Decontamination solution and its use for denaturation, modification, degradation, solubilisation and removal of proteins, nucleic acid molecules and microorganisms

Abstract

The invention concerns a three component system comprising surface-active substances, vitamins and metal ions for efficient destruction and removal of contaminating proteins, nucleic acids and microorganisms from surfaces like for example laboratory benches, floors, equipment and instruments. These non-corrosive and non-toxic solutions for removal of proteins, nucleic acids and microorganisms are applied by spraying, rubbing or immersion of contaminated surfaces thereby destroying, solubilizing inactivating and removing proteins and nucleic acids. In that way also microorganisms are killed with high efficiency and at the same time all genetic information is inactivated.

Claims

1. A method comprising: providing a decontamination solution comprising: at least a synergistic mixture of a) at least one water-soluble vitamin in concentration from 10 mM to 100 mM, b) at least one metal ion in concentration from 1 mM to 100 mM, and c) at least on surface-active substance in concentration from 0.1% to 10% (weight) in relation to the total solution, wherein the synergistic mixture of the decontamination solution is configured to degrade DNA (deoxyribonucleic acid), protein and RNA (ribonucleic acid) on a surface of equipment, and treating the surface of the equipment that is contaminated by proteins, nucleic acid molecules and/or microorganisms comprising proteins and nucleic acid molecules with said decontamination solution resulting in a treated surface, wherein the decontamination solution degrades protein and any DNA and any RNA on the treated surface, and wherein the microorganisms are killed by degrading the proteins and any DNA and any RNA of the microorganisms on the treated surface.

2. The method according to claim 1, wherein the solution is adjusted via a buffer system to a pH value ranging between 2 to 8.5.

3. The method of claim 1, wherein the DNA and the RNA is completely degraded after a residence time of the decontamination solution on the treated surface of 0.5 to 2 minutes at room temperature.

4. A decontamination solution comprising a synergistic mixture, wherein said mixture comprises: 100 mM vitamin C, 10 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20; 100 mM vitamin C, 10 mM FeCl.sub.3, and 0.2% octoxynol 9; 50 mM vitamin C, 5 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20; 10 mM vitamin C, 10 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20; 100 mM vitamin C, 5 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20; 100 mM Na-ascorbate, 5 or 10 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20; 100 mM thiamin, 5 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20; 100 mM Na-ascorbate, 10 mM ZnCl.sub.2, 0.2% octoxynol 9; 100 mM Na-ascorbate, 10 mM FeCl.sub.3, 0.2% octoxynol 9; 100 mM vitamin C, 10 mM ZnCl.sub.2, and 0.2% octoxynol 9; or 100 mM vitamin C, 10 mM ZnCl.sub.2, 0.2% octoxynol 9, and 0.2% polysorbate 20.

5. The decontamination solution of claim 4, wherein said mixture comprises 100 mM vitamin C, 10 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20.

6. The decontamination solution of claim 4, wherein said mixture comprises 100 mM vitamin C, 10 mM FeCl.sub.3, and 0.2% octoxynol 9.

7. The decontamination solution of claim 4, wherein said mixture comprises 50 mM vitamin C, 5 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20.

8. The decontamination solution of claim 4, wherein said mixture comprises 10 mM vitamin C, 10 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20.

9. The decontamination solution of claim 4, wherein said mixture comprises 100 mM vitamin C, 5 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20.

10. The decontamination solution of claim 4, wherein said mixture comprises 100 mM Na-ascorbate, 5 or 10 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20.

11. The decontamination solution of claim 4, wherein said mixture comprises 100 mM thiamin, 5 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20.

12. The decontamination solution of claim 4, wherein said mixture comprises 100 mM Na-ascorbate, 10 mM ZnCl.sub.2, 0.2% octoxynol 9.

13. The decontamination solution of claim 4, wherein said mixture comprises 100 mM Na-ascorbate, 10 mM FeCl.sub.3, 0.2% octoxynol 9.

