CN115820343A - Nucleic acid scavenger, preparation method and application thereof - Google Patents
Nucleic acid scavenger, preparation method and application thereof Download PDFInfo
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- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 125
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- 238000002360 preparation method Methods 0.000 title claims abstract description 6
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- 235000019333 sodium laurylsulphate Nutrition 0.000 description 8
- 230000000052 comparative effect Effects 0.000 description 6
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- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 5
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- 239000000460 chlorine Substances 0.000 description 3
- 229910052801 chlorine Inorganic materials 0.000 description 3
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
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- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 2
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- -1 fatty alcohol ether sulfonates Chemical class 0.000 description 2
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- ONDPHDOFVYQSGI-UHFFFAOYSA-N zinc nitrate Chemical compound [Zn+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ONDPHDOFVYQSGI-UHFFFAOYSA-N 0.000 description 2
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- 241000711466 Murine hepatitis virus Species 0.000 description 1
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- XTVVROIMIGLXTD-UHFFFAOYSA-N copper(II) nitrate Chemical compound [Cu+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O XTVVROIMIGLXTD-UHFFFAOYSA-N 0.000 description 1
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- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of nucleic acid pollution removal, and discloses a nucleic acid remover, which aims to solve the problems that in the prior art, a nucleic acid remover can corrode metal and has a certain toxic action on a human body, and does not have a product for removing aerosol nucleic acid molecules, wherein the nucleic acid remover comprises a liquid A and a liquid B, and the liquid A comprises the following components in percentage by mass: 3% -7.5% of hydrogen peroxide, 5% -10% of absolute ethyl alcohol and the balance of water; the pH value of the solution B is 9-12, and the solution B comprises the following components: 10-100 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 2-10mmol/L metal ion salt, 0.001-0.002 mass percent of surfactant, alkali and water. The nucleic acid scavenger can remove nucleic acid in a wide spectrum, has high scavenging efficiency, does not corrode metal, is harmless to human body, can be used for removing nucleic acid by a suspension method, and can also be used for removing nucleic acid in aerosol. The invention also provides a preparation method of the nucleic acid scavenger.
Description
Technical Field
The invention relates to the technical field of nucleic acid pollution removal, and particularly relates to a nucleic acid remover, and a preparation method and application thereof.
Background
Nucleic acids are a generic term for biopolymers deoxyribonucleic and ribonucleic acids, and are present in all life forms known to date. The specific nucleic acid sequences of different organisms are different, and the detection of the specific nucleic acid sequences by Polymerase Chain Reaction (PCR) is widely applied to the fields of in vitro diagnosis, animal and plant commodity quarantine, forensic identification and the like. However, the PCR operation process is susceptible to false positive results caused by nucleic acid contamination, and the detection accuracy is seriously influenced. Therefore, it is necessary to develop a scheme for removing nucleic acid contamination to ensure the reliability of the detection result. The traditional method for removing the nucleic acid pollution of the environment and equipment mainly adopts alcohol or 84 disinfectant as a nucleic acid remover for wiping, or adopts an ultraviolet lamp for irradiation disinfection. However, the capability of removing nucleic acid pollution by alcohol and the 84 disinfectant is relatively weak, and the 84 disinfectant belongs to a chlorine-containing disinfectant, has certain toxicity to people and animals, and has strong corrosion and decoloration effects on skin, metal and colored textiles. The ultraviolet irradiation is time-consuming and labor-consuming, the action principle of the ultraviolet irradiation is that the pyrimidine base in the PCR product forms a dimer, so that the extension is terminated, and the scheme is only effective on long fragments of more than 500bp and has little effect on short fragments.
Nucleic acid scavengers available on the market for degrading short-chain nucleic acid molecules are enzyme-degradable nucleic acid scavengers, such as "a nucleic acid scavenger based on a virus blocking agent and its use" published under the publication No. CN115181616a in chinese patent literature, which comprises the following components: viral blocking proteins, nucleases; optionally a stabilizer; optionally a solvent; the recombinant fusion protein is a virus blocker and comprises a structural domain specifically combined with coronavirus S-spinous-process protein. Such nucleic acid scavengers may cause protein contamination while degrading nucleic acids with nucleases, and there are some non-spray type nucleic acid scavengers which degrade nucleic acid molecules by a suspension method, and the elimination effect of aerosol nucleic acid molecules is not detailed. For the accuracy of nucleic acid detection and the safety of laboratory personnel, it is necessary to invent a harmless, non-corrosive and highly efficient broad-spectrum nucleic acid scavenger.
