Nucleic acid scavenging agent, application and nucleic acid scavenging method

Abstract

The invention belongs to the field of chemistry, and discloses a nucleic acid scavenger which comprises trichloroisocyanuric acid or trichloroisocyanurate and metal ions, the metal ions are silver ions or copper ions. The nucleic acid scavenger is combined with metal ions, and the purpose of rapidly and thoroughly removing nucleic acid can be achieved. Meanwhile, the invention also provides application of the nucleic acid scavenger as a bactericide and a nucleic acid removal method.

Description

Technical Field

[0001] This invention relates to the field of chemistry, and more particularly to a nucleic acid scavenging agent, its uses, and a method for nucleic acid scavenging. Background Technology

[0002] In this field, nucleic acid clearance is generally carried out according to one or more of the following three principles. Denaturation and depolymerization: Denaturing agents such as guanidine salts can break the interactions such as hydrogen bonds and base stacking forces within nucleic acid molecules, thereby destroying the double helix structure or higher-order structure of nucleic acids, turning them into single-stranded or random linear molecules, making the structure of nucleic acids loose, and creating conditions for subsequent clearance steps.

[0003] Enzymatic degradation: The added nucleases play a crucial role. DNase can recognize specific sites in the DNA molecule, cleaving phosphodiester bonds and progressively degrading long DNA chains into small deoxyribonucleotide fragments; RNase has a similar effect on RNA, degrading it into ribonucleotide fragments. These smaller fragments are more easily metabolized or removed in subsequent processing.

[0004] Chemical modification and precipitation: Some components can chemically modify nucleic acids, altering their properties. For example, certain chemicals can bind to nucleic acids, causing them to precipitate under specific conditions. The precipitated nucleic acids can then be separated from the solution using methods such as centrifugation and filtration, thereby achieving the goal of removing nucleic acids from the system.

[0005] In simple terms, nucleic acid removal agents remove nucleic acids from samples (such as biological samples, surfaces of experimental equipment, etc.) by destroying the nucleic acid structure, degrading nucleic acids with enzymes, or separating nucleic acids by precipitation.

[0006] In this field, bactericides and nucleic acid scavengers are often equated, but in reality, their working principles are quite different. Bactericides typically work by disrupting cell membrane structures or denaturing microbial proteins. Nucleic acid scavengers, in particular, have higher requirements.

[0007] The literature on existing nucleic acid scavenging agents can be found as follows: Patent application CN115786039A, entitled "A Silver-Based Nucleic Acid Scavenger and Its Preparation Method and Application," describes a method for scavenging nucleic acids by using nano-silver / silver salts and auxiliary oxidants. The method involves breaking the phosphodiester bonds of nucleic acids, thereby degrading them. The auxiliary oxidant is one or more of sodium hypochlorite, potassium hypochlorite, potassium permanganate, potassium perchlorate, sodium dichromate, potassium dichromate, hydrogen peroxide, peracetic acid, ammonium persulfate, sodium percarbonate, sodium perborate, and potassium perborate, with a concentration of 0.5%-7%. Preferably, the auxiliary oxidant is selected from one or more of potassium permanganate, hydrogen peroxide, sodium perborate, and potassium perborate. More preferably, the auxiliary oxidant is selected from hydrogen peroxide and / or sodium perborate.