14. The decontamination solution of claim 4, wherein said mixture comprises 100 mM vitamin C, 10 mM ZnCl.sub.2, and 0.2% octoxynol 9.

15. A method comprising: providing a decontamination solution comprising a synergistic mixture, wherein said mixture comprises: 100 mM vitamin C, 10 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20; 100 mM vitamin C, 10 mM FeCl.sub.3, and 0.2% octoxynol 9; 50 mM vitamin C, 5 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20; 10 mM vitamin C, 10 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20; 100 mM vitamin C, 5 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20; 100 mM Na-ascorbate, 5 or 10 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20; 100 mM thiamin, 5 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20; 100 mM Na-ascorbate, 10 mM ZnCl.sub.2, 0.2% octoxynol 9; 100 mM Na-ascorbate, 10 mM FeCl.sub.3, 0.2% octoxynol 9; 100 mM vitamin C, 10 mM ZnCl.sub.2, and 0.2% octoxynol 9; or 100 mM vitamin C, 10 mM ZnCl.sub.2, 0.2% octoxynol 9, and 0.2% polysorbate 20.

16. The method of claim 15, wherein said mixture comprises 100 mM vitamin C, 10 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20.

17. The method of claim 15, wherein said mixture comprises 100 mM vitamin C, 10 mM FeCl.sub.3, and 0.2% octoxynol 9.

18. The method of claim 15, wherein said mixture comprises 50 mM vitamin C, 5 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20.

19. The method of claim 15, wherein said mixture comprises 10 mM vitamin C, 10 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20.

20. The method of claim 15, wherein said mixture comprises 100 mM vitamin C, 5 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20.

21. The method of claim 15, wherein said mixture comprises 100 mM Na-ascorbate, 5 or 10 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20.

22. The method of claim 15, wherein said mixture comprises 100 mM thiamin, 5 mM FeCl.sub.3, 0.2% octoxynol 9, and 0.2% polysorbate 20.

23. The method of claim 15, wherein said mixture comprises 100 mM Na-ascorbate, 10 mM ZnCl.sub.2, 0.2% octoxynol 9.

24. The method of claim 15, wherein said mixture comprises 100 mM Na-ascorbate, 10 mM FeCl.sub.3, 0.2% octoxynol 9.

25. The method of claim 15, wherein said mixture comprises 100 mM vitamin C, 10 mM ZnCl.sub.2, and 0.2% octoxynol 9.

26. The method of claim 15, wherein said mixture comprises 100 mM vitamin C, 10 mM ZnCl.sub.2, 0.2% octoxynol 9, and 0.2% polysorbate 20.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) The invention is illustrated by nonrestrictive figures, examples and tables shown in the following part:

(2) In that case FIGS. 1 to 5 show the efficient degradation of DNA molecules by the three component system in comparison with known other solutions of prior art.

(3) FIG. 6 demonstrates the blockage of PCR amplification of DNA molecules after the treatment with the new three component system.

(4) FIG. 7 shows the standard test with RNaseA for enzyme inactivation with the new three component system.

(5) FIG. 8. shows the efficient degradation of genomic DNA and extra chromosomal genetic material inside microorganisms by the new three component system.

(6) Table 1 shows a test for the anti-microbial action of the new three component system.

(7) Table 2 shows the preferred basic composition and the preferred mixtures for the three component system containing detergents, vitamins and metal ions.