Disclosure of Invention
The invention provides a nucleic acid scavenger for overcoming the problems that the nucleic acid scavenger in the prior art can corrode metal and has certain toxic effect on human body, and does not have a product for eliminating aerosol nucleic acid molecules. The nucleic acid scavenger can be used for removing nucleic acid molecules by suspension method and aerosol method. The peroxide content in the components is not high, and the components do not contain chlorine-containing compounds, so that the metal surface is not corroded, and the composition has no toxic or harmful effect on human bodies; the nucleic acid molecules are subjected to nonspecific destruction, and the broad spectrum property is realized. The invention also provides a preparation method of the nucleic acid scavenger and application of the nucleic acid scavenger.
In order to achieve the purpose, the invention adopts the following technical scheme:
a nucleic acid scavenger comprises solution A and solution B,
the liquid A comprises the following components in percentage by mass: 3% -7.5% of hydrogen peroxide, 5% -10% of absolute ethyl alcohol and the balance of water; the pH value of the solution B is 9-12, and the solution B comprises the following components: 10-100 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 2-10mmol/L metal ion salt, 0.001-0.002 mass percent of surfactant, alkali and water.
The hydrogen peroxide and the absolute ethyl alcohol in the solution A are used as inactivators in the nucleic acid scavenger, the hydrogen peroxide generates hydroxyl radicals to inactivate nucleic acid molecules, and the ethyl alcohol can break nucleic acid chains. The metal ions provided by the metal ion salt in the solution B can catalyze hydrogen peroxide to generate hydroxyl radicals, the 4-hydroxyethyl piperazine ethanesulfonic acid plays an auxiliary role in the action process of the hydrogen peroxide and the metal ions and can further inactivate nucleic acid molecules, and the action of the metal ions and the hydrogen peroxide in the solution A enables the concentration of the hydrogen peroxide in the solution A to have a good nucleic acid removing effect under the condition of low concentration, so that the toxic effect on a human body caused by the use of the hydrogen peroxide with high concentration is avoided. The 4-hydroxyethyl piperazine ethanesulfonic acid has high solubility in alkaline solution and good auxiliary effect on hydrogen peroxide and metal ions, so that the pH of the solution B also needs to be adjusted by using alkali, and the solution B is still alkaline overall after being mixed with the solution A under the pH and does not corrode metals. The surfactant can increase the wetting effect of the nucleic acid scavenger on the nucleic acid to be scavenged in the contaminated material, thereby increasing the rate of decomposition of the nucleic acid by the nucleic acid scavenger.
Preferably, the metal ion salt is one or more of water-soluble copper salt, water-soluble zinc salt and water-soluble ferrous salt.
Cu 2+ 、Zn 2+ And Fe 2+ Can effectively catalyze the decomposition of the hydrogen peroxide.
Preferably, the metal ion salt is one or more of copper sulfate, copper nitrate, zinc sulfate, zinc nitrate and ferrous sulfate.
Preferably, the alkali is one or more of sodium hydroxide and potassium hydroxide.
Preferably, the surfactant is an alkali resistant surfactant.
Alkali-resistant surfactants such as sodium lauryl sulfate, fatty alcohol ether sulfonates and fatty alcohol ether carboxylates can function well in improving the permeation and wetting effects of the nucleic acid scavenger in alkaline systems.
More preferably, the surfactant is sodium lauryl sulfate.
A method of preparing a nucleic acid scavenger comprising the steps of:
(1) Mixing hydrogen peroxide and absolute ethyl alcohol, adding water, and uniformly mixing to obtain a solution A;
(2) Dissolving 4-hydroxyethyl piperazine ethanesulfonic acid in water, adding alkali to adjust the pH value to 9-12, sequentially adding metal ion salt and surfactant, and uniformly mixing to obtain solution B.
Preferably, in the step (2), 4-hydroxyethyl piperazine ethanesulfonic acid is dissolved in water, an alkali-containing aqueous solution is added to adjust the pH to 9-12, and the alkali concentration in the alkali-containing aqueous solution is 0.1-1mol/L.
The application of the nucleic acid scavenger in removing nucleic acid pollution by a suspension method comprises the following steps: mixing the nucleic acid scavenger A solution and B solution in the ratio of (1-1.5): 1, immersing the contaminated equipment into the mixed solution for 10-20min, and cleaning the surface of the equipment to remove the nucleic acid scavenger.
