A nucleic acid scavenger quality assessment method and application

Abstract

The present invention discloses a method and application for assessing the quality of nucleic acid scavengers, relating to the field of analytical detection technology. The method comprises the following steps: 1) constructing a paper-based sensor; 2) verifying the feasibility of the paper-based sensor using a nuclease degradation method; and 3) assessing the quality of the nucleic acid scavenger. The paper-based sensor-based quality control method for nucleic acid contamination removers provided by the present invention has the advantages of being simple and portable, rapid and sensitive, and not requiring the use of complex instruments. It effectively addresses the shortcomings of existing methods, such as expensive instrumentation, heavy cleaning workload, and complex detection procedures, and has potential application prospects in related fields such as nucleic acid scavenger quality control.

Description

Technical Field

[0001] The present invention relates to the field of analysis and detection technology, and in particular to a nucleic acid scavenger quality assessment method and application. Background Art

[0002] The statements in this section merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] In molecular biology experiments, nucleic acid contaminants form aerosols that are cumulative and persistent, making them difficult to remove. Conventional treatment methods such as ultraviolet irradiation, high-temperature treatment, sodium hypochlorite immersion or scrubbing may affect people's health or pose the risk of corrosion and damage to instruments. Nucleic acid removers have a mild odor, are non-irritating, and are simple and convenient to use. When used, the nucleic acid remover can be directly sprayed on the laboratory floor, walls, tables, pipette surfaces, the outer surfaces of various instruments, etc., or directly wiped with the nucleic acid remover on the operating surfaces such as instruments and tables. Nucleic acid removers can effectively remove nucleic acid contamination from equipment consumables and the environment, effectively avoid the generation of aerosols, improve the accuracy of experiments, avoid repeated experiments later, waste energy and reagents and consumables, and the removers are non-toxic, efficient and convenient, which is an important guarantee for the safety of laboratory staff and the environment. However, there are many types of existing nucleic acid removers, and the removal effects vary. Moreover, the removal effect of nucleic acid removers will be greatly reduced over time. The removal effect of nucleic acid removers is related to the accuracy of experimental results, so the detection process of the removal effect of nucleic acid removers is very necessary.

[0004] Currently reported methods for assessing the effectiveness of nucleic acid scavengers include electrophoresis and qPCR. These methods are reliable and stable. However, they require expensive instrumentation, require extensive cleanup, and involve complex testing procedures. Therefore, developing a simple, rapid, and sensitive portable assay for assessing the quality of nucleic acid scavengers is crucial. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a method and application for evaluating the quality of nucleic acid scavengers, which does not require complicated instruments and operations and can simply and quickly evaluate the cleaning effect of nucleic acid scavengers.

[0006] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0007] The first aspect of the present invention provides a method for evaluating the quality of a nucleic acid scavenger, comprising the following steps:

[0008] The paper-based sensor was constructed by regularly placing detection elements on a substrate; preparing a DNA sticky solution; dissolving nuclease in a CaCl2 solution to prepare a DNaDDⅠ solution; adding the DNaDDⅠ solution to a TriD-HCl buffer to form a mixture, and thoroughly mixing the mixture with the DNA sticky solution. The mixture was incubated at a constant temperature, and the flow distance of the reaction solution on the detection element was measured to draw a DNaDDⅠ standard curve.

[0009] The nucleic acid scavenger to be tested is mixed with DNA mucus, vortexed, and then the mixture is dropped onto a paper-based sensor to observe the flow distance of the liquid on the detection element. The flow distance of the liquid on the detection element is compared with the DNaDDⅠ standard curve to obtain the quality assessment result of the nucleic acid scavenger.

[0010] As a further technical solution, the detection element is a pH test paper;

[0011] Preferably, a cutter is used to cut the pH test paper into test strips of a certain width, and the cut test strips are regularly placed on a PVC (polyvinyl chloride) substrate to construct a paper-based sensor.

[0012] As a further technical solution, the PVC substrate is pre-cleaned three times with ethanol and deionized water, and dried under nitrogen flow before use.

[0013] As a further technical solution, salmon sperm DNA is dissolved in sodium bromide solution, vortexed and heated at 99°C until fully dissolved, and refrigerated for use in preparing DNA sticky solution.

