Nucleic acid scavenger and application thereof

Abstract

The invention discloses a nucleic acid scavenger and application thereof. The scavenging agent is divided into a bottle A and a bottle B. The component of the bottle A is a sodium hypochlorite solution with the available chlorine content of 3%-10%, and the sodium hypochlorite solution contains sodium chloride with the mass percent concentration of 3%-5%, sodium hydroxide with the mass percent concentration of 0.5%-1% and sodium bicarbonate with the mass percent concentration of 0.5%-1%. The bottle B comprises the following components: zinc sulfate with the mass percent concentration of 0.5%-4.5% and dodecyl dimethyl amine oxide with the volume percent concentration of 2%-10%; the solvent is purified water. A PCR result of the invention shows that under a similar control ct value, a nucleic acid sample after removal has no ct value, the removal effect is better, and compared with the prior art that only one nucleic acid of the feline mycoplasma pneumoniae is subjected to removal verification, the nucleic acid used for removal verification has more types and is more common in a molecular laboratory, and the method has a wide application prospect. And the nucleic acid removal effect is better guaranteed.

Description

Technical Field

[0001] The invention belongs to the field of biological products and relates to a nucleic acid remover and application thereof. Background Art

[0002] Currently, the common methods for removing nucleic acid contamination in molecular laboratories include spraying sodium hypochlorite disinfectant, spraying alcohol disinfectant, ultraviolet irradiation and ventilation. The first three are only effective in removing large fragments of nucleic acid, but less effective in removing small fragments of nucleic acid. In addition, none of the above methods can completely remove nucleic acid contamination in the short term, and the removal of contamination usually requires a longer period of time. A variety of nucleic acid removers have been introduced for the purpose of quickly and effectively removing nucleic acid contamination. Currently, there are two main principles for nucleic acid removers on the market. One is based on enzymes, which decompose nucleic acids through enzymes. This method is relatively mild and harmless to the human body, but the development of enzyme products is more difficult, and the prices are generally high. At the same time, the storage conditions have certain restrictions. Currently, there are few such products on the market. The other is based on chemical reagents, which remove nucleic acids through physical and chemical reactions, or by destroying the structure of nucleic acids to decompose them, or by precipitating nucleic acids to make them easy to erase. The development of such products is relatively simple, and the price is low and easy to preserve. Currently, the market is dominated by such products, but such products are mainly chemical reagents, which are not as mild and safe as enzyme reagents, and are easy to cause harm to the human body and the environment. Therefore, controlling the reagent components and concentrations is a difficult hurdle.

[0003] Many existing similar products have the disadvantages of poor nucleic acid removal effect, or can only remove DNA or RNA alone, or are relatively expensive. The purpose of the present invention is to develop a nucleic acid remover that can effectively remove nucleic acid pollution including DNA and RNA, and has simple reagent components, low price, and is easy to mass produce. Summary of the invention

[0004] The purpose of the present invention is to provide a nucleic acid remover and application thereof.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A nucleic acid remover, the remover is divided into two bottles, A and B, the components of bottle A are sodium hypochlorite solution with an effective chlorine content of 3%-10%, sodium chloride with a mass percentage concentration of 3%-5%, sodium hydroxide with a mass percentage concentration of 0.5%-1%, and sodium bicarbonate with a mass percentage concentration of 0.5%-1%; the components of bottle B are zinc sulfate with a mass percentage concentration of 0.5%-4.5% and dodecyl dimethyl amine oxide with a volume percentage concentration of 2%-10%; the solvents are all purified water.

[0007] As a preferred embodiment of the present invention, the scavenger is divided into two bottles, A and B. The components of bottle A are sodium hypochlorite solution with an effective chlorine content of 3%, sodium chloride with a mass percentage concentration of 3%, sodium hydroxide with a mass percentage concentration of 0.6%, and sodium bicarbonate with a mass percentage concentration of 0.6%. The components of bottle B are zinc sulfate with a mass percentage concentration of 3.5% and dodecyl dimethyl amine oxide with a volume percentage concentration of 5%. The solvents are all purified water.

[0008] The nucleic acid remover is used for removing nucleic acid in vitro.

[0009] The nucleic acid remover is used for removing nucleic acid on the surface of an object.

[0010] The nucleic acid remover is used in preparing a product for removing nucleic acid from the surface of an object.

[0011] A product for removing nucleic acid from the surface of an object, comprising the nucleic acid remover and a carrier.

