0.5% laurylsarcosyl in 0.5 M EDTA. Mixed for 40 seconds on a vortex until the sediment was completely broken. A homogeneous suspension of the washed and decalcified bone powder was poured into a clean 50 ml test tube so that the glass ball remained in the first test tube. Centrifuged at 1500 g for 5 min. The supernatant was removed. Then, 4 ml of lysis solution No. 2 was added to the precipitate: 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 2.5 mm MgCl ₂ ; 0.45% Tween 20 (Kornienko I.V., Kharlamov S.G. Methods for the study of human DNA. DNA isolation and its quantification in the aspect of forensic medical examination of material evidence of biological origin: educational and methodological manual. Rostov n / D: SFedU, 2012. - 216 p.). Thoroughly mixed. Incubated at 43°C in a thermoshaker at 160 rpm. within 12 hours. Centrifuged at 4000 g for 10 min.

The supernatant was transferred into separate sterile 50 ml tubes. The volume of the mixture of phenol/chloroform/isoamilol (25: 24: 1) was added to the supervision to the contents of the tube. Thoroughly mixed on a vortex for at least 10 seconds. The contents of the tubes were centrifuged at 4000 g for 10 min. The upper aqueous phase was carefully transferred into sterile 50 ml tubes without affecting the interphase. Extracted with phenol/chloroform/isoamylol twice. An equal volume of n-butanol or chloroform was added to the aqueous solution with DNA. Thoroughly mixed on a vortex for at least 10 seconds. The contents of the tubes were centrifuged at 4000 g for 5 min. The top layer of butanol (or chloroform) was removed, leaving an aqueous solution with DNA in the test tube.

For additional purification and concentration of DNA preparations, an AmiconUltra-4, ultracel30k device was used in accordance with the user manual.

Далее проводили генотипирование исследуемых образцов с использованием наборов реагентов для мультиплексного анализа 19-и STR-маркеров (D3S1358, ТН01, D12S391, D1S1656, D10S1248, D22S1045, D2S441, D7S820, D13S317, FGA, ТРОХ, D18S51, D16S539, D8S1179, CSF1PO, D5S818 , VWA, D21S11, SE33) and a fragment of the human amelogenin gene (Amelogenin) according to the test systems “Cordis Plus” and Cordis “Expert” (Gordiz, Russia) in accordance with the user's instructions. The conditions for conducting PCR and genotyping DNA drugs are described in example 1 of the present invention. The genotyping results are shown in Fig. 7.

On the electrophoregram of Fig. 7a is presented a genetic profile of a bone -sample contaminated by the cell culture without preliminary processing proposed in the present invention of DKR. This profile is mixed in nature, as it contains up to four alleles in the studied loci. At the same time, this profile contains genetic features characteristic of both the genotype of the bone material (Fig. 7C) and the genotype of the HeLa cell culture (Fig. 7D) (alleles are highlighted). Processing of contaminated bone powder using DCR allowed us to obtain an authentic bone profile without impurities: on the electrophoregram (Fig. 7B), the genotype contains alleles characteristic of the bones of this skeleton. The control of the purity of the reagents is reflected in the absence of allelic peaks in the profile on the electrophoregram of Fig. 7D (blank sample).

The data obtained indicate that the pre-treatment of bone powder samples proposed in the present invention DKR contributes to the removal of the introduced cell line and allows you to get a clean profile of the studied bone sample. The treatment of crushed bone objects (powder, chips) in this example of the present invention with the help of DCR is considered as a method of decontamination purification in the process of sample preparation of samples before decalcification and DNA extraction from them.

Example 5

We studied the effect on the genetic material of the pathogen of the new coronavirus infection of SARS-COV-2 liquid composition of the DCR No. 4, offered in the present invention, as well as commercial disinfectants: “Asept Pro” (“Acea, Russia), Farmspt” (“Pharmsept”) (Uralhimfarm - plus, Russia), “Forispot” (alpha, Russia), hand antiseptic “Don pharmacy” (GUP RO “Rostovoblhpharmation”, Russia) and “Coronosept Dr. Arsenin" (NATUROTHERAPY, Russia). The experimental study was carried out in eight repetitions.

1. Composition of the liquid composition DKR No. 4 (DKR-1: 1% Sodium dodecyl sulfate (SDS), 5 mM copper(II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O); DKR-2: 1% hydrogen peroxide (H ₂ O ₂ ), 0.05% sodium benzoate (C ₆ H ₅ COONa)): a mixture of DKR-1 solutions: 100 mg sodium dodecyl sulfate, 12.5 mg copper (II) sulfate pentahydrate, brought to 10 ml with sterile deionized water, and DKR-2: 330 μl of 30% aqueous hydrogen peroxide (H ₂ O ₂ ) and 5 mg sodium benzoate (C ₆ H ₅ COONa), adjusted to 10 ml with sterile deionized water.