[0008] Patent application CN118421404A, entitled "A Nucleic Acid and Nuclease Contamination Removal Agent, its Preparation Method, and its Application," describes a method for removing nucleic acid contamination through the use of alkaline agents and oxidizing agents. The oxidizing agent is at least one selected from hypochlorous acid, compounds containing hypochlorite ions, hydrogen peroxide, and sodium trichloroisocyanurate, with a concentration of 0.11-0.15%. The alkaline agent imparts alkaline properties to the removal agent, enabling the degradation of RNA components sensitive to alkaline conditions. It also promotes the breaking of hydrogen bonds within DNA molecules, leading to denaturation into single strands. This accelerates the degradation of single-stranded DNA and RNA by the oxidizing agent. The oxidizing agent primarily utilizes its strong oxidizing properties to disrupt the chemical structure of nucleic acids, thereby degrading DNA and RNA. Simultaneously, its strong oxidizing properties also disrupt the structure and function of nuclease proteins, rendering them inactive. Through the synergistic action of the oxidizing agent and alkaline agent on nucleic acid molecules, the internal structure of the molecules can be rapidly destroyed, further degrading them into smaller molecules, achieving highly efficient removal of nucleic acid contamination.

[0009] Patent application CN116042326A, entitled "A Nucleic Acid Contamination Removal Agent," describes a nucleic acid contamination removal agent comprising B vitamins, sodium hydroxide, magnesium sulfate / zinc sulfate, sodium chloride, potassium chloride, sodium trichloroisocyanurate, Na₂HPO₄, KH₂PO₄, potassium carbonate, Tween, ethanol, and deionized water. Its mechanism involves the active ingredient molecules breaking DNA or RNA through contact with air to generate reactive oxygen species and through secondary derivatives acting on phosphodiester bonds, thus exhibiting a certain degree of selective removal efficacy. The concentration of sodium trichloroisocyanurate used is 0.001%. Meanwhile, this scheme uses a high concentration of vitamin B. As is well known, vitamin B12 can accelerate nucleic acid degradation, and sodium trichloroisocyanurate is used to maintain the activity of vitamin B12. From this scheme, it can be seen that the main means of nucleic acid clearance is the combined action of B vitamins with magnesium sulfate / zinc sulfate and potassium carbonate to degrade nucleic acids. Its formula is complex and the total concentration of effective ingredients is high.

[0010] As can be seen from the above records, sodium trichloroisocyanurate has been used in the field of nucleic acid removal. However, the problem with the above scheme is that the formula is relatively complex and requires the use of buffer solutions or alkaline agents to make the performance of sodium trichloroisocyanurate obvious. Summary of the Invention

[0011] The purpose of this invention is to provide a nucleic acid scavenger that binds to metal ions to achieve rapid and thorough nucleic acid removal.

[0012] In addition, the present invention also provides the use of the nucleic acid scavenger as a bactericide and a method for nucleic acid scavenging.

[0013] To achieve the above objectives, this application discloses a nucleic acid scavenging agent, comprising trichloroisocyanuric acid or trichloroisocyanurate, and metal ions; The metal ions are silver ions or copper ions.

[0014] The formulation of this invention is simple, and the purpose of rapidly removing nucleic acids can be achieved by using metal ions and trichloroisocyanuric acid or trichloroisocyanurate.

[0015] In the above-mentioned nucleic acid scavenging agent, the concentration of trichloroisocyanuric acid or trichloroisocyanurate in the nucleic acid scavenging agent is 500~1000ppm (equivalent to 0.05~0.1wt% by weight). Preferably, the concentration of trichloroisocyanuric acid or trichloroisocyanurate in the nucleic acid scavenger is 500~625ppm (equivalent to 0.05~0.0625wt%).

[0016] In the above-mentioned nucleic acid scavenger, the concentration of metal ions in the nucleic acid scavenger is 50~100ppm (equivalent to 0.005~0.01wt%).

[0017] The aforementioned nucleic acid scavenging agent further includes an auxiliary oxidant, which is one or more combinations of hydrogen peroxide, sodium hypochlorite, and potassium persulfate.

[0018] In the above-mentioned nucleic acid scavenging agent, the concentration of the auxiliary oxidant in the nucleic acid scavenging agent is 0.005-0.01wt%.

[0019] In the above-mentioned nucleic acid scavenging agent, the solvent of the nucleic acid scavenging agent is water; In the above-mentioned nucleic acid scavenging agent, the trichloroisocyanurate is sodium trichloroisocyanurate and / or potassium trichloroisocyanurate.