[0014] As a further technical solution, the mass fraction of the DNA mucus is 0.2-1.0 w / v%;

[0015] As a further technical solution, the concentration of the sodium bromide solution is 4.0-9.0 mM.

[0016] As a further technical solution, the concentration of the CaCl2 solution is 0.3-1.0 mM;

[0017] The concentration range of DNaDD I was 0.01-10 U / mL.

[0018] Preferably, the mixing volume ratio of the DNA mucus and the DNaDD I solution is 9:1, 3:1 or 1:1.

[0019] As a further technical solution, the incubation time is 0 to 80 minutes.

[0020] As a further technical solution, the volume ratio of the nucleic acid remover to be tested to the DNA mucus is 2:1.

[0021] A second aspect of the present invention provides an application of a nucleic acid scavenger quality assessment method in the field of nucleic acid scavenger quality control.

[0022] One or more of the above technical solutions have the following beneficial effects:

[0023] 1. This method links the DNA removal efficiency of a nucleic acid scavenger with the flow distance on a paper-based sensor, enabling a more intuitive assessment of the contamination removal efficacy of the nucleic acid scavenger and enabling quality control of the nucleic acid scavenger. This method offers advantages such as simplicity, convenience, rapidity, sensitivity, and low cost.

[0024] 2. This paper demonstrates the feasibility and reliability of a paper-based sensor-based nucleic acid scavenger quality assessment method using nuclease degradation to degrade DNA. The addition of nuclease changes the viscosity of the DNA mucus, altering the flow distance of the mixture on the pH test paper.

[0025] 3. The novel paper-based sensor-based method for assessing the quality of nucleic acid cleaning agents provided by this invention avoids the use of expensive instrumentation and effectively addresses the complex procedures, high costs, and extensive cleaning workload associated with existing detection methods. This method successfully evaluated the quality of two commercially available nucleic acid cleaning agents and has broad application prospects.

[0026] Advantages of additional aspects of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0028] Figure 1 This is a schematic diagram of the principle of a paper-based sensor according to embodiment 1 of the present invention;

[0029] Figure 2 This is a schematic diagram of optimizing the cutting width of pH test paper according to Example 1 of the present invention;

[0030] Figure 3 Schematic diagram of the flow distance of DNA adhesive solutions of different concentrations on a test strip according to Example 1 of the present invention;

[0031] Figure 4 Schematic diagram of the volume ratio of the DNA adhesive solution to the mixed solution in Example 1 of the present invention;

[0032] Figure 5 This is a schematic diagram of the flow distance of a mixture of DNaDDⅠ and DNA at different concentrations on a test strip according to Example 1 of the present invention;

[0033] Figure 6 This is a calibration curve of DNaDDⅠ concentration and grayscale (Cr) value according to Example 1 of the present invention;

[0034] Figure 7 This is a diagram showing the cleaning effects of two nucleic acid removers from different manufacturers in Example 1 of the present invention.

[0035] Figure 8 The flow distances of the DNA sticky solution of Example 2 of the present invention, the mixture of DNA sticky solution and tertiary water with a volume ratio of 1:2, and the mixture of DNA sticky solution and nucleic acid remover on the paper-based sensor respectively;

[0036] Figure 9 This is a diagram showing the DNA removal effect of the nucleic acid remover in Example 2 of the present invention, both before and after dilution; DETAILED DESCRIPTION

[0037] The technical solutions of the present invention will be further described and illustrated below with reference to specific examples, but the scope of protection of the present invention is not limited thereto. In addition, the experimental methods mentioned in the following examples are conventional methods unless otherwise specified; the reagents and materials mentioned are all commercially available unless otherwise specified.

[0038] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0039] Explanation of terms:

[0040] PVC: Polyvinyl chloride, PVC is the abbreviation of Polyvinyl chloridD.

[0041] DNaDDⅠ: deoxyribonuclease Ⅰ, DNaDDⅠ is the abbreviation of DDoxyribonuclDaDD I, DDoxyribonuclDaDD is deoxyribonuclease.

