Magnetic Bead Exogenous Nucleic Acid Pollution Removal Reagent and Removal Method

Abstract

The present invention relates to the field of molecular biotechnology, and in particular to a magnetic bead exogenous nucleic acid contamination removal reagent and a removal method. The magnetic bead exogenous nucleic acid contamination removal reagent provided by the present invention comprises solution A and solution B, wherein solution A comprises a surfactant and an oxidant, and solution B comprises an alkali, a salt and a lysate. The magnetic bead exogenous nucleic acid contamination removal reagent provided by the present invention has a simple and economical operation method, is safe and gentle, and has high efficiency, can achieve the purpose of removing nucleic acid contamination while ensuring that the treated magnetic beads maintain their original performance, and is suitable for large-scale processing.

Description

Technical Field

[0001] The present invention relates to the field of molecular biotechnology, and particularly to a reagent and method for removing exogenous nucleic acid contamination from magnetic beads. Background Art

[0002] Biological contamination is a common problem in many fields of science, medicine, and industry. Especially in medical services and the food industry, it is necessary to determine whether there are residues of potential pathogenic bacteria such as bacteria, viruses, and fungi. Although a completely sterile environment is impossible to achieve, sometimes this biological contamination may have a significant impact on measurement results. The market space for pathogenic microorganism detection is huge. The gene extraction of pathogenic microorganisms is an important step in clinical specimen testing and also an essential way for clinical diagnosis and treatment of infectious diseases. Before nucleic acid amplification, rapid and effective nucleic acid extraction is an essential step. Currently, the main nucleic acid extraction methods are column extraction and magnetic bead method. However, column extraction is not convenient for mechanization in practical applications, it is difficult to achieve high throughput, and it requires a large amount of labor cost and time cost. Therefore, when using the magnetic bead method for nucleic acid extraction, it is particularly important to ensure the removal of exogenous nucleic acid contamination from magnetic beads, which is also the problem to be solved by the present invention.

[0003] Common nucleic acid contaminations include: contamination of amplification products, contamination of extracted nucleic acids, contamination of positive controls, contamination of foreign nucleic acids, etc. Regarding how to handle these contaminated nucleic acids, many treatment methods have been proposed by researchers, such as nuclease degradation method, ultraviolet irradiation method, chloride oxidation method, high-temperature treatment method, sodium hypochlorite soaking method or scrubbing method. The existing methods for removing nucleic acids are not completely suitable for treating magnetic beads with functional groups on their surfaces. The nuclease method can hydrolyze nucleic acids through incubation for a certain time and at a certain temperature, but it will introduce protein contamination on the surface of the magnetic beads. Although the enzyme can be inactivated by heat incubation, the protein will still adhere to the surface of the magnetic beads and is difficult to completely separate, increasing the possibility of other contaminations. The ethylene oxide sterilization process is slow, expensive, and will release residual toxic gases, which is very likely to pose a safety hazard to researchers. In addition, magnetic beads are not suitable for ethylene oxide sterilization due to the functional groups on their surfaces, which may cause structural damage to the functional groups and thus affect the performance of the magnetic beads.

[0004] Therefore, it is urgent to seek a simple, effective method that does not affect the performance of magnetic beads. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a reagent and method for removing exogenous nucleic acid contamination from magnetic beads. The reagent and method for removing exogenous nucleic acid contamination from magnetic beads provided by the present invention are simple, economical, safe, mild, and highly efficient, and are suitable for large-scale treatment.

[0006] The present invention provides a nucleic acid removal reagent combination, including: Solution A and Solution B, wherein:

[0007] Solution A is composed of water, 0.1 wt% - 5 wt% surfactant, and 0.5 wt% - 4.5 wt% oxidant;

[0008] Solution B is composed of water, 0.5 - 1.5 M base, 0.05 - 0.2 M salt, and 0.25 - 1 M lysis solution.