2. "Asept Pro" ("Asept Pro") manufactured by LLC "ACEYA", registered in the Eurasian Economic Community, No. KZ.16.01.98.002.E.000270.04.19 dated 05.04.2019 (Moscow).

3. “Pharmacept” (“Pharmsept”), production of Uralhimfarm - Plus LLC, state registration number No. RU.77.99.88.002.E.007106.06.15 dated 06/18/2015 (Chelyabinsk).

4. Forispot, manufactured by OOO NPK Alfa, state registration number NQRU.77.99.88.002.E.003495.08.18 dated August 14, 2018 (Rostov-on-Don).

5. Antiseptic for hands "Pharmacy of the Don", produced by the State Unitary Enterprise RO "Rostovoblfarmatsiya" (Rostov-on-Don).

6. CORONOSEPT Dr. Arsenin, produced by CJSC NATUROTHERAPY (Noginsk).

The composition of liquid compositions of commercial disinfectants is not disclosed by manufacturers.

The selection of biological material was carried out by a medical worker in the medical office of the Rostov Research Institute of Microbiology and Parasitology of Rospotrebnadzor, using personal protective equipment. Swabs were performed with rotational movements from the surface of the tonsils, palatine arches and posterior walls of the nasopharynx and oropharynx in patients with a new coronavirus infection (pathogen SARS-CoV-2) using special dry sterile probes. The effect on the genetic material of the causative agent of the new coronavirus infection SARS-CoV-2 of the liquid composition of DKR No. 4 was studied on biological material from 14 patients, and commercial disinfectants - from 8 patients.

500 μl of the reagent for storing and transporting respiratory strokes “Transport environment for storing and transporting respiratory strokes” (Interlab Shebsis type) to sterile Espendorf type sterile tubes were dug up with sterile tips. Swabs with completed washings were placed in test tubes with the transport medium. At the same time, swabs from the nasal cavity and oropharynx were combined in one test tube. The ends of the probes were broken off so that they allowed to tightly close the test of the test tube.

The tubes with contents were briefly shaken on a vortex. With sterile tips, 50 µl of the transport medium with swabs from the nasal cavity and oropharynx were dropped into sterile 1.5 ml Eppendorf tubes. The studied reagents were added to the samples in the following ratio:

1. 50 μl of sample + 50 μl composition (1): 40 μl DKR-1 + 10 μl DKR-2

2. 50 µl sample + 50 µl Asept Pro (ATSEYA, Russia)

3. 50 μl of a sample + 50 μl "Pharmsept" ("Pharmsept") (Uralhimfarm - plus, Russia)

4. 50 μl of a sample + 50 μl "Forospot" (Alpha, Russia)

5. 50 µl sample + 50 µl Hand antiseptic "Pharmacy of the Don" (GUP RO "Rostovoblfarmatsiya", Russia)

6. 50 μl of a sample + 50 μl "Coronosept Dr. Arsenin "(Naturotherapy, Russia)

7. 50 μl of a sample + 50 μl of deionized water (control)

Samples were incubated for 30 minutes at room temperature (22-25 ° C). Immediately after incubation, RNA of the SARS-CoV-2 pathogen was isolated using the RIBO-prep kit (manufactured by InterLabService).

To do this, 300 μl of solution for lysis were added to 1.5 ml tubes, then 100 μl of prepared samples were added after 30 minutes of incubation. The contents of the tubes were thoroughly mixed on a vortex, centrifuged for 5 sec in a microcentrifuge to remove drops from the inner surface of the lid, and heated for 5 min at 65°C in a thermostat. 400 µl of the precipitation solution was added to the test tubes, mixed on a vortex. The tubes were centrifuged in a microcentrifuge for 5 min at 13,000 rpm. The supernatant was carefully removed without touching the precipitate. 500 μl of wash solution 3 were added to the tubes, the lids were tightly closed, the precipitate was carefully washed by turning the tubes 3-5 times. Centrifuged at 13,000 rpm for 1-2 min in a microcentrifuge. Carefully, without capturing the precipitate, the supernatant liquid was taken. 200 μl of wash solution 4 was added to the tubes, the lids were tightly closed, and the precipitate was carefully washed by turning the tubes 3-5 times. Centrifuged at 13,000 rpm for 1-2 min in a microcentrifuge. Carefully, without capturing the precipitate, the supernatant liquid was taken. The test tubes were placed in a thermostat at a temperature of 65°C for 5 min to dry the precipitate (the lids of the tubes were left open). 50 µl of buffer RNA were added to the tubes. Mixed on a vortex. Placed in a thermostat at a temperature of 65°C for 5 min, periodically shaking on a vortex. The tubes were centrifuged at 13,000 rpm for 1 min in a microcentrifuge. Next, the supernatant was examined.