[0012] As a preferred embodiment of the present invention, the carrier is a non-woven fabric.

[0013] A method for removing nucleic acid from the surface of an object comprises contacting the nucleic acid remover with the surface of the object containing nucleic acid.

[0014] As a preferred embodiment of the present invention, the nucleic acid remover is used to soak the surface of an object containing nucleic acid for 15 to 30 minutes.

[0015] The nucleic acid scavenger component of the present invention comprises sodium hypochlorite, sodium hydroxide, sodium bicarbonate, sodium chloride, dodecyl dimethylamine oxide and water, wherein the sodium hypochlorite can oxidize the amino heterocycle in the base to denature it, thereby destroying the primary and secondary conformations of the nucleic acid; the two alkaline substances, sodium hydroxide and sodium bicarbonate, can destroy the phosphodiester bond and hydrogen bond of DNA, especially the phosphodiester bond of RNA, which is very easy to degrade in an alkaline solution, and the alkaline environment is also helpful to maintain the stability of sodium hypochlorite; the zinc sulfate has a certain auxiliary effect on the decomposition of nucleic acid; the sodium chloride can maintain the stability of sodium hypochlorite; the dodecyl dimethylamine oxide can remove organic matter that may exist on the surface of nucleic acid, so that it is completely exposed to the scavenger, and can enhance the solubility of other components.

[0016] Beneficial effects: It can be seen from the PCR results of the present invention that, under similar control ct values, the nucleic acid samples cleared by the present invention have no ct values, while the nucleic acid samples cleared by the prior art still have ct values, which shows that the present invention has better clearance effect, and compared with the prior art, which only performs clearance verification on one nucleic acid of Mycoplasma felis pneumoniae, the present invention uses more types of nucleic acids for clearance verification and is more common in molecular laboratories, and the nucleic acid clearance effect is more guaranteed. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Amplification results of internal standard DNA and human plasma circulating DNA (the high value in the figure is only used as a reference for whether there is any abnormality in PCR amplification, and the ct value is not compared), where A is the internal standard DNA channel and B is the human plasma circulating DNA channel.

[0018] Figure 2 2019-nCoV-PCR-positive control amplification results

[0019] Figure 3 Amplification results of internal standard DNA and human plasma circulating DNA (the high value in the figure is only used as a reference for whether there is any abnormality in PCR amplification, and the ct value is not compared; 1-1, 1-2, and 1-3 are the test results of the removal effect of the nucleic acid remover of formula five), where A is the internal standard DNA channel and B is the human plasma circulating DNA channel.

[0020] Figure 4 2019-nCoV-PCR-positive control amplification results

[0021] Figure 5 Agarose gel electrophoresis results DETAILED DESCRIPTION

[0022] Example 1: Preparation of nucleic acid scavenger

[0023] Formula 1: A nucleic acid remover, liquid A contains the following ingredients: sodium hypochlorite solution with an effective chlorine content of 10%, sodium chloride with a mass percentage concentration of 3%, sodium hydroxide with a mass percentage concentration of 0.6%, sodium bicarbonate with a mass percentage concentration of 0.6%, and the solvent is purified water; liquid B contains the following ingredients: zinc sulfate with a mass percentage concentration of 3.5%, dodecyl dimethyl amine oxide with a volume percentage concentration of 5%, and the solvent is purified water.

[0024] Formula 2: A nucleic acid remover, liquid A contains the following ingredients: sodium hypochlorite solution with an effective chlorine content of 7.5%, sodium chloride with a mass percentage concentration of 3%, sodium hydroxide with a mass percentage concentration of 0.6%, and sodium bicarbonate with a mass percentage concentration of 0.6%, and the solvent is purified water; liquid B contains the following ingredients: zinc sulfate with a mass percentage concentration of 3.5%, dodecyl dimethyl amine oxide with a volume percentage concentration of 5%, and the solvent is purified water.

[0025] Formula 3: A nucleic acid remover, liquid A contains the following ingredients: sodium hypochlorite solution with an effective chlorine content of 5%, sodium chloride with a mass percentage concentration of 3%, sodium hydroxide with a mass percentage concentration of 0.6%, sodium bicarbonate with a mass percentage concentration of 0.6%, and the solvent is purified water; liquid B contains the following ingredients: zinc sulfate with a mass percentage concentration of 3.5%, dodecyl dimethyl amine oxide with a volume percentage concentration of 5%, and the solvent is purified water.