[0020] In addition, the present invention also discloses the use of the nucleic acid scavenging agent described above in the preparation of bactericides.

[0021] Finally, the present invention also discloses a nucleic acid removal method, which involves contacting the item to be removed with any of the nucleic acid removal agents described above to remove the nucleic acid.

[0022] The beneficial effects of this application are: This invention experimentally verifies that when low-concentration sodium trichloroisocyanurate cannot degrade nucleic acids, adding trace amounts of metal elements can significantly promote the nucleic acid degradation effect of sodium trichloroisocyanurate.

[0023] Compared with the prior art, the significant advantages of the present invention are: 1. Simple formula; 2. An auxiliary oxidant is not a necessary condition for this invention; 3. No additional pH control of the solution is required; 4. High nucleic acid degradation efficiency. Detailed Implementation

[0024] The present invention will now be clearly and completely described in conjunction with embodiments thereof. It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0025] Part 1: Nucleic Acid Degradation Experiments Using Different Concentration Gradients of Sodium Trichloroisocyanurate 1.1 Sample Preparation 80% sodium trichloroisocyanurate was diluted with water to form a concentration gradient of 1:800 to 1:4000 (by weight) to form a sodium trichloroisocyanurate solution. 1.2 Detection Reagents and Methods Porcine pseudorabies live vaccine (trade name Keweining), batch number 20240654, was purchased from Wuhan Keqian Biotechnology Co., Ltd. The vaccine lyophilized powder was diluted with physiological saline, and the viral fluid was extracted using an automated nucleic acid extractor (Shenzhen Xinsiwei Singu32, MTQ064V). Primers and probes were synthesized according to the national standard "Method for Fluorescent PCR Detection of Pseudorabies Virus" (GB / T35911-2018) to detect PRV- gH The patent application, with publication number CN 115369096 A, entitled "A Method for Calculating Nucleic Acid Concentration of Porcine Virus in Concentrated Water," relates to a method for calculating nucleic acid concentration of porcine viruses in concentrated water.

[0026] The specific procedure is as follows: Mix 100 μL of nucleic acid with 900 μL of nucleic acid scavenging agent at different dilutions, incubate at 25°C for 20 minutes, and then detect the nucleic acid concentration before and after the reaction.

[0027] 1.3 Test results are referenced in Table 1; Table 1 Test Results Sample number Dilution factor Ct value PRV nucleic acid concentration (copies / μL) 1 1:800 none 0 2 1:1000 none 0 3 1:1200 none 0 4 1:1350 none 0 5 1:1400 none 0 6 1:1450 none 0 7 1:1500 none 0 8 1:1550 none 0 9 1:1600 36.254 35.4 10 1:1650 34.135 140.4 11 1:1750 32.84 325.8 12 1:1800 33.199 258.0 13 1:2000 28.52 5398.9 14 1:4000 26.285 23074.1 15 0 26.012 27553.6

[0028] Note: Sample 15 refers to 100 μL of nucleic acid and 900 μL of sterile water mixed together; in Table 1, no CT value indicates that the sample test is negative, and the nucleic acid concentration in the sample is 0 or very low, exceeding the detection threshold.

[0029] Results analysis: 1. As can be seen from the above test results, when the dilution factor reaches 1550, complete removal of nucleic acid can be achieved.

[0030] 2. As can be seen from the combination of sample 13 and sample 15, sample 15 is a blank sample, indicating that the sample is basically invalid after the dilution factor reaches 1:1600.