The detection of SARS-CoV-2 coronavirus RNA was carried out by reverse transcription (RT) and real-time polymerase chain reaction (RT-PCR) using a specialized test system RU No. RZN 2020/9948 dated April 01, 2020 (DNA-Technology, Russia).

They prepared a mixture of OT-PCR-buffer with the TAQ/RT enzyme. Mixed in a separate test tube:

15 × (N+1) µl RT-PCR buffer,

0.5 × (N+1) µl of Taq/RT enzyme,

Where n is the number of marked test tubes, taking into account “k-” and “k+”.

The tube was shaken for 3-5 sec on a microcentrifuge-vortex and centrifuged for 1-3 sec on a microcentrifuge-vortex. Added to each labeled tube, without damaging the paraffin layer, 15 μl of a mixture of RT-PCR buffer with Taq/RT enzyme. The lids of the vials were closed. The test tubes with test and control samples were shaken for 3-5 sec on a microcentrifuge-vortex and centrifuged for 1-3 sec on a microcentrifuge-vortex. 10 μl of test samples, negative and positive control samples were added to the respective test tubes without damaging the paraffin layer. The tubes were centrifuged for 3-5 sec on a microcentrifuge-vortex. All tubes were installed in a DTprime detecting cycler unit (DNA-technology, Russia) and RT-PCR was performed taking into account the volume of the reaction mixture equal to 40 µl. The RealTime PCR software was launched in the “Instrument operation” mode. PCR was carried out according to the following enzymatic amplification program (Table 6):

Fluorescence measurements were set for the FAM, HEX, ROX, and Cy5 channels at 64°C (Table 7).

Detection of RNA of SARS-CoV-2 coronavirus, which causes a new coronavirus infection COVID-19, was also carried out by RT and RT-PCR using a kit of reagents for detecting RNA of SARS-CoV-2 coronavirus by RT-PCR-RT "RealBest RNA SARS-CoV -2" (Vector Best, Russia).

50 µl of the isolated RNA solution was added to the tubes with the prepared lyophilized reaction mixture for RT-PCR. The tubes were sealed with optical film. The tubes were placed in a mini-shaker. The contents of the test tubes were stirred at a speed of 1200 rpm for 1 min. The test tubes were placed in a DTprime detecting amplifier unit (DNA technology, Russia). Spent the reaction RT-PCR in accordance with the program specified in table. 8. Channels for detection of the results of amplification of the cDNA of the STV and cDNA of the infectious agent were chosen:

"Rchokh"-for registration of the signal of the KDNK SARS-COV-2;

"FAM" - for the registration of the signal of the KDNK VKO.

The analyzed sample was considered positive if for this sample the CT value through the Rox channel was smaller or equal to 40.

The results of DCR tests on their effect on the genetic material (RNA) of the causative agent of a new coronavirus infection (SARS-CoV-2) using the RT method and PCR-RT using a specialized test system RU No. RZN 2020/9948 dated April 01, 2020 (DNA -Technology, Russia) are given in Table. 9 and FIG. 8A-B

The obtained data show that incubation of swabs from the nasopharynx and oropharynx of patients with a new coronavirus infection (pathogen SARS-CoV-2) with DCR No. 4 for 30 minutes leads to complete degradation of the SARS-CoV-2 virus genome in 5 out of 6 samples. In one case (sample 4), a decrease in the concentration of the genetic material of the virus by 776 ( ^29.6 ) times was detected.

Comparative results of studies on the impact of DKR No. 4 and commercial disinfectants Asept Pro, Pharmsept, Forispot, Antiseptic for the hands of Pharmacy Don and CORONOSEPT Dr. Arsenin") on the genetic material (RNA) of the causative agent of a new coronavirus infection by RT-PCR-RT using the RealBest RNA SARS-CoV-2 reagent kit (Vector Best, Russia) are given in Table. 10.