[0009] Compared with other reagents, in Solution A and Solution B of the present invention, each component can be used more effectively in combination. Among them, in Solution A, the surfactant and the oxidant cooperate, and can more effectively break the hydrogen bonds and phosphodiester bonds of nucleic acids compared with other reagent combinations, thereby destroying the structure of nucleic acids; in Solution B, the alkaline environment and salts cooperate to jointly cause the breakage of the primary structure of nucleic acids and affect the solubility of nucleic acids. A certain proportion of lysis solution components are also added to ensure the effect of removing contamination. While degrading nucleic acids, the reagent provided by the present invention can also maintain the stability of magnetic beads. By using Solution A and Solution B in combination to cross - process the sample, it is not only safe and mild but also fully removes residual exogenous nucleic acids, and can ensure the stability of the performance of magnetic beads when removing exogenous nucleic acid contamination from magnetic beads, thereby obtaining more accurate and sensitive technical effects.

[0010] Preferably, the surfactant includes at least one of Tween 20, betaine, sodium deoxycholate, and Triton.

[0011] Preferably, the oxidant includes at least one of hydrogen peroxide and sodium peroxide.

[0012] Preferably, the base includes at least one of sodium hydroxide and potassium hydroxide. Experimental exploration shows that the alkaline environment in Solution B plays a relatively important role in removing nucleic acid contamination from magnetic beads.

[0013] Preferably, the salt includes at least one of sodium chloride and potassium chloride.

[0014] Preferably, the lysis solution includes at least one of guanidine isothiocyanate and guanidine hydrochloride.

[0015] In some embodiments, Solution A is composed of water, 5 wt% Tween 20, and 3 wt% hydrogen peroxide, and Solution B is composed of water, 1.5 M sodium hydroxide, 0.05 M sodium chloride, and 0.25 M guanidine isothiocyanate.

[0016] In some embodiments, Solution A is composed of water, 3 wt% betaine, 1.5 wt% Triton, and 4.5 wt% sodium peroxide, and Solution B is composed of water, 0.5 M potassium hydroxide, 0.2 M sodium chloride, and 1 M guanidine hydrochloride.

[0017] In some embodiments, Solution A is composed of water, 0.1 wt% sodium deoxycholate, 3 wt% Triton, and 0.5 wt% hydrogen peroxide, and Solution B is composed of water, 1 M potassium hydroxide, 0.1 M potassium chloride, and 0.8 M guanidine isothiocyanate.

[0018] Experiments show that within the above concentration ranges, both Solution A and Solution B exhibit the best ability to remove exogenous nucleic acid contamination from magnetic beads.

[0019] In some embodiments, Solution A is composed of water, 2.5 wt% Triton, and 3 wt% hydrogen peroxide, and Solution B is composed of water, 0.8 M sodium hydroxide, 0.15 M sodium chloride, and 0.75 M guanidine isothiocyanate.

[0020] Experiments show that at this concentration, magnetic beads treated with Solution B can simultaneously meet the requirements of reducing residual exogenous nucleic acids, maintaining the extraction performance of magnetic beads, and ensuring the suspension of magnetic beads.

[0021] The present invention also provides the application of the described nucleic acid removal reagent combination in removing exogenous nucleic acid contamination from magnetic beads.

[0022] The present invention provides a method for removing nucleic acids from magnetic beads, which is carried out using the described nucleic acid removal reagent combination.

[0023] Preferably, the method includes the following steps:

[0024] Add Solution A to the magnetic beads, mix well and wash, then remove the waste liquid;

[0025] Then add Solution B, mix well and wash, and obtain clean magnetic beads after removing the waste liquid.

[0026] More preferably, add 5 volumes of Solution A to the magnetic beads, remove the waste liquid after washing for 20 minutes; then add 5 volumes of Solution B to the magnetic beads and wash for 20 minutes.

[0027] In some embodiments, Solution A and Solution B are alternately washed repeatedly 2 times.