[0026] Formula 4: A nucleic acid remover, liquid A contains the following ingredients: sodium hypochlorite solution with an effective chlorine content of 3%, sodium chloride with a mass percentage concentration of 3%, sodium hydroxide with a mass percentage concentration of 0.6%, sodium bicarbonate with a mass percentage concentration of 0.6%, and the solvent is purified water; liquid B contains the following ingredients: zinc sulfate with a mass percentage concentration of 3.5%, dodecyl dimethyl amine oxide with a volume percentage concentration of 5%, and the solvent is purified water.

[0027] Formula 5: A nucleic acid remover, liquid A contains the following ingredients: sodium hypochlorite solution with an effective chlorine content of 1%, sodium chloride with a mass percentage concentration of 3%, sodium hydroxide with a mass percentage concentration of 0.6%, and sodium bicarbonate with a mass percentage concentration of 0.6%, and the solvent is purified water; liquid B contains the following ingredients: zinc sulfate with a mass percentage concentration of 3.5%, dodecyl dimethyl amine oxide with a volume percentage concentration of 5%, and the solvent is purified water.

[0028] Preparation method of liquid A: First dissolve sodium chloride, sodium hydroxide and sodium bicarbonate in purified water, and then add sodium hypochlorite solution after they are fully dissolved.

[0029] Preparation method of liquid B: first dissolve dodecyl dimethyl amine oxide in purified water and mix thoroughly, then add zinc sulfate to dissolve and mix.

[0030] Example 2: Nucleic acid scavenger removal effect at different sodium hypochlorite concentrations (PCR detection)

[0031] The nucleic acid scavengers of Formula 1, Formula 2, and Formula 3 described in Example 1 were used to treat the human plasma circulating DNA added with the internal standard DNA of the circulating DNA quantitative detection kit and the positive control RNA of the Shengxiang new coronavirus 2019-nCoV nucleic acid detection kit to verify their effect of clearing nucleic acids. The specific steps are as follows:

[0032] (1) Sample preparation: Add the internal standard to human plasma at a ratio of internal standard: plasma = 1:39 and mix well. Use a DNA / RNA purification kit to extract DNA as a DNA sample for later use. The positive control of the 2019-nCoV nucleic acid detection kit does not need to be extracted and is thawed before use as an RNA sample for later use.

[0033] (2) Sample processing: Take 10 μL of each of the nucleic acid remover AB solutions of Formula 1, Formula 2, and Formula 3 and add them to 20 μL of DNA sample and RNA sample as experimental samples. Take 20 μL of nuclease-free water and add it to 20 μL of DNA and RNA sample as control samples. Mix well by pipetting and let stand for 20 minutes to simulate the removal of nucleic acid contamination by the nucleic acid remover. Three parallel samples are prepared for each of the DNA and RNA experimental samples and the control samples.

[0034] (3) Sample extraction and amplification: The processed samples were extracted and purified using a DNA / RNA purification kit to obtain DNA and RNA amplification template samples. The DNA samples were then amplified using a circulating DNA quantitative detection kit and a Roche LightCycler 480 fluorescent quantitative PCR instrument, and the RNA samples were amplified using a new coronavirus 2019-nCoV nucleic acid detection kit and a Biori fluorescent quantitative PCR instrument. The kit components used for amplification are shown in Tables 1 and 2, the amplification system is shown in Tables 3 and 4, the amplification program settings are shown in Tables 5 and 6, and the nucleic acid removal effects of the nucleic acid remover at different sodium hypochlorite concentrations are shown in Tables 7, 8 and Figure 1 , 2 .

[0035] Table 1 Components of circulating DNA quantitative detection kit

[0036]

[0037]

[0038] Table 2 Components of the 2019-nCoV nucleic acid detection kit

[0039]

[0040] Table 3 Human plasma circulating DNA amplification system (prepared according to the instructions)

[0041]

[0042] Table 4 2019-nCoV-PCR-positive control amplification system (prepared according to the instructions)

[0043]

[0044]

[0045] Table 5 Human plasma circulating DNA amplification program settings (set according to the instructions)

[0046]

[0047] Note: FAM channel is the internal standard channel, and VIC channel is the circulating DNA channel.

