A laboratory nucleic acid aerosol contamination scavenger and method of formulation thereof

Abstract

The application discloses a laboratory nucleic acid aerosol pollution removing agent, which comprises chemical removing agents and physical removing agents which are mutually cooperative and have multi-level removing effects; the chemical removing agents can crack structures and comprise hydrogen peroxide and guanidine hydrochloride, the hydrogen peroxide accounts for 2-10 wt% of the total mass of the removing agent, the guanidine hydrochloride accounts for 1-5 wt% of the total mass of the removing agent, the physical removing agent is magnesium chloride hexahydrate, the magnesium chloride hexahydrate accounts for 5-15 wt% of the total mass of the removing agent, and the remaining part is filled to 1000 ml by using deionized water, the nucleic acid pollution in equipment consumables and the environment can be effectively removed, false positives in experiments are avoided, the application is high in efficiency and convenient, and laboratory results are more accurate.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, specifically to a laboratory nucleic acid aerosol contaminant remover and its preparation method. Background Technology

[0002] Nucleic acid aerosol contamination is a serious problem in laboratory and biosafety fields. In molecular biology, biomedical research, clinical diagnostics, and epidemic monitoring, nucleic acid sample processing and analysis are routine laboratory procedures, which may generate nucleic acid aerosol contamination, posing significant safety hazards. Traditional methods for removing nucleic acid aerosol contamination mainly include high-temperature inactivation, ultraviolet irradiation, chemical disinfectants, and high-efficiency filtration. However, these methods have some limitations: 1. High-temperature inactivation and ultraviolet irradiation: These require high-temperature and ultraviolet equipment, are complex to operate, are not suitable for all laboratory environments, and may affect the integrity and usability of nucleic acid samples; 2. Chemical disinfectants: Commonly used disinfectants may harm laboratory equipment and personnel, and are also environmentally unfriendly; 3. High-efficiency filtration: Although high-efficiency filtration can remove nucleic acid aerosols, the filters need to be replaced regularly, are costly, and cannot handle already deposited nucleic acid contamination. Therefore, finding a simple, efficient, non-toxic, harmless nucleic acid remover that can improve experimental accuracy is urgently needed. Summary of the Invention

[0003] To solve the above-mentioned technical problems, the present invention provides a laboratory nucleic acid aerosol contaminant remover and its preparation method. The remover prepared by the present invention is non-toxic, harmless, time-saving, labor-saving, efficient and convenient.

[0004] The technical solution of the present invention is as follows:

[0005] This invention provides a laboratory nucleic acid aerosol contaminant remover, comprising a chemical remover and a physical remover that work synergistically and at multiple levels. The chemical remover is capable of breaking down structures and includes hydrogen peroxide and guanidine hydrochloride, wherein the hydrogen peroxide accounts for 2-10 wt% of the total mass of the remover, and the guanidine hydrochloride accounts for 1-5 wt% of the total mass of the remover. The physical remover is magnesium chloride hexahydrate, which accounts for 5-15 wt% of the total mass of the remover.

[0006] Preferably, it also includes surfactants, salts, alkalis, B vitamins, and ethanol.

[0007] Preferably, the surfactant is TrionX-100, accounting for 1-5 wt% of the total mass of the cleaning agent.

[0008] Preferably, the salt is a mixture of sodium chloride and sodium citrate, wherein the mass ratio of sodium chloride to sodium citrate is 1:2 to 1:20; and the salt accounts for 10%-25% of the total mass of the cleaning agent.

[0009] Preferably, the alkali is sodium hydroxide, accounting for 0.5-2 wt% of the total mass of the cleaning agent.

[0010] Preferably, the B vitamins account for 0.1-1 wt% of the total mass of the scavenger.

[0011] Preferably, the ethanol accounts for 5-20 wt% of the total mass of the scavenging agent.

[0012] Preferably, the pH value of the laboratory nucleic acid aerosol contaminant remover is between 7.0 and 8.5.

[0013] This invention also provides a method for preparing a laboratory nucleic acid aerosol contaminant remover, comprising the following steps:

[0014] (1) Add sodium citrate, magnesium chloride hexahydrate, hydrogen peroxide, guanidine hydrochloride, TrionX-100, B vitamins, sodium chloride, sodium hydroxide, and ethanol in sequence according to the concentration ratio and stir thoroughly.

[0015] (2) Use deionized water to bring the volume of the above mixture to 1000ml.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] 1. The laboratory nucleic acid aerosol contaminant remover provided by this invention exhibits excellent performance in laboratory nucleic acid-related operations. Through reasonable formulation and synergistic effect of multiple components, the remover can efficiently remove nucleic acid aerosol contamination, which not only ensures the accuracy of experimental results but also effectively reduces potential safety risks. The preparation method is simple and economical and suitable for various laboratory environments.