Due to insufficient mixing of the reaction mixtures in samples Nos. 4, 6 and 7 during the procedure for the impact of DCR No. 4 on the genome of the SARS-CoV-2 pathogen, repeated studies were carried out for these objects. Additionally, the commercial disinfectant Forispot (Alfa, Russia) was studied as a potentially decontaminating agent.

Comparative results of repeated DCR No. 4 and the commercial disinfectant "Forispot" in relation to the degradation effect on the genetic material (RNA) of the causative agent of a new coronavirus infection by RT-PCR-RT using the RealBest RNA SARS-CoV-2 reagent kit (Vector Best, Russia) are given in Table. 11 and FIG. 9.

Incubation of swabs from the nasopharynx and oropharynx of patients with a new coronavirus infection with DCC No. 4 for 30 minutes led to complete degradation of the SARS-CoV-2 virus genome in 2 out of 8 samples. In 7.1% of samples (1 out of 14), DCR #4 led to a decrease in the concentration of the genetic material of the SARS-CoV-2 virus by 776 times. In the remaining 42.9% of samples (6 out of 14), incubation of swabs from the nasopharynx and oropharynx of patients with a new coronavirus infection with DCC No. 4 led to a significant decrease in the concentration of the genetic material of the virus:

1. in sample 2 - 122294.5 (2 ^16.9 ) times;

2. in sample 3 - 6813667.0 ( ^222.7 ) times;

3. in sample 5 - 9635980.2 (2 ^23.2 ) times;

4. in sample 6 - 4705.1 (2 ^12.2 ) times;

5. in sample 7 - 2965820.8 ( ^221.5 ) times;

6. in sample 8 - 1482910.4 (2 ^20.5 ) times.

The data obtained indicate that the incubation of swabs from the nasopharynx and oropharynx of patients with a new coronavirus infection with commercial disinfectants Asept Pro, Pharmsept, Forispot, Antiseptic for hands of the Don Pharmacy, CORONOSEPT Dr. Arsenin ”for 30 minutes showed lower effectiveness compared to DCR No. 4 for the degradation of the pathogen genome of a new coronavirus infection. Thus, none of the above commercial disinfectants has led to the complete destruction of the genome of the SARS-CoV-2 virus.

Claims (94)