The invention can be used for removing nucleic acid pollution by a suspension method, the mixed solution of the solution A and the solution B of the nucleic acid remover can destroy the polluted nucleic acid on the equipment soaked in the mixed solution, the speed of removing the polluted nucleic acid is high, and the required soaking time is short. After soaking, the surface of the device is left with the deactivator which is not reacted with the nucleic acid, and the device needs to be cleaned to remove part of the deactivator, so as to avoid the damage of the deactivator to the nucleic acid sample to be detected when the device is used for detecting the nucleic acid again.
Preferably, the step includes washing the surfaces of the device with a neutralizing agent comprising sodium thiosulfate.
Sodium thiosulfate can react with residual hydrogen peroxide, deactivating the nucleic acid scavenger.
More preferably, the neutralizing agent is a tryptone physiological saline solution containing the following components in mass fraction: 1.5-2% of sodium thiosulfate, 0.4-0.5% of lecithin and 0.4-0.5% of tween-80.
The application of the nucleic acid scavenger in scavenging the nucleic acid pollution of the aerosol comprises the following steps: spraying the liquid B of the nucleic acid scavenger in the air, spraying the liquid A of the nucleic acid scavenger in the air after the liquid B is sprayed, standing the liquid A after the liquid A is sprayed, and standing the liquid for more than 1h.
The invention can also be used for removing nucleic acid in aerosol, and when in use, the solution B and the solution A are sprayed in sequence, so that the removing effect is good.
Preferably, the spraying amount of the B liquid is (8-15) mL/m based on the air volume in the step 3 The spraying amount of the solution A is (8-15) mL/m 3 。
Preferably, after the liquid A is sprayed and stands still, the liquid B and the liquid A are sprayed for 2 to 3 times.
If the pollution of the nucleic acid in the aerosol is serious, the aerosol can be repeatedly sprayed for 2-3 times to improve the clearing effect.
Therefore, the invention has the following beneficial effects: (1) Can carry out nonspecific destruction on nucleic acid molecules, has broad spectrum and high removal efficiency; (2) The peroxide content in the nucleic acid scavenger is not high, and the nucleic acid scavenger does not contain chlorine-containing compounds, does not corrode metal surfaces to cause damage to instruments, and has no toxic or harmful effect on human bodies; (3) The nucleic acid scavenger can be used for removing nucleic acid by suspension method or removing nucleic acid in aerosol.
Detailed Description
The invention is further described below with reference to specific embodiments.
Example 1
A nucleic acid scavenger prepared by the steps of:
(1) Adding hydrogen peroxide and absolute ethyl alcohol into a beaker, mixing, adding purified water for dilution, and uniformly stirring to obtain a solution A, wherein the mass fraction of the hydrogen peroxide in the solution A is 3%, and the mass fraction of the absolute ethyl alcohol is 5%;
(2) Adding 4-hydroxyethyl piperazine ethanesulfonic acid and purified water into a beaker, adding 0.2mol/L sodium hydroxide solution to adjust the pH value to 10, sequentially adding copper sulfate pentahydrate and sodium dodecyl sulfate, and uniformly mixing to obtain a solution B, wherein the concentration of the 4-hydroxyethyl piperazine ethanesulfonic acid in the solution B is 50mmol/L, the concentration of the copper sulfate is 2mmol/L, and the mass fraction of the sodium dodecyl sulfate is 0.001%.
Example 2
A nucleic acid scavenger is prepared by steps different from those of example 1 in that the mass fraction of hydrogen peroxide in solution A is 6%, the mass fraction of absolute ethyl alcohol is 10%, the concentration of 4-hydroxyethylpiperazine ethanesulfonic acid in solution B is 30mmol/L, the concentration of copper sulfate is 4mmol/L, and the mass fraction of sodium lauryl sulfate is 0.0015%.
Example 3
A nucleic acid scavenger prepared by a procedure different from that of example 1 in that ferrous sulfate heptahydrate is used in place of copper sulfate pentahydrate in solution B.
Comparative example 1
A nucleic acid scavenger prepared by a procedure different from that of example 2 except that no sodium hydroxide solution was added in the procedure (2).
The solution B obtained in the comparative example 1 contains white floccules, which shows that 4-hydroxyethyl piperazine ethanesulfonic acid cannot be dissolved in the solution B under the condition of not adjusting the pH of the solution B, so that the solution B cannot be fully mixed with metal ions and hydrogen peroxide in use, and the technical effect of assisting the metal ions to catalyze the hydrogen peroxide to generate free radicals is further realized.