[0018] 2. The multi-component composition of the laboratory nucleic acid aerosol contaminant removal agent provided by this invention gives it wide applicability. It can be adjusted as needed to meet the removal requirements of different operations. The components contained in the removal agent are all common chemicals, which are relatively low in cost and environmentally friendly.

[0019] 3. The pH range of the laboratory nucleic acid aerosol contaminant removal agent provided by this invention is reasonably designed and is suitable for operation under various laboratory acid and alkaline conditions. The flexibility of the concentration allows the removal agent to be adjusted according to the degree of contamination and specific application conditions to ensure the best effect.

[0020] 4. The presence of hydrogen peroxide and guanidine hydrochloride in the laboratory nucleic acid aerosol contaminant provided by this invention enables it to degrade nucleic acid molecules. Hydrogen peroxide decomposes nucleic acid, and guanidine hydrochloride destroys the nucleic acid structure, thereby effectively removing nucleic acid aerosol contamination.

[0021] 5. The laboratory nucleic acid aerosol contaminant removal agent provided by the present invention contains magnesium chloride hexahydrate, which has the function of removing particulate matter. These particulate matter may carry nucleic acid aerosol contamination. By removing particulate matter through the removal agent, nucleic acid contamination is indirectly removed.

[0022] 6. The laboratory nucleic acid aerosol contaminant removal agent provided by this invention achieves multi-level and comprehensive removal of nucleic acids, including physical removal, chemical degradation and structural destruction. These mechanisms work together to ensure the efficient removal of nucleic acid aerosol contamination.

[0023] At the physical removal level, magnesium chloride hexahydrate removes particulate matter that may carry nucleic acid contamination, thus achieving physical removal of particulate matter.

[0024] At the level of chemical degradation, the presence of hydrogen peroxide and guanidine hydrochloride in the scavenger provides a chemical means for the degradation of nucleic acid molecules. Hydrogen peroxide can decompose nucleic acids into smaller fragments, while guanidine hydrochloride destroys the structure of nucleic acid molecules, causing them to lose their function.

[0025] At the level of structural damage, the combined action of hydrogen peroxide and guanidine hydrochloride disrupts the original structure of nucleic acid molecules, thereby causing them to lose their biological activity.

[0026] 7. The laboratory nucleic acid aerosol contaminant remover of the present invention has a shelf life of more than 12 months, which makes it easy for laboratories to maintain the storage and use of the remover, reducing resource waste and costs. Attached Figure Description

[0027] Figure 1 The graph shows the PCR experimental results of nucleic acid clearance ability for the positive control (without nucleic acid clearing agent), Example 1, Example 2 and Example 3;

[0028] Figure 2 The graph shows the qPCR experimental results of nucleic acid clearance ability for the positive control (without nucleic acid clearing agent), Example 1, Example 2 and Example 3;

[0029] Figure 3 The graph shows the results of the stability test after 1 month of storage for the positive control (without nucleic acid scavenging agent), Example 1, Example 2, and Example 3 (with scavenging agent).

[0030] Figure 4 The graph shows the results of the stability test after 6 months of storage for the positive control (without nucleic acid scavenging agent), Example 1, Example 2, and Example 3 (with scavenging agent).

[0031] Figure 5 The graph shows the results of the stability test after 12 months of storage for the positive control (without nucleic acid scavenging agent), Example 1, Example 2, and Example 3 (with scavenging agent). Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0033] The pH range of the laboratory nucleic acid aerosol contaminant remover in this invention is between 7.0 and 8.5, making it suitable for operation under various laboratory acid and alkaline conditions.

[0034] Example 1: The laboratory nucleic acid aerosol contaminant removal agent provided in this example includes the following components by mass fraction: 2% hydrogen peroxide, 1% guanidine hydrochloride, 5% magnesium chloride hexahydrate, with the remainder supplemented by deionized water;

[0035] Example 2: The laboratory nucleic acid aerosol contaminant removal agent provided in this example includes the following components by mass fraction: 6% hydrogen peroxide, 3% guanidine hydrochloride, 10% magnesium chloride hexahydrate, with the remainder supplemented by deionized water;

[0036] Example 3: The laboratory nucleic acid aerosol contaminant removal agent provided in this example includes the following components by mass fraction: 10% hydrogen peroxide, 5% guanidine hydrochloride, 15% magnesium chloride hexahydrate, with the remainder supplemented by deionized water;

[0037] Example 4: The laboratory nucleic acid aerosol contaminant removal agent provided in this example comprises the following components by mass fraction: 10 wt% sodium citrate, 5 wt% magnesium chloride hexahydrate, 2 wt% hydrogen peroxide, 1 wt% guanidine hydrochloride, 1 wt% TrionX-100, 0.1 wt% B vitamins, 1 wt% sodium chloride, 0.5 wt% sodium hydroxide, and 5 wt% ethanol, with the remainder supplemented by deionized water;