[0048] Table 6 2019-nCoV-PCR-positive control amplification program settings (set according to the instructions)

[0049]

[0050] Table 7 The removal effect of nucleic acid removers on internal standard DNA and human plasma circulating DNA at different sodium hypochlorite concentrations

[0051]

[0052]

[0053] Table 8 The removal effect of nucleic acid removers on 2019-nCoV-PCR-positive control RNA at different sodium hypochlorite concentrations

[0054]

[0055] It can be seen from the above DNA and RNA removal result table that the nucleic acid removers of formulas one, two, and three with effective chlorine contents of 10%, 7.5%, and 5% can completely remove the internal standard DNA and human plasma circulating DNA, and the 2019-nCoV-PCR-positive control RNA can completely remove the ORF1ab gene and the N gene. Although the internal standard gene cannot be completely removed, the ct value of the internal standard gene has obviously changed greatly, all close to 40. This proves that the nucleic acid removers of the above three formulas can effectively remove DNA and RNA contamination when the cleaning time is 20 minutes. From an economic point of view, it is better to choose the nucleic acid remover of formula three with an effective chlorine content of 5%. Example 3: The removal effect of the nucleic acid remover after further reducing the sodium hypochlorite concentration and increasing the cleaning time (PCR detection)

[0056] The results obtained in Example 2 can be used to determine that there is still room for further reduction in the concentration of sodium hypochlorite, a nucleic acid scavenger. Therefore, in this experiment, the sample treatment time of the nucleic acid scavenger was extended from 20 minutes to 30 minutes, hoping to solve the problem that the standard gene in the RNA sample cannot be completely removed. This example uses the nucleic acid scavengers of Formula 4 and Formula 5 described in Example 1 to treat human plasma circulating DNA added with the standard DNA in the circulating DNA quantitative detection kit and the positive control RNA of the Shengxiang new coronavirus 2019-nCoV nucleic acid detection kit. The treatment time is 30 minutes, and the sample processing and detection methods are the same as in Example 2. The test results are shown in Tables 9, 10 and Figure 3 , 4 .

[0057] Table 9 The removal effect of nucleic acid removers on internal standard DNA and human plasma circulating DNA at different sodium hypochlorite concentrations

[0058]

[0059] Table 10 The effect of nucleic acid removers on 2019-nCoV-PCR-positive control RNA removal at different sodium hypochlorite concentrations

[0060]

[0061] It can be seen from the above DNA and RNA removal result table that the formula four nucleic acid remover with an effective chlorine content of 3% can completely remove the internal standard DNA, human plasma circulating DNA and 2019-nCoV-PCR-positive control RNA under the condition of 30 minutes of removal. However, the formula five nucleic acid remover with an effective chlorine content of 1% cannot completely remove the above DNA and RNA even if the removal time reaches 30 minutes. As can be seen from the results of this embodiment, when the removal time is 30 minutes, the formula four nucleic acid remover with an effective chlorine content of 3% has an excellent effect, and the economy is better than that of formula three, which is the best choice for this reagent formula.

[0062] Example 4: Verification of nucleic acid scavenger removal ability (electrophoresis)

[0063] The nucleic acid scavenger of formula 4 was used to perform a clearance experiment on human plasma circulating DNA samples added with internal standard DNA in the circulating DNA quantitative detection kit. The operation was the same as in Example 2. Four experimental samples were made in parallel, and two control samples were made in parallel. After the treatment, two of the experimental samples were extracted and purified using a DNA / RNA purification kit. Then, all the above experimental samples and control samples were subjected to agarose gel electrophoresis to verify the clearance ability of the nucleic acid scavenger. The results are as follows: Figure 5 As shown, lane 1 is a marker, lanes 2 and 3 are extracted experimental samples, lanes 4 and 5 are unextracted experimental samples, and lanes 6 and 7 are unextracted control samples. Figure 5 It can be seen from the electrophoresis results that, except for the control sample lane, there are no nucleic acid bands in the other experimental sample lanes, proving that the formula four nucleic acid remover has completely decomposed and removed the DNA sample.

[0064] Example 5: Effect of residual nucleic acid remover on PCR

[0065] Because nucleic acid removers are mostly used in molecular laboratories, in addition to verifying their effect of removing nucleic acids, it is also necessary to consider whether their residues will affect subsequent PCR experiments. If they do, it is necessary to consider how to solve them. Therefore, the residual experiment of this embodiment is designed for inspection. This embodiment uses the formula four nucleic acid removers described in Example 1. After residual treatment, the human plasma circulating DNA added to the internal standard DNA of the circulating DNA quantitative detection kit and the positive control RNA of the Shengxiang new coronavirus 2019-nCoV nucleic acid detection kit are mixed with the residues and then PCR is performed to observe whether it will affect the experimental results. The reagent residue simulation and sample processing method are as follows.