1. Composition for removing DNA and/or RNA-containing biological material in the form of a mixture of two solutions: decontamination solution-1 (DKR-1) and decontamination solution-2 (DKR-2) in a ratio of 1:1 to 35:1, Where DKR-1: 0.5-2% of sodium predecyl sulfate (Sodium Dodecyl Sulfate, SDS), 1-5 mM copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 1-15 mM Tris (hydroxymethyl) aminomethane (Tris, (HOCH ₂ ) ₃ CNH ₂ ), 0.1-1 vol. % glycerin, deionized water - up to 10 ml and DKR-2: 0.1-3% hydrogen peroxide (H ₂ O ₂ ), 0.01-0.1% sodium benzoate (C ₆ H ₅ COONa), Deionized water - up to 10 ml. 2. The composition according to clause 1, where DKR-1 contains: 100 mg of sodium dodecyl sulfate (Sodium Dodecyl Sulfate, SDS), 12.5 mg copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 3.63 mg Tris (hydroxymethyl) aminomethane (Tris, (HOCH ₂ ) ₃ CNH ₂ ), 50 µl of glycerin, up to 10 ml of sterile deionized water and DKR-2 contains: 330 µl 30% aqueous hydrogen peroxide (H ₂ O ₂ ), 5 mg sodium benzoate (C ₆ H ₅ COONa), up to 10 ml of sterile deionized water. 3. Composition for removing DNA and/or RNA-containing biological material in the form of a mixture of two solutions: decontamination solution-1 (DKR-1) and decontamination solution-2 (DKR-2) in a ratio from 1:1 to 35:1, Where DKR-1: 0.5-2% Sodium dodecyl sulfate (SDS), 1-5 mM copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 1-15 mM Tris (hydroxymethyl) aminomethane (Tris, (HOCH ₂ ) ₃ CNH ₂ ), 1-3 mM iron (II) sulfate heptahydrate (FeSO ₄ × 7H ₂ O), deionized water - up to 10 ml and DKR-2: 0.1-3% hydrogen peroxide (H ₂ O ₂ ), 0.01-0.1% sodium benzoate (C ₆ H ₅ COONa), Deionized water - up to 10 ml. 4. The composition according to paragraph 3, where DKR-1 contains: 100 mg of sodium dodecyl sulfate (Sodium Dodecyl Sulfate, SDS), 12.5 mg copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 3.63 mg Tris (hydroxymethyl) aminomethane (Tris, (HOCH ₂ ) ₃ CNH ₂ ), 5.6 mg iron (II) sulfate heptahydrate (FeSO ₄ × 7H ₂ O), up to 10 ml of sterile deionized water and DKR-2 contains: 330 µl 30% aqueous hydrogen peroxide (H ₂ O ₂ ), 5 mg sodium benzoate (C ₆ H ₅ COONa), up to 10 ml of sterile deionized water. 5. The composition for removing DNA and/or RNA-containing biological material in the form of a mixture of two solutions: decontamination solution-1 (DKR-1) and decontamination solution-2 (DKR-2) in the ratio of 1: 1 to 35: 1, Where DKR-1: 0.5-2% of sodium predecyl sulfate (Sodium Dodecyl Sulfate, SDS), 1-5 mM copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 1-15 mM Tris (hydroxymethyl) aminomethane (Tris, (HOCH ₂ ) ₃ CNH ₂ ), 1-3 mM iron (II) sulfate heptahydrate (FeSO ₄ × 7H ₂ O), 0.1-1 vol. % glycerin, Deionized water - up to 10 ml and DKR-2: 0.1-3% hydrogen peroxide (H ₂ O ₂ ), 0.01-0.1% sodium benzoate (C ₆ H ₅ COONa), deionized water - up to 10 ml. 6. The composition according to clause 5, where DKR-1 contains: 100 mg of sodium dodecyl sulfate (Sodium Dodecyl Sulfate, SDS), 12.5 mg copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), 3.63 mg Tris (hydroxymethyl) aminomethane (Tris, (HOCH ₂ ) ₃ CNH ₂ ), 5.6 mg iron (II) sulfate heptahydrate (FeSO ₄ × 7H ₂ O), 50 µl of glycerin, up to 10 ml sterile deionized water and DKR-2 contains: 330 µl 30% aqueous hydrogen peroxide (H ₂ O ₂ ), 5 mg sodium benzoate (C ₆ H ₅ COONa), up to 10 ml of sterile deionized water. 7. Composition for removing DNA and/or RNA-containing biological material in the form of a mixture of two solutions: decontamination solution-1 (DKR-1) and decontamination solution-2 (DKR-2) in a ratio of 1:1 to 35:1, Where DKR-1: 0.5-2% Sodium dodecyl sulfate (SDS), 1-5 mM copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), deionized water - up to 10 ml and DKR-2: 0.1-3% hydrogen peroxide (H ₂ O ₂ ), 0.01-0.1% sodium benzoate (C ₆ H ₅ COONa), deionized water - up to 10 ml. 8. Composition according to claim 7, where DKR-1 contains: 100 mg Sodium dodecyl sulfate (SDS), 12.5 mg copper (II) sulfate pentahydrate (CuSO ₄ × 5H ₂ O), up to 10 ml sterile deionized water and DKR-2 contains: 330 µl 30% aqueous hydrogen peroxide (H ₂ O ₂ ), 5 mg sodium benzoate (C ₆ H ₅ COONa), up to 10 ml of sterile deionized water. 9. The composition according to any one of paragraphs. 1-8, where the ratio of the mixture of two decontamination solutions to the sample of biological material is 1:1. 10. The composition according to any one of paragraphs. 1-8, where the biological material is a cell culture or cells. 11. Composition according to claim 10, where the ratio of the mixture of two decontamination solutions to the sample of biological material is 25.8:1. 12. The composition according to any one of paragraphs. 1-8, where the biological material is a DNA or RNA preparation. 13. Composition according to claim 12, where the ratio of DKR-1 to DKR-2 is from 8.5:1 to 9:1. 14. The composition according to any one of paragraphs. 1-8, where the biological material is an amplicon. 15. Composition according to claim 14, where the ratio of DKR-1 to DKR-2 is from 3.7:1 to 6:1. 16. The composition according to any one of paragraphs. 1-8, where a sample of biological material is represented by a DNA and / or RNA-containing preparation / smear / trace. 17. Composition according to claim 16, where the ratio of DKR-1 to DKR-2 is 4:1. 18. Composition according to any one of paragraphs. 1-8, where the ratio of DCR-1 to DCR-2 is 32.25:1, and the sample of biological material is represented by a cell culture suspension. 19. The composition according to any one of paragraphs. 1-8, where the object to be decontaminated is bone material. 20. The composition according to claim 19, where the bone material is selected from: bone, bone fragment, bone chips, bone powder.

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