DESCRIPTION OF ADVANTAGEOUS AND PREFERRED EMBODIMENTS OF THE INVENTION

(8) FIGS. 1 to 5 show the efficient degradation of DNA molecules by the new three component system in comparison with known other solutions. Identical aliquots of DNA plasmids (YEp351) were treated for 2 minutes with the listed solutions. Afterwards the DNA samples were denatured and the single-stranded DNA molecules were separated by gel electrophoresis on an agarose gel (1%). After staining with ethidium bromide the listed pictures were produced. The control shows intact plasmid DNA after treatment with sterile water. Introduction of nicks into the DNA strand results in a reduction of the size and molecular weight of the respective DNA molecules. This effect can be identified in the gel by comparison with the control and the molecular weight marker. In each sample 5 μg DNA were present in 5 μl sterile Tris buffer (1 mM; pH 8.0) and were treated for 2 minutes at room temperature with 5 μl of the listed solutions. Subsequently the samples were mixed with 5 μl 100 mM Tris (pH 12) and bromphenol blue marker and were denatured for 5 minutes at 95° C. The denatured samples were immediately cooled to 4° C. and identical aliquots of 1 μg DNA were loaded per gel lane. DNA molecules were stained with ethidium bromide after gel electrophoresis in a 1% agarose gel and photographed.

(9) FIG. 1 shows the comparison of nucleic acid degradation by vitamins and metal ions alone and by the three component system containing vitamins, metal ions and detergents. (M: Marker DNA 1 kb ladder; C: Control: DNA+5 μl sterile H.sub.2O; 1: 5 mM FeCl.sub.3; 2: 1 mM FAD; 3: 1 mM FAD+1 mM FeCl.sub.3; 4: 100 mM NAD; 5: 100 mM NAD+5 mM FeCl.sub.3; 6: 100 mM thiamin; 7: 100 mM thiamin+5 mM FeCl.sub.3; 8: 100 mM vitamin C; 9: 100 mM vitamin C+5 mM FeCl.sub.3; 10: 100 mM Na-ascorbate; 11: 100 mM Na-ascorbate+5 mM FeCl.sub.3; 12: 100 mM ascorbic acid+5 mM FeCl.sub.3). All samples contained 0.2% Triton X-100 and 0.2% Tween 20.

(10) FIG. 2 shows the test for nucleic acid degradation by mixtures containing vitamin C, metal ions and detergents. L: 1 Kb Ladder; M: Marker DNA Lambda EcoRI/HindIII; 1: 10 mM vitamin C; 2: 100 mM vitamin C; 3: 10 mM FeCl.sub.3; 4: 100 mM vitamin C+10 mM FeCl.sub.3; 5: 10 mM ZnCl.sub.2; 6: 100 mM vitamin C+10 mM ZnCl.sub.2; 7: DNA-OFF™; C: Control: 5 μl sterile H.sub.2O). All samples contained 0.2% Triton X-100 and 0.2% Tween 20.

(11) FIG. 3 shows the comparison of nucleic acid degradation by mixtures containing ascorbic acid or Na-ascorbate. (M: Marker DNA Lambda EcoRI/HindIII; C: Control: 5 μl sterile H.sub.2O; 1: DNA-OFF™; 2: 100 mM HAc+10 mM FeCl.sub.3+0.2% Triton X-100; 3: 100 mM ascorbic acid+0.2% TritonX-100; 4: 100 mM ascorbic acid+10 mM FeSO.sub.4+0.2% TritonX-100; 5: 100 mM ascorbic acid+ZnCl.sub.2+0.2% TritonX-100; 6: 100 mM Na-ascorbate+0.2% TritonX-100; 7: 100 mM Na-ascorbate+10 mM FeCl.sub.3+0.2% Triton X-100; 8: 100 mM Na-ascorbate+ZnCl.sub.2+0.2% Triton X-100).