[0038] Example 5: The laboratory nucleic acid aerosol contaminant removal agent provided in this example comprises the following components by mass fraction: 20 wt% sodium citrate, 15 wt% magnesium chloride hexahydrate, 10 wt% hydrogen peroxide, 5 wt% guanidine hydrochloride, 5 wt% TrionX-100, 1 wt% B vitamins, 5 wt% sodium chloride, 2 wt% sodium hydroxide, and 20 wt% ethanol, with the remainder supplemented by deionized water;

[0039] Example 6: The laboratory nucleic acid aerosol contaminant removal agent provided in this example comprises the following components by mass fraction: 15 wt% sodium citrate, 10 wt% magnesium chloride hexahydrate, 6 wt% hydrogen peroxide, 3 wt% guanidine hydrochloride, 3 wt% TrionX-100, 0.5 wt% B vitamins, 3 wt% sodium chloride, 1 wt% sodium hydroxide, and 13 wt% ethanol, with the remainder supplemented by deionized water.

[0040] To verify the beneficial effects of Examples 1 to 3 above, the following experiments were conducted:

[0041] I. Nucleic Acid Clearance Capacity Experiment

[0042] 1. PCR experimental verification

[0043] Take eight-tube sets for the experiment and divide the experiment into four groups. Each group is repeated three times. The reaction system for the experimental group and the control group is as follows: add the following to the eight-tube sets: 0.3 μL Taq enzyme, 2.5 μL 10× buffer, 2 μL dNTP, 2.5 μL magnesium ions, 0.2 μL upstream primer, 0.2 μL downstream primer, 12.3 μL ultrapure water, and 5 μL template. Mix thoroughly. No nucleic acid scavenging agent is added to wells 1-3, nucleic acid scavenging agent is added to wells 4-6 (Example 1), nucleic acid scavenging agent is added to wells 7-9 (Example 2), and nucleic acid scavenging agent is added to wells 10-12 (Example 3). Centrifuge briefly.

[0044] 2. PCR amplification program: 35 cycles

[0045] 95℃ 5min 95℃ 30s 60℃ 1min 72℃ 20s 72℃ 15min

[0046] qPCR reaction system:

[0047] Take eight-tube sets for the experiment and divide the experiment into four groups. Each group is repeated three times. The reaction system for the experimental group and the control group is as follows: add the following to the eight-tube sets: 0.3 μL Taq enzyme, 2.5 μL 10× buffer, 2 μL dNTP, 2.5 μL magnesium ions, 0.2 μL upstream primer, 0.2 μL downstream primer, 0.1 μL probe, 12.2 μL ultrapure water, and 5 μL template, and mix thoroughly. No nucleic acid scavenging agent is added to wells 1-3, nucleic acid scavenging agent is added to wells 4-6 (Example 1), nucleic acid scavenging agent is added to wells 7-9 (Example 2), and nucleic acid scavenging agent is added to wells 10-12 (Example 3). Centrifuge briefly.

[0048] qPCR amplification program: 45 cycles

[0049] 95℃ 2.5min 95℃ 10s 60℃ 30s

[0050] 3. Experimental verification of nucleic acid clearance ability

[0051] After the PCR amplification experiment, the results were verified by agarose gel electrophoresis, and the results are as follows: Figure 1 As shown, Figure 1The wells numbered 1-12 at the top center are the well positions. Wells 1-3, which did not contain nucleic acid scavenging agent, showed bright bands, indicating that nucleic acid was not cleared and the gene could be expressed normally. In Example 1, wells 4-6, wells 7-9, and wells 10-12 were filled with nucleic acid scavenging agent; in Example 3, wells 4-12 were filled with nucleic acid scavenging agent. The results showed that no bands were observed in wells 4-12, indicating that nucleic acid was degraded and the gene could not be expressed normally. The experimental results prove that nucleic acid scavenging agent can effectively clear nucleic acid.

[0052] After the qPCR experiment, the amplification curves were analyzed, and the results were as follows: Figure 2 As shown, compared with the control group without nucleic acid scavenging agent, no amplification curves were detected in Examples 1, 2, and 3, which respectively added nucleic acid scavenging agent, indicating that nucleic acids were degraded and no gene expression was detected. The experimental results demonstrate that nucleic acid scavenging agent can effectively remove nucleic acids.