[0028] In some embodiments, after the last wash of Solution B and removal of the waste liquid, add 5 volumes of sterilized water to the magnetic beads, mix well and wash for 20 minutes to obtain clean magnetic beads.

[0029] The magnetic bead exogenous nucleic acid contamination removal reagent provided by the present invention includes solution A and solution B. Among them, solution A includes a surfactant and an oxidant, and solution B includes an alkali, a salt, and a lysis solution. Compared with the prior art, the magnetic bead exogenous nucleic acid contamination removal reagent and operation method of the present invention are simple, economical, safe, mild, and efficient. It can achieve the purpose of removing nucleic acid contamination while ensuring that the treated magnetic beads maintain their original performance, and is suitable for large-scale treatment. It was found in the experiment that the average CT value of the magnetic bead exogenous nucleic acid contamination removal reagent before treatment was 22.40, the average CT value without adding an alkaline reagent was 29.35, and no pollution residue was detected after adding an alkaline reagent for treatment, indicating that adding an alkaline reagent to the magnetic bead exogenous nucleic acid contamination removal reagent can effectively improve the nucleic acid removal effect on the surface of the magnetic beads. Description of the Drawings

[0030] Figure 1 Amplification diagram showing the detection of exogenous residues on magnetic beads;

[0031] Figure 2 Amplification diagrams showing before and after the removal of exogenous residues on magnetic beads and after treatment with scavengers A and B;

[0032] Figure 3 Amplification diagram showing the detection of the extraction performance of magnetic beads before and after the removal of residues and after treatment with scavengers A and B. Detailed Embodiments

[0033] The present invention provides a magnetic bead exogenous nucleic acid contamination removal reagent and a removal method. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate changes and combinations to the methods and applications in this article without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0034] In the present invention, the alkali can be an alkali commonly used in the art, such as sodium hydroxide, calcium hydroxide, potassium hydroxide, etc. In this article, the alkali, alkaline reagent, and alkaline solution have the same meaning and can be used interchangeably; the salt refers to a salt commonly used in the art, such as sodium chloride, potassium chloride, calcium sulfate, sodium carbonate, sodium bicarbonate, etc. In this article, the salt, salt reagent, and salt solution have the same meaning and can be used interchangeably;

[0035] The test materials used in the present invention are all ordinary commercially available products and can be purchased in the market.

[0036] The magnetic bead exogenous nucleic acid contamination removal reagent provided by the present invention mainly includes:

[0037] Nucleic acid contamination removal solution A (referred to as solution A) contains 0.1 wt% - 5 wt% surfactant, 0.5 wt% - 4.5 wt% oxidant, and the balance is made up with sterilized water. Among them, the surfactant includes but is not limited to Tween 20, betaine, sodium deoxycholate, and Triton. The oxidant includes but is not limited to hydrogen peroxide and sodium peroxide.

[0038] Nucleic acid contamination removal solution B (referred to as solution B) contains 0.5 - 1.5 M alkaline reagent, 0.05 - 0.2 M salt reagent, 0.25 - 1 M lysis solution, and the balance is made up with sterilized water. The alkaline reagent includes but is not limited to sodium hydroxide and potassium hydroxide. The salt reagent includes but is not limited to sodium chloride and potassium chloride. The lysis solution includes but is not limited to guanidine isothiocyanate and guanidine hydrochloride.

[0039] The method for treating magnetic beads nucleic acid contamination provided by the present invention is operated as follows:

[0040] Take an appropriate amount of magnetic beads a, magnetize them on a magnetic stand for 2 minutes, then discard the supernatant. First, add 5 times the volume of nucleic acid contamination removal solution A, mix well by shaking, and then place it on a vertical mixer and mix at an appropriate speed for 20 minutes. After that, place it on the magnetic stand and magnetize for 2 minutes, then discard nucleic acid contamination removal solution A;

[0041] Then add 5 times the volume of nucleic acid contamination removal solution B, mix well by shaking, and then place it on a vertical mixer and mix at an appropriate speed for 20 minutes. After that, place it on the magnetic stand and magnetize for 2 minutes, then discard nucleic acid contamination removal solution B; Then alternately add 5 times the volume of nucleic acid contamination removal solution A or B, repeat 2 times. Finally, discard nucleic acid contamination removal solution B, then add 5 times the volume of sterilized water, mix well by shaking, and then place it on a vertical mixer and mix at an appropriate speed for 20 minutes. After that, place it on the magnetic stand and magnetize for 2 minutes, then pour out the waste liquid, repeat 2 times, and finally add an equal amount of magnetic bead preservation solution.

[0042] The following further elaborates the present invention in combination with embodiments:

[0043] Example 1: Detection of exogenous residue of magnetic beads

[0044] Take 100 μL of magnetic beads a, b, and c with a concentration of 50 mg / mL and put them into a centrifuge tube. Magnetize them on a magnetic stand for 2 minutes, take out the supernatant of the preservation solution, and directly add the supernatant into the qPCR detection system. The primer is a universal bacterial primer (16S rRNA), the sample loading amount is 5 μL, and sterilized water is used as a negative control. The results are shown in the following table.

[0045]

[0046] As can be seen from the above table, there are obvious bacterial residues in different magnetic beads.

[0047] Example 2: Method for removing nucleic acid contamination from magnetic beads

[0048] A method for removing nucleic acid contamination from magnetic beads is as follows:

[0049] First, prepare a nucleic acid decontamination solution A containing the following components: 5 wt% Tween 20, 3 wt% hydrogen peroxide, and the balance made up with sterilized water; prepare a nucleic acid decontamination solution B containing the following components: 1.5 M sodium hydroxide, 0.05 M sodium chloride, 0.25 M guanidine isothiocyanate, and the balance made up with sterilized water.

[0050] Select magnetic bead a in Example 1 as the representative magnetic bead for removing exogenous nucleic acid. Take 1 mL of magnetic bead a, magnetically attract it on a magnetic stand for 2 minutes, then discard the supernatant. First, add 5 mL of nucleic acid decontamination solution A, mix well by shaking, and then place it on a vertical mixer and mix at an appropriate speed for 20 minutes. After that, place it on a magnetic stand and magnetically attract it for 2 minutes, then discard nucleic acid decontamination solution A; then add 5 mL of nucleic acid decontamination solution B, mix well by shaking, and then place it on a vertical mixer and mix at an appropriate speed for 20 minutes. After that, place it on a magnetic stand and magnetically attract it for 2 minutes, then discard nucleic acid decontamination solution B; then alternately add 5 mL of nucleic acid decontamination solution A or B, repeat 2 times. Finally, discard nucleic acid decontamination solution B, then add 5 mL of sterilized water, mix well by shaking, and then place it on a vertical mixer and mix at an appropriate speed for 20 minutes. After that, place it on a magnetic stand and magnetically attract it for 2 minutes, then pour out the waste liquid, repeat 2 times. Finally, add 1 mL of magnetic bead preservation solution.

[0051] In addition, we also carried out the treatment using nucleic acid decontamination solution A or B alone, each treatment for 1.5 hours. After that, place it on a magnetic stand and magnetically attract it for 2 minutes, then discard nucleic acid decontamination solution A or B. A total of 3 washes were carried out, then washed with water three times, and finally added the same magnetic bead preservation solution as above.

[0052] Take 100 μL of magnetic bead a before and after treatment and put it into a centrifuge tube, magnetically attract it on a magnetic stand for 2 minutes, take out the supernatant of the preservation solution, and directly add the supernatant to the qPCR detection system. The primer is a universal bacterial primer (16S rRNA), the sample loading amount is 5 μL, and sterilized water is used as a negative control. The results are shown in the following table.

[0053]

[0054] As can be seen from the above table, after treatment with nucleic acid decontamination solution A or B, magnetic bead a has different degrees of reduction, but there is still some residue. After cross-treatment with A and B, the nucleic acid residue is completely removed. In terms of time, the part treated with A and B cross 3 times takes 2 hours, while the part treated with A or B alone takes 4.5 hours, but the effect is not as good as the cross-treatment.

[0055] Example 3: Detection of the residue of magnetic beads for removing nucleic acid contamination

[0056] First, prepare a nucleic acid contamination-removing solution A, which contains the following components: 3 wt% betaine, 1.5 wt% Triton, 4.5 wt% sodium peroxide, and the balance is made up with sterilized water; prepare a nucleic acid contamination-removing solution B, which contains the following components: 0.5 M potassium hydroxide, 0.2 M sodium chloride, 1 M guanidine hydrochloride, and the balance is made up with sterilized water.

[0057] Select the magnetic bead a in Example 1 as the representative magnetic bead for exogenous nucleic acid removal treatment. Take 1 mL of magnetic bead a, magnetically attract it on a magnetic stand for 2 minutes, then discard the supernatant. First, add 5 mL of nucleic acid contamination-removing solution A, mix well by shaking, and then place it on a vertical mixer and mix at an appropriate speed for 20 minutes. After that, place it on the magnetic stand and magnetically attract it for 2 minutes, then discard nucleic acid contamination-removing solution A; then add 5 mL of nucleic acid contamination-removing solution B, mix well by shaking, and then place it on a vertical mixer and mix at an appropriate speed for 20 minutes. After that, place it on the magnetic stand and magnetically attract it for 2 minutes, then discard nucleic acid contamination-removing solution B; then alternately add 5 mL of nucleic acid contamination-removing solution A or B, repeat 2 times. Finally, discard nucleic acid contamination-removing solution B, then add 5 mL of sterilized water, mix well by shaking, and then place it on a vertical mixer and mix at an appropriate speed for 20 minutes. After that, place it on the magnetic stand and magnetically attract it for 2 minutes, then pour out the waste liquid, repeat 2 times. Finally, add 1 mL of magnetic bead preservation solution. The results are shown in the following table.

[0058]

[0059] As can be seen from the above table, compared with the untreated ones, when the nucleic acid contamination-removing solution A and solution B prepared in this example are used to treat the magnetic bead a, the nucleic acid residues on the surface of the magnetic bead are completely removed.

[0060] Example 4: Performance detection of nucleic acid contamination-removing magnetic beads

[0061] Take the untreated magnetic bead a in Example 1 and the magnetic bead a after being treated in Example 3, and use them with the corresponding kit for pseudovirus nucleic acid extraction, and detect the nucleic acid before and after extraction.

[0062]

[0063] After the magnetic bead a is cross-treated with the nucleic acid contamination-removing solutions A and B, its performance is equivalent to that of the original magnetic bead.

[0064] Example 5: Comparative cases of nucleic acid scavengers

[0065] First, a nucleic acid contamination removal solution A was prepared, containing the following ingredients: 0.1wt% sodium deoxycholate, 3wt% Triton, 0.5wt% hydrogen peroxide, and the balance was made up with sterile water; a nucleic acid contamination removal solution B was prepared, containing the following ingredients: 1M potassium hydroxide, 0.1M potassium chloride, 0.8M guanidine isothiocyanate, and the balance was made up with sterile water. The treatment was carried out in the manner of combined cleaning in Example 2.

[0066] In this embodiment, a contrast reagent is also provided for nucleic acid treatment, such as nucleic acid remover C, which is composed of the following components: 20 wt% guanidine thiocyanate, 0.5 M citric acid, 5 mL / L peracetic acid, 0.5 M Zn 2+ , 0.03M EDTA, 5wt% cocoyl glucoside. Prepare nucleic acid remover C, and perform treatment according to the single cleaning method in Example 2.

[0067] The results are shown in the following table.

[0068]

[0069] Compared with the untreated state, the magnetic beads a were treated with the nucleic acid removal solution A and solution B prepared in this example. The magnetic beads showed that the nucleic acid residues were completely removed, while the nucleic acid remover of the comparison reagent had a worse removal effect than that of this example.

[0070] Example 6: Exploration of important components in nucleic acid removers

[0071] First, a nucleic acid contamination removal solution B1 containing no alkaline reagent was prepared, containing the following ingredients: 0.05M sodium chloride, 0.25M guanidine isothiocyanate, and the balance was made up with sterile water. Compared with the nucleic acid contamination removal solution B containing the following ingredients: 1.5M sodium hydroxide, 0.05M sodium chloride, 0.25M guanidine isothiocyanate, a nucleic acid contamination removal solution A was prepared, containing the following ingredients: 5wt% Tween 20, 3wt% hydrogen peroxide. It was used for the treatment of the above-mentioned magnetic beads, and the treatment operation method was the same as in Example 3, and the results are shown in the following table.

[0072]

[0073] It can be seen from the above table that removing the alkaline reagent in the nucleic acid removal solution B will reduce the effect of nucleic acid removal on the surface of magnetic beads, indicating that the alkaline environment plays a relatively important role in the removal of nucleic acid contamination from magnetic beads.

[0074] Example 7: Testing of the formula of the nucleic acid removal solution B based on alkaline reagent

[0075] Prepare Solution B containing alkaline reagents at different concentrations. Taking sodium hydroxide as an example, prepare it while keeping the components of 0.15 M sodium chloride and 0.75 M guanidine isothiocyanate unchanged (as shown in the following table). Combine it with Solution A containing 2.5 wt% Triton, 3 wt% hydrogen peroxide and water to perform the treatment of removing exogenous nucleic acids from magnetic beads. The results are shown in the following table.

[0076] solution alkali reagent concentration nucleic acid residue magnetic bead extraction performance taking all factors into consideration B0 0M NaOH yes qualified contamination exists, not feasible B1 0.5M NaOH no qualified feasible B2 0.8M NaOH no qualified feasible, magnetic bead suspension becomes better B3 1M NaOH no qualified feasible B4 1.5M NaOH no qualified feasible B5 2M NaOH no unqualified magnetic beads stick to the wall and extraction becomes worse

[0077] Considering various factors such as the residual exogenous nucleic acids on magnetic beads, the extraction performance of magnetic beads and the suspension of magnetic beads, the formulation of Solution B prepared with 0.8 M sodium hydroxide is relatively optimal.

[0078] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Nucleic acid scavenging reagent, characterized in that, it comprises: Solution A and Solution B, wherein Solution A is composed of water, 5 wt% Tween 20 and 3 wt% hydrogen peroxide, and Solution B is composed of water, 1.5 M sodium hydroxide, 0.05 M sodium chloride and 0.25 M guanidine isothiocyanate; or Solution A is composed of water, 3 wt% betaine, 1.5 wt% Triton and 4.5 wt% sodium peroxide, and Solution B is composed of water, 0.5 M potassium hydroxide, 0.2 M sodium chloride and 1 M guanidine hydrochloride; or Solution A is composed of water, 0.1 wt% sodium deoxycholate, 3 wt% Triton and 0.5 wt% hydrogen peroxide, and Solution B is composed of water, 1 M potassium hydroxide, 0.1 M potassium chloride, 0.8 M guanidine isothiocyanate; or Solution A is composed of water, 2.5 wt% Triton and 3 wt% hydrogen peroxide, and Solution B is composed of water, 0.8 M sodium hydroxide, 0.15 M sodium chloride and 0.75 M guanidine isothiocyanate.

2. Use of the nucleic