(12) FIG. 4 shows the comparison of nucleic acid degradation by mixtures containing mineralic acids, ascorbic acid or Na-ascorbate (pH 6 to 8.5). (M: Marker DNA Lambda EcoRI/HindIII C: Control: DNA+5 μl sterile H.sub.2O; 1: RNase-OFF™ (sample 1); 2: RNase-OFF™ (sample 2); 3: DNA-OFF™ (sample 1); 4: DNA-OFF™ (sample 2); 5: 0.5 M H.sub.3PO.sub.4; 6: 0.5 M HNO.sub.3; 7: 100 mM ascorbic acid+10 mM FeCl.sub.3; 8: 10 mM ascorbic acid+10 mM FeCl.sub.3; 9: 100 mM Na-ascorbate+10 mM FeCl.sub.3; 10: mixture like in 9 only with pH 6; 11: mixture like in 9 only with pH 7.1; 12: mixture like in 9 only with pH 8; 13: mixture like in 9 only with pH 8.5). The samples number 5 to 13 contained 0.2% Triton X-100 and 0.2% Tween 20.

(13) FIG. 5 shows an example for the new buffer system containing Na-carbonate and malic (hydroxy-succinic) acid in comparison with mineralic acid. The basic mixture contains 50 mM ascorbic acid, 5 mM FeCl.sub.3 and 0.2% Triton X-100 and 0.2% Tween 20. (M: Marker DNA 1 kb Ladder), C: Control: DNA+5 μl sterile H.sub.2O; 1: mixture with pH 10; 2: mixture with pH 9; 3: mixture with pH 7; 4: mixture with pH 6; 5: mixture with pH 4; 6: 0.5 M H.sub.3PO.sub.4 (pH 1.5) with 0.2% Triton X-100 and 0.2% Tween 20.

(14) FIG. 6 shows the blockage of PCR amplification for DNA molecules after treatment with the new three component system (mixture A: 100 mM vitamin C+10 mM FeCl.sub.3+0.2% Triton X-100 and 0.2% Tween 20). Different amounts (0.1 to 1 ng) of a DNA sample were dried in PCR tubes. PCR tubes containing the dried DNA were treated for 20 seconds with the listed solutions. Subsequently the tubes were washed two-times with 100 μl of sterile, distilled water. Finally the tubes were filled with a 50 μl PCR reaction mixture and the PCR reaction was performed. The PCR reaction mixture contained pair of primers for the amplification of the control DNA (scIMP2 gene of yeast) and the test DNA (scPCP1 gene of yeast). The control DNA (1 ng) indicates a successful PCR reaction. A band of the test DNA demonstrates that intact DNA molecules for this gene are still present. In case of complete removal or blockage of the test DNA there shall not be any amplified DNA band in the gel.

(15) DNA was stained with ethidium bromide after gel electrophoresis in a 1% agarose gel and the gel was photographed. As a positive control the conventional DNA-OFF™ was used. Sterile water (H.sub.2O) served as a negative control.

(16) FIG. 7 shows the test of prior art for RNaseA enzyme inactivation in comparison with the new three component system. Identical aliquots of 10 μg RNaseA were dried in Eppendorf tubes. Afterwards each tube was treated with 1 ml of the listed solutions, vortexed for 20 seconds and finally incubated for 5 minutes at room temperature. Subsequently each tube was washed two-times with 1 ml of sterile water. Afterwards 5 μg of total RNA from E. coli were added into the tubes and incubated for 30 minutes at 37° C. Subsequently total RNA samples were mixed with formamide/bromphenol blue buffer and denatured at 95° C. for 5 minutes. Afterwards the complete 5 μg total RNA sample was loaded onto an agarose gel (1.2%) and separated by gel electrophoresis. After staining with ethidium bromide the documented picture was taken. The untreated controls represent intact total RNA. In the presence of active RNaseA these RNA molecules are degraded. In case of successful, complete inactivation of RNaseA the total RNA molecules will also remain intact.