Comparative example 2
A nucleic acid scavenger is prepared by the steps different from those of example 2 in that in step (2), purified water is added into a beaker, 0.1mol/L sodium hydroxide solution is added to adjust the pH value to 10, copper sulfate pentahydrate and sodium dodecyl sulfate are sequentially added, and the mixture is uniformly mixed to obtain a solution B, wherein the concentration of copper sulfate in the solution B is 4mmol/L, and the mass fraction of the sodium dodecyl sulfate is 0.0015%.
Example 4
The nucleic acid scavenger is applied to the removal of nucleic acid pollution by a suspension method, and comprises the following steps:
(1) Preparing tryptone physiological saline solution (TPS) containing 2 percent of sodium thiosulfate, 0.5 percent of lecithin and 0.5 percent of Tween-80 as a neutralizer;
(2) The solution a and the solution B obtained in example 1 were mixed at a ratio of 1:1 to obtain a mixed solution;
(3) Taking 5 groups of reaction tubes as a group (1), a group (2), a group (3), a positive control group and a negative control group, wherein 1mL of nucleic acid quality control substance, 2mL of nucleic acid scavenger and 2mL of deionized water are added into the reaction tube of the group (1), 1mL of nucleic acid quality control substance, 2mL of nucleic acid scavenger and 2mL of neutralizer are added into the reaction tube of the group (2), 1mL of nucleic acid quality control substance, 2mL of deionized water and 2mL of neutralizer are added into the reaction tube of the group (3), 1mL of nucleic acid quality control substance and 4mL of deionized water are added into the reaction tube of the positive control group, and 5mL of deionized water is added into the reaction tube of the negative control group;
(4) The components in each group of reaction tubes are mixed and reacted for 10min, then a sample is taken for carrying out fluorescence quantitative PCR detection, the fluorescence quantitative PCR amplification system is shown in table 1, and the instrument setting parameters are shown in table 2.
TABLE 1 fluorescent quantitative PCR amplification System
TABLE 2 fluorescent quantitative PCR amplification reaction conditions
The results are shown in Table 3.
TABLE 3 test results of example 1
As can be seen from Table 3, the present invention can rapidly and efficiently remove nucleic acids. (3) The Ct value of the group is similar to that of the positive control, which shows that the neutralizing agent does not affect the detection of nucleic acid, while the Ct value of the group (2) is increased by 8-11 compared with the positive control, but is lower than that of the group (1), which shows that the neutralizing agent has the effect of neutralizing the strong oxidant in the system.
And respectively mixing the solution A and the solution B obtained in the examples 2-3 and the comparative example 2 in a ratio of 1:1, mixing 0.9mL of the mixed nucleic acid remover with 0.1mL of the nucleic acid quality control product for 20min, sampling, and carrying out fluorescent quantitative PCR detection, wherein a fluorescent quantitative PCR amplification system is shown in Table 1, instrument setting parameters are shown in Table 2, and detection results are shown in Table 4.
TABLE 4 test results of examples 2-3 and comparative examples 1-2
As is apparent from Table 4, the nucleic acid scavengers obtained in examples 2 to 3 were effective in decomposing nucleic acids when mixed with the nucleic acids for 20min, and the scavenging effect of comparative examples 1 and 2 was weaker than that of example 2, indicating that 4-hydroxyethylpiperazine ethanesulfonic acid can assist in the action of hydrogen peroxide and metal ions.
Example 5
A nucleic acid scavenger is prepared by steps different from those of example 1 in that the mass fraction of hydrogen peroxide in solution A is 7.5%, the mass fraction of absolute ethyl alcohol is 10%, the concentration of 4-hydroxyethylpiperazine ethanesulfonic acid in solution B is 100mmol/L, the concentration of copper sulfate is 4mmol/L, and the mass fraction of sodium lauryl sulfate is 0.001%.