[0066] (1) Reagent residue simulation: Take 15 μL of each of the four nucleic acid removers AB and AB and add them to an EP tube as the experimental sample, and take 30 μL of nuclease-free water and add it to an EP tube as the control sample. After adding, place the EP tube in an EP tube rack and let it stand for 30 minutes to simulate the removal of surface nucleic acids by the nucleic acid remover. Make 12 parallel sets of experimental samples and 6 parallel sets of control samples. After 30 minutes, aspirate all the liquid in the tube with a pipette and discard it.

[0067] (2) Experimental sample processing: The experimental samples were divided into two groups, each with six EP tubes. One group was unwashed samples and no processing was performed, and the other group was washed samples. 1000 μL of nuclease-free water was added to each of the six EP tubes containing washed samples. After the tube caps were sealed, the tubes were placed on a vortex shaker for 10 seconds, and then briefly centrifuged and all the liquid inside was aspirated to simulate the use of a wet cloth to wipe off the residual cleaning agent on the surface.

[0068] (3) Nucleic acid sample processing: 30 μL / tube of DNA or RNA sample, add nucleic acid sample to the previously treated EP tube, DNA sample to three unwashed experimental sample EP tubes, three washed experimental sample EP tubes and three control sample EP tubes, RNA sample to the corresponding EP tube in the same manner as DNA, pipette several times and let stand for 5 minutes. Samples were directly put on the machine for PCR detection without extraction, the detection method was the same as in Example 2, and the detection results are shown in Tables 11 and 12.

[0069] Table 11 Effects of nucleic acid remover residues on PCR of internal standard DNA and human plasma circulating DNA at different cleaning times

[0070]

[0071]

[0072] Table 12 Removal effect of nucleic acid remover residue on 2019-nCoV-PCR-positive control RNA at different cleaning times

[0073]

[0074] From the ct value results in the above table, it can be seen that the residual nucleic acid remover will affect the PCR detection of DNA and RNA, but the impact on different kits is different. The difference between the sample ct value after simulated wiping and the control ct value is very small, which shows that the residual nucleic acid remover has basically no effect on PCR after wiping once. Therefore, it is recommended to wipe the surface of the object sprayed with the remover with a wet cloth after using this remover.

Claims

1. A nucleic acid remover, characterized in that The scavenger is divided into two bottles, A and B. The components of bottle A are sodium hypochlorite solution with an effective chlorine content of 3%-10%, sodium chloride with a mass percentage concentration of 3%-5%, sodium hydroxide with a mass percentage concentration of 0.5%-1%, and sodium bicarbonate with a mass percentage concentration of 0.5%-1%; the components of bottle B are zinc sulfate with a mass percentage concentration of 0.5%-4.5% and dodecyl dimethyl amine oxide with a volume percentage concentration of 2%-10%; the solvents are all purified water.

2. The nucleic acid remover according to claim 1, characterized in that The scavenger is divided into two bottles, A and B. The components of bottle A are sodium hypochlorite solution with an effective chlorine content of 3%, sodium chloride with a mass percentage concentration of 3%, sodium hydroxide with a mass percentage concentration of 0.6%, and sodium bicarbonate with a mass percentage concentration of 0.6%. The components of bottle B are zinc sulfate with a mass percentage concentration of 3.5% and dodecyl dimethyl amine oxide with a volume percentage concentration of 5%. The solvents are all purified water.

3. Use of the nucleic acid remover according to claim 1 or 2 in removing nucleic acids in vitro.

4. Use of the nucleic acid remover according to claim 1 or 2 in removing nucleic acids from the surface of an object.

5. Use of the nucleic acid remover according to claim 1 or 2 in the preparation of a product for removing nucleic acid from the surface of an object.

6. A product for removing nucleic acid from the surface of an object, characterized in that The invention comprises the nucleic acid remover according to claim 1 or 2 and a carrier.

7. The product according to claim 6, characterized in that The carrier is non-woven fabric.

8. A method for removing nucleic acid from the surface of an object, characterized in that The nucleic acid remover according to claim 1 or 2 is brought into contact with the surface of an object containing nucleic acid.

9. The method according to claim 8, characterized in that The nucleic acid remover according to claim 1 or 2 is used to immerse the surface of an object containing nucleic acid for 15 to 30 minutes.

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