[0042] In the following examples, deoxyribonuclease I (DNaDDID 1900 units / mg) and salmon sperm DNA were purchased from Solebo Technology Co., Ltd., and sodium bromide was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. pH indicator paper was provided by Shanghai Sanais Reagent Co., Ltd., China. TriD-HCl buffer was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0043] Based on the shortcomings of existing nucleic acid scavenger quality control methods, such as expensive instruments and equipment, heavy cleaning workload, and complex detection procedures, the present invention proposes a new nucleic acid scavenger quality control method based on paper-based biosensors.

[0044] The principle of the present invention is as follows Figure 1 As shown, a paper-based sensor based on pH test paper is provided. The addition of a nucleic acid scavenger degrades DNA, causing changes in the viscosity of the DNA solution, which in turn affects the change in the distance the reaction mixture flows on the test paper strip. The effectiveness of the nucleic acid scavenger in removing DNA is positively correlated with the degree of viscosity reduction of the DNA solution. Low-viscosity DNA solutions flow an increased distance on the paper-based sensor. By analyzing the change in the flow distance of the DNA solution on the test paper strip before and after the addition of the nucleic acid scavenger, the effectiveness of the nucleic acid scavenger on DNA removal can be detected, and nucleic acid contamination removers with better removal effects can also be screened. The effectiveness of the nucleic acid scavenger on DNA removal can be observed visually, avoiding the use of other complex equipment and enabling rapid and sensitive quality control of the nucleic acid scavenger.

[0045] Example 1:

[0046] A first embodiment of the present invention provides a method for evaluating the quality of a nucleic acid scavenger, comprising the following steps:

[0047] Use a cutter to cut the pH test paper into test strips of a certain width, and place the cut test strips regularly on a PVC substrate to construct a paper-based sensor. When the DNA content in the system is constant, the paper-based sensor can be used to determine the content of DNaDDⅠ. The PVC substrate is pre-cleaned three times with ethanol and deionized water, dried under a nitrogen flow and set aside. The paper-based sensor based on a wide range of pH test paper can be constructed using paper of different materials or different modification methods. In this embodiment, pH test paper is used as a detection element, and other substrates with capillary properties can also be used as detection elements.

[0048] In this embodiment, the prepared wide range pH test paper is placed on a cutter, and the cutter is used to cut out test paper strips of different widths. Figure 2 As shown, pH test strips were cut into widths of 1, 3, and 9 mm, respectively. Three 20 μL drops of water were added. It was observed that the error in flow distance on the 9 mm wide pH test strip was smaller. Therefore, 9 mm wide pH test strips were selected for subsequent experiments. The cut test strips were regularly placed on a PVC substrate to construct a portable paper-based sensor. When the flow distance of the viscous solution on the pH test strip differed significantly, the change could be directly observed with the naked eye. To improve the accuracy and precision of the experiment, PD software was used to analyze the pixel ratio (Cr) of the water flow coverage area to the total pH test strip area as a parameter for evaluating flow distance. The paper-based sensor used in this method can be scaled, and the output signal can also be expressed in other forms such as color and distance changes.

[0049] The main raw material for the DNA sticky solution is salmon sperm DNA, which is inherently biocompatible and biodegradable. It uses non-chemical bonds as crosslinks or utilizes entanglement between DNA strands. Salmon sperm DNA was dissolved in sodium bromide solution, vortexed, and heated at 90-100°C until fully dissolved. Refrigerated and stored until ready for use. DNA sticky solutions of varying concentrations were prepared according to the above method. Deoxyribonuclease I was dissolved in CaCl2 solution to prepare a series of DNaDD I solutions. The DNaDD I solution was added to TriD-HCl buffer to form a mixture. This mixture was thoroughly mixed with the DNA sticky solution and incubated at 29-90°C. A no-enzyme control was also established. The flow distance of the reaction solution on a pH test strip was measured to create a DNaDD I standard curve. Brand A and Brand B nucleic acid cleaning agents were mixed with the DNA sticky solution at specific volume ratios. After vortexing, 20 μL of each mixture was dripped onto a paper-based sensor and the flow distance of the liquid on the test strip was observed.