Example 6
The nucleic acid scavenger is applied to scavenging nucleic acid pollution of aerosol, and comprises the following steps:
(1) The mouse hepatitis virus nucleic acid is put into a fluorescent quantitative PCR instrument for amplification;
(2) Adding 50uL of amplified nucleic acid product into 50mL of deionized water, putting into a spray can, uniformly spraying in a laboratory with the height of 2.72 m, the length of 7.7 m and the width of 6.08 m, and closing doors and windows;
(3) Randomly taking 10 points by using a disposable sampling cotton swab to collect samples (A01-A05 is an object table, A06-A10 is a culture dish for natural sedimentation), adding each sample cotton swab into 1mL of deionized water, soaking for 60s, taking out the cotton swab as a template, and detecting according to a fluorescent quantitative PCR amplification system;
(4) Spraying the solution B of the nucleic acid scavenger prepared in example 5 into a laboratory in the form of 10 mL/m3, and after the solution B is sprayed, fully spraying the solution A of the nucleic acid scavenger into the laboratory in the form of 10 mL/m 3;
(5) And (4) standing for 1h and 2h after the liquid A is sprayed, sampling and detecting by using a disposable sampling cotton swab in the sampling mode in the step (3).
The fluorescent quantitative PCR amplification system is shown in Table 5, the fluorescent quantitative PCR instrument amplification reaction conditions are shown in Table 6, and the detection results are shown in Table 7.
TABLE 5 fluorescent quantitative PCR amplification System
TABLE 6 fluorescent quantitative PCR amplification reaction conditions
TABLE 7 test results
As can be seen from Table 7, the Ct value of the aerosol after 1 hour of treatment with the nucleic acid scavenger was increased by 6 or more compared with the Ct value before scavenging, which indicates that the amount of nucleic acid in the original aerosol was 104 times the amount of nucleic acid in the aerosol after degradation by the nucleic acid scavenger prepared in example 5, and the nucleic acid scavenger prepared in example 5 had the effect of scavenging nucleic acid in the aerosol.
Claims (10)
1. A nucleic acid scavenger is characterized by comprising a solution A and a solution B,
the liquid A comprises the following components in percentage by mass: 3% -7.5% of hydrogen peroxide, 5% -10% of absolute ethyl alcohol and the balance of water;
the pH value of the solution B is 9-12, and the solution B comprises the following components: 10-100 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 2-10mmol/L metal ion salt, 0.001-0.002 mass percent of surfactant, alkali and water.
2. The nucleic acid scavenger according to claim 1, wherein the metal ion salt is one or more of a water-soluble copper salt, a water-soluble zinc salt and a water-soluble ferrous salt.
3. The nucleic acid scavenger according to claim 1, wherein the base is one or more of sodium hydroxide and potassium hydroxide.
4. A nucleic acid scavenger according to claim 1 wherein the surfactant is an alkali resistant surfactant.
5. A process for the preparation of a nucleic acid scavenger according to any one of claims 1 to 4, comprising the steps of:
(1) Mixing hydrogen peroxide and absolute ethyl alcohol, adding water, and uniformly mixing to obtain a solution A;
(2) Dissolving 4-hydroxyethyl piperazine ethanesulfonic acid in water, adding alkali to adjust the pH value to 9-12, sequentially adding metal ion salt and surfactant, and uniformly mixing to obtain solution B.
6. The method for producing a nucleic acid scavenger according to claim 5, wherein the step (2) is a step of dissolving 4-hydroxyethylpiperazine ethanesulfonic acid in water, and adding an alkali-containing aqueous solution to adjust the pH to 9 to 12, wherein the alkali concentration in the alkali-containing aqueous solution is 0.1 to 1mol/L.
7. Use of the nucleic acid scavenger according to any one of claims 1 to 4 for the suspension decontamination of nucleic acids, characterized in that it comprises the steps of: mixing the nucleic acid scavenger A solution and B solution in the ratio of (1-1.5): 1, immersing the contaminated equipment into the mixed solution for 10-20min, and cleaning the surface of the equipment to remove the nucleic acid scavenger.
8. The use of a nucleic acid scavenger according to claim 7 in a suspension method for the removal of nucleic acid contamination, wherein the steps comprise washing the surfaces of the material with a neutralising agent comprising sodium thiosulphate.
9. Use of the nucleic acid scavenger according to any one of claims 1 to 4 for the aerosol decontamination of nucleic acids, comprising the steps of: spraying the liquid B of the nucleic acid scavenger in the air, spraying the liquid A of the nucleic acid scavenger in the air after the liquid B is sprayed, standing for more than 1h after the liquid A is sprayed.
10. The use of the nucleic acid scavenger for scavenging nucleic acid contamination of aerosol according to claim 9, wherein the amount of the B solution sprayed in the step is (8-15) mL/m in terms of air volume 3 The spraying amount of the solution A is (8-15) mL/m 3 。
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