[0031] Note: Due to fluctuations in the test results, the CT value of the blank control group may sometimes be lower than that of other dilution ratios. This is a reasonable phenomenon. Repeating the experiment multiple times and taking the average value can eliminate this phenomenon. Part Two: Study on the Nucleic Acid Clearance Effects of Sodium Trichloroisocyanurate and Metal Ions at Different Concentration Gradients 2.1 Sample Preparation Sodium trichloroisocyanurate was diluted with water to a concentration gradient of 1:1000 to 1:2000 (by weight), and metal ions were added to a final concentration of 0.005 to 0.01% to form a sodium trichloroisocyanurate solution. 1.2 Detection Method Porcine pseudorabies live vaccine (trade name Keweining), batch number 20240654, was purchased from Wuhan Keqian Biotechnology Co., Ltd. Physiological saline was added to the lyophilized vaccine powder, and the viral fluid was extracted using an automated nucleic acid extractor (Shenzhen Xinsiwei Singu32, MTQ064V). Primers and probes were synthesized according to the national standard "Method for Fluorescent PCR Detection of Pseudorabies Virus" (GB / T35911-2018) to detect PRV- gH Gene.

[0032] The specific procedure is as follows: Mix 100 μL of nucleic acid with 900 μL of nucleic acid scavenging agent at different dilutions, incubate at 25°C for 20 minutes, and then detect the nucleic acid concentration before and after the reaction.

[0033] 1.3 Test Results The test results are shown in Table 2. Table 2. Formulation and Test Results Serial Number Sodium trichloroisocyanurate dilution factor Ag+ / ppm Cu2+ / ppm CT1 CT2 CT average PRV nucleic acid concentration (copies / μL) 1 1:2000 50 0 none none none 0 2 1:2000 100 0 none none none 0 3 1:2000 0 50 none none none 0 4 1:2000 0 100 none none none 0 5 1:1500 100 0 none none none 0 6 1:1000 100 0 none none none 0 7 1:1500 50 50 none none none 0 8 1:1000 50 50 none none none 0 9 1:1500 0 0 none none none 0 10 1:1000 0 0 none none none 0 11 1:2000 0 0 26.343 25.987 26.165 24945.6 12 0 100 0 25.809 26.066 25.938 28920.4 13 0 0 100 26.324 25.879 26.102 25996.6 14 0 0 0 25.457 26.145 25.801 31603.2

[0034] Results analysis: Based on the test results in Table 2 above, the following conclusions can be drawn: 1. As can be seen from samples 12 to 14, Ag ions and Cu ions at a concentration of 0.01% have virtually no effect on nucleic acid removal; 2. As can be seen from samples 5 to 10, there is no antagonism between Ag ions and Cu ions and sodium trichloroisocyanurate; 3. As can be seen from samples 1 to 4, as well as samples 11 and 13, Ag ions and Cu ions can significantly promote the nucleic acid clearance effect of sodium trichloroisocyanurate.

[0035] The results above show that when sodium trichloroisocyanurate is diluted to a large factor, trace amounts of metal elements can significantly promote the nucleic acid clearance effect of sodium trichloroisocyanurate.

Claims

1. A nucleic acid scavenger, characterized in that, trichloroisocyanuric acid or trichloroisocyanuric acid salt, and metal ions; the metal ions are silver ions or copper ions.

2. The nucleic acid scavenger of claim 1, wherein, The concentration of trichloroisocyanuric acid or trichloroisocyanuric acid salt in the nucleic acid scavenger is 500-1000 ppm.

3. The nucleic acid scavenger of claim 1, wherein The concentration of metal ions in the nucleic acid scavenger is 50-100 ppm.

4. The nucleic acid scavenger of claim 1, wherein The solvent of the nucleic acid scavenger is water.

5. The nucleic acid scavenger of any one of claims 1 to 4, wherein, The trichloroisocyanuric acid salt is sodium trichloroisocyanurate and / or potassium trichloroisocyanurate.

6. Use of the nucleic acid scavenger according to any one of claims 1 to 5 for the preparation of a sterilizing agent.

7. A method of nucleic acid scavenging, comprising, Contacting the nucleic acid scavenger according to any one of claims 1 to 5 with an article to be scavenged and thereby removing nucleic acids.