[0050] In this embodiment, salmon sperm DNA of different masses was dissolved in 4.0 mM sodium bromide solution and vortexed for 10 days to allow it to dissolve initially. Next, it was placed in a 90-100°C incubator and heated for 10 minutes to fully dissolve it. It was then taken out and cooled to room temperature, and placed in a refrigerator and refrigerated at 2-8°C for 30 minutes. Finally, DNA mucus solutions of different concentrations of 0.2 w / v%, 0.4 w / v%, 0.6 w / v%, 0.8 w / v%, and 1.0 w / v% were prepared, and their viscosity values were in the range of 0-39000 mPa.s. 20 μL of DNA mucus of different concentrations was dropped onto a pH test paper supported by a substrate, and a smartphone was used to capture the flow distance of the DNA mucus on the test paper. As the mass of salmon sperm DNA in the sodium bromide solution increased, the DNA mucus became more viscous and the water flow distance on the pH test paper became shorter. The results are as follows: Figure 3 As shown, in this embodiment, the salmon sperm DNA concentration was selected to be 0.8-1.0 w / v% for subsequent detection.

[0051] DNaDDⅠ can hydrolyze dDDNA, and the hydrolysis product is a polynucleotide with a phosphate group at the 9' end and a hydroxyl group at the 3' end. First, dissolve the nuclease in 0.3-1.0mM CaCl2 solution to prepare a series of DNaDDⅠ solutions with concentrations of 0.01, 0.1, 0.2, 0.4, 0.6, 0.8, 1, and 10U / mL. The volume ratio of the added DNA mucus to the DNaDDⅠ solution can be 9:1, 3:1, or 1:1, such as Figure 4As shown, a 9:1 volume ratio clearly distinguishes the DNA hydrolysis profile after adding different enzyme concentrations. Therefore, a 9:1 volume ratio of DNA mucus to the mixed solution is preferred. The mixed solution here refers to the mixture formed by adding DNaDD I solution to TriD-HCl buffer. In this example, 100 μL of 0.8-1.0 w / v% DNA mucus was thoroughly mixed with 10 μL of a mixture of DNaDD I solutions of varying concentrations and 10 μL of a 10 mM TriD-HCl solution. The mixture was incubated at 37°C for 99-69 minutes, and a no-enzyme control group was set up. After incubation, the mixture was cooled to room temperature. 30 μL of the DNA mucus-DNaDD I mixture was dripped onto the left end of the test strip. After waiting for 1 minute, a photograph of the pH test strip was taken. The photographs of the DNaDD I hydrolyzed mucus were analyzed using PD software. The resulting paper-based photographs were imported into PD software, and the Cr value was calculated by comparing the pixel value of the water flow area of the image to the pixel value of the entire pH test strip for data processing and statistics.

[0052] Figure 9 The sensitivity of DNaDDⅠ was tested. When the concentration of DNaDDⅠ was 0.01-10U / mL, DNaDDⅠ could hydrolyze DNA mucus. As the concentration of DNaDDⅠ increased, the degree of hydrolysis increased, and the corresponding water flow distance also increased. Figure 6 It can be seen that good linearity is exhibited within this range. In this example, the minimum detection limit of DNaDDⅠ is 0.1 U / mL, which verifies the feasibility of the paper-based sensor.

[0053] Take the nucleic acid remover to be tested and mix it with DNA mucus in a certain volume ratio. After vortexing, take 20μL of the mixed liquid and drop it onto the paper-based sensor to observe the flow distance of the liquid on the test strip; compare the flow distance of the liquid on the test strip with the DNaDDⅠ standard curve to obtain the nucleic acid remover quality assessment result. In this embodiment, two different brands of nucleic acid removers A and B are added to the DNA mucus solution respectively, wherein the volume ratio of the nucleic acid contamination remover to the DNA mucus solution is 2:1. Figure 7 As shown, the nucleic acid remover can achieve good removal effects, and Brand B nucleic acid contamination remover is better than Brand A. It can be understood that this application protects a method for evaluating the quality of nucleic acid removers. Regardless of the brand, efficacy, and formula of the nucleic acid remover used, as long as the method is used to reduce the viscosity of DNA mucus, it should be covered by the scope of protection of this invention.