(17) (C: Control of total RNA; 1: RNase-OFF (A); 2: RNase-OFF (B); 3: ascorbic acid mixture with 100 mM vitamin C+10 mM FeCl.sub.3+0.5% SDS; 4: empty lane; 5: H.sub.2O; 6: 10 μg RNase A; 7: empty lane; C: Control of total RNA)

(18) FIG. 8 shows the efficient degradation of genomic DNA and extra-chromosomal genetic material inside microorganisms by the new three component system. A recombinant Escherichia coli strain with an extra-chromosomal plasmid (YEp351) was grown over night in LB, amp medium. Aliquots of 5 μl from the E. coli suspension were treated with 5 μl lysozyme solution (1 mg/ml) for 5 minutes and subsequently incubated with 5 μl of the listed solutions (1-5) for additional 5 minutes. After addition of bromphenol blue the samples were loaded into the gel slots and separated by electrophoresis of the DNA molecules. Only for sample number 5 with the three component system a massive degradation of the DNA molecules is identifiable. For the control with sterile water (C) and for sample 3 and 4 lysis of the cells is observed and therefore the extra-chromosomal plasmid DNA is released and can migrate into the gel. For samples 1 and 2 one observes a precipitation of the lysate and the DNA and therefore consequently all DNA molecules remain at the bottom of the gel slot.

(19) (M: Marker 1 Kb Ladder; C: Control with H.sub.2O; 1: 70% Ethanol; 2: 0.5% Bacillozid™; 3: 0.5% SDS; 4: 0.5% Na-Azide+0.5% SDS; 5: 100 mM vitamin C+10 mM FeCl.sub.3+0.5% SDS; C: Control: 5 μl sterile H.sub.2O)

(20) Table 1 shows the test for antimicrobial action of the new three component system.

(21) Freshly grown cultures of the listed microorganisms were adjusted to a cell number of 10.sup.6 in a 50 μl volume and mixed in a ratio of 1:1 with 50 μl of water, 70% ethanol or the three component system (100 mM ascorbic acid, 10 mM FeCl.sub.3 and 0.5% SDS). After an incubation time of 2 minutes the 100 μl samples containing the microorganisms were plated onto the respective growth media. After an incubation period of 1-3 days at 28° C. (Saccharomyces cerevisiae and Candida parapsilosis) or 37° C. (Escherichia coli and Bacillus subtilis) the number of grown colonies was determined. In test samples with sterile water all microorganisms survived. Test samples with 70% ethanol or the three component system did not show any living cell colony, indicating that under these conditions all microorganisms were killed.

(22) Table 2 summarizes the preferred composition of the solution with the three component system comprising surface-active substances, vitamins and metal ions for removal of DNA molecules from surfaces and equipments.

List with Explanation of the Abbreviations in the Figures

(23) amp: ampicillin Bacillozid™: commercial anti-bacterial solution DNase-OFF™: commercial solution for inactivation of DNA EtBr: ethidium bromide FAD: Flavine adenine dinucleotide C: Control M: Molecular weight marker PCR: Polymerase Chain Reaction RNase-OFF™: commercial solution for inactivation of RNases RNaseA: Ribonuclease A (from bovine pancreas) RT: Room Temperature sc: Saccharomyces cerevisiae scIMP2: Saccharomyces cerevisiae gene for Inner Membrane Protease 2 scPCP1: Saccharomyces cerevisiae gene for Processing of Cytochrome c Peroxidase SDS: Sodiumdodecylsulfate TX: TritonX-100 (non-ionic detergent) YEp351: Yeast Episomal plasmid

(24) TABLE-US-00001 TABLE 1 Test for the anti-microbial action of the new three component system. 70% three component microorganisms H.sub.2O ethanol system Escherichia 10.sup.6 0 0 coli Bacillus 10.sup.6 0 0 subtilis Saccharomyces 10.sup.6 0 0 cerevisiae Candida 10.sup.6 0 parapsilosis

(25) TABLE-US-00002 TABLE 2 Preferred composition of solutions containing the three component system comprising surface-active substances, vitamins and metal ions for the removal of DNA molecules from surfaces and equipment. composition of the solutions pH range: 2.0 to 8.5 vitamins: 1 mM to 100 mM metal ions: 1 mM to 50 mM detergents: 0.1% to 5%