[0053] II. Stability Test

[0054] The nucleic acid scavenging agent was prepared according to requirements and subjected to a 12-month stability test (the same procedures as for PCR experiments). The results of the nucleic acid scavenging agent stability test are as follows: Figure 3 As shown, wells 1-3 are the control group, without the addition of nucleic acid scavenging agent. Obvious bands can be seen on agarose gel, indicating that the nucleic acid was not degraded and the gene could be expressed normally. Wells 4-6 are the experimental results of nucleic acid scavenging agent Example 1 after 1 month, wells 7-9 are the experimental results of nucleic acid scavenging agent Example 2 after 1 month, and wells 9-12 are the experimental results of nucleic acid scavenging agent Example 3 after 1 month. The results show that no bands were observed in wells 4-12, indicating that the nucleic acid was degraded and the gene could not be expressed normally. The experimental results prove that nucleic acid scavenging agents Example 1, Example 2, and Example 3 can effectively clear nucleic acid after one month of storage.

[0055] like Figure 4 As shown, wells 1 and 8 are markers, and wells 3, 4, and 6 are control groups without nucleic acid scavenging agent. Clear bands are visible on the agarose gel, indicating that nucleic acids were not degraded and genes were expressed normally. Well 2 shows the results of the nucleic acid scavenging agent in Example 1 after 6 months, well 5 shows the results of the nucleic acid scavenging agent in Example 2 after 6 months, and well 7 shows the results of the nucleic acid scavenging agent in Example 3 after 6 months. The results show that no bands were observed in wells 2, 5, and 7, indicating that nucleic acids were degraded and genes were not expressed normally. The experimental results prove that the nucleic acid scavenging agents in Examples 1, 2, and 3 can effectively clear nucleic acids after 6 months of storage.

[0056] like Figure 5As shown, wells 1 and 8 are markers, and wells 3, 4, and 6 are control groups without nucleic acid scavenging agent. Clear bands are visible on the agarose gel, indicating that nucleic acids were not degraded and genes were expressed normally. Well 2 shows the results of the nucleic acid scavenging agent in Example 1 after 12 months, well 5 shows the results of the nucleic acid scavenging agent in Example 2 after 12 months, and well 7 shows the results of the nucleic acid scavenging agent in Example 3 after 12 months. The results show that no bands were observed in wells 2, 5, and 7, indicating that nucleic acids were degraded and genes were not expressed normally. The experimental results prove that the nucleic acid scavenging agents in Examples 1, 2, and 3 can effectively remove nucleic acids after 12 months of storage. The experimental results show that the nucleic acid scavenging agent can still degrade nucleic acids after being stored at room temperature for twelve months.

Claims

1. A laboratory nucleic acid aerosol contaminant remover, characterized in that: The system includes chemical and physical scavengers that work synergistically and at multiple levels to remove pollutants. The chemical scavengers, capable of breaking down structures, include hydrogen peroxide and guanidine hydrochloride. The hydrogen peroxide accounts for 2-10 wt% of the total mass of the scavenger, and the guanidine hydrochloride accounts for 1-5 wt% of the total mass of the scavenger. The physical scavenger is magnesium chloride hexahydrate, which accounts for 5-15 wt% of the total mass of the scavenger. The remainder is replenished with deionized water. The pH value of the laboratory nucleic acid aerosol contaminant remover is between 7.0 and 8.

5.

2. The laboratory nucleic acid aerosol contaminant remover as described in claim 1, characterized in that: It also includes surfactants, salts, alkalis, B vitamins, and ethanol.

3. The laboratory nucleic acid aerosol contaminant remover as described in claim 2, characterized in that: The surfactant is Triton X-100, accounting for 1-5 wt% of the total mass of the cleaning agent.

4. The laboratory nucleic acid aerosol contaminant remover as described in claim 3, characterized in that: The salt is a mixture of sodium chloride and sodium citrate, wherein the mass ratio of sodium chloride to sodium citrate is 1:2 to 1:20; the salt accounts for 10%-25% of the total mass of the cleaning agent.

5. The laboratory nucleic acid aerosol contaminant remover as described in claim 4, characterized in that: The alkali is sodium hydroxide, accounting for 0.5-2 wt% of the total mass of the cleaning agent.

6. The laboratory nucleic acid aerosol contaminant remover according to claim 5, characterized in that: The B vitamins comprise 0.1-1 wt% of the total mass of the scavenger.

7. The laboratory nucleic acid aerosol contaminant remover according to claim 6, characterized in that: The ethanol accounts for 5-20 wt% of the total mass of the scavenging agent.

8. A method for preparing a laboratory nucleic acid aerosol contaminant remover according to claim 7, characterized in that: Includes the following steps: (1) Add sodium citrate, magnesium chloride hexahydrate, hydrogen peroxide, guanidine hydrochloride, Triton X-100, B vitamins, sodium chloride, sodium hydroxide, and ethanol in sequence according to the concentration ratio and stir thoroughly. (2) Use deionized water to bring the volume of the above mixture to 1000 mL.

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