[0054] It should be noted that the nuclease is used as an example to verify the feasibility of this method. This example is merely an example, including but not limited to this one. Similar and similar principles that can achieve the same effect are still within the scope of protection of this invention. This method can be used for DNA removal based on different principles, including nuclease degradation methods. The method of this embodiment can also be applied to other nucleic acid-containing viscous solutions to achieve nucleic acid removal effect detection.

[0055] Example 2:

[0056] The second embodiment of the present invention provides an application of the nucleic acid scavenger quality assessment method described in the first embodiment in the field of nucleic acid scavenger quality control or molecular laboratory cleaning.

[0057] First, dissolve salmon sperm DNA at a mass concentration of 1.0% in sodium bromide solution and heat at 90-100°C for 9-19 minutes to prepare a DNA sticky solution of the corresponding viscosity. Next, take 20 μL of the DNA sticky solution alone, a mixture of DNA sticky solution and ultrapure water at a volume ratio of 1:2, and a mixture of DNA sticky solution and nucleic acid remover, and dropwise add them to the left end of the pH test strip. Figure 8 As shown in the figure, after adding the nucleic acid scavenger, the flow distance of the mixed solution on the test strip is particularly significant. Again, the nucleic acid scavenger was diluted 0 times, 10 times, and 100 times, and added to the DNA sticky solution respectively. Figure 9 As shown in the figure, the undiluted nucleic acid remover has the best effect on DNA removal. This method can effectively achieve the quality control of nucleic acid removers.

[0058] Each step of the nucleic acid scavenger quality assessment method involved in the above embodiment 2 corresponds to that of the method embodiment 1. For the specific implementation method, please refer to the relevant description part of embodiment 1.

[0059] Although the above describes the specific embodiments of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art on the basis of the technical solution of the present invention without any creative work are still within the scope of protection of the present invention.

Claims

1. A method for evaluating the quality of a nucleic acid scavenger, characterized in that: The following steps are involved: The detection elements are regularly placed on the substrate to construct a paper-based sensor; Prepare DNA sticky solution; Dissolve nuclease in CaCl2 solution to prepare DNase I solution; Add DNase I solution to Tris-HCl buffer to form a mixture, and thoroughly mix the mixture with DNA mucus. Incubate at a constant temperature, measure the flow distance of the reaction solution on the detection element, and draw a DNase I standard curve. The nucleic acid scavenger to be tested is mixed with DNA mucus, vortexed, and then the mixture is dropped onto a paper-based sensor. The flow distance of the liquid on the detection element is observed. The flow distance of the liquid on the detection element is compared with the DNase I standard curve to obtain the nucleic acid scavenger quality assessment result. Dissolve salmon sperm DNA in sodium bromide solution, vortex and heat at 90-100°C until fully dissolved, and refrigerate until ready to prepare DNA sticky solution; The mass fraction of the DNA mucus is 0.2 w / v%-1.0 w / v%; The concentration range of the DNase I was 0.01-10 U / mL; The mixing volume ratio of the DNA mucus and DNase I solution is 5:1, 3:1 or 1:1; The volume ratio of the nucleic acid remover to the DNA mucus is 2:

1.

2. The nucleic acid scavenger quality assessment method according to claim 1, wherein: The detection element is pH test paper.

3. The method for evaluating the quality of a nucleic acid scavenger according to claim 2, wherein: Use a cutter to cut the pH test paper into test strips of a certain width, and place the cut test strips regularly on a PVC substrate to construct a paper-based sensor.

4. The method for evaluating the quality of a nucleic acid scavenger according to claim 1, wherein: The PVC substrate was pre-cleaned three times with ethanol and deionized water, and dried under nitrogen flow before use.

5. The method for evaluating the quality of a nucleic acid scavenger according to claim 1, wherein: The concentration of the sodium bromide solution is 4.0-5.0 mM.

6. The method for evaluating the quality of a nucleic acid scavenger according to claim 1, wherein: The concentration of the CaCl2 solution is 0.3-1.0 mM.

7. The method for evaluating the quality of a nucleic acid scavenger according to claim 1, wherein: The incubation time is 0 to 80 minutes.

8. Use of the nucleic acid scavenger quality assessment method according to any one of claims 1 to 7 in the field of nucleic acid scavenger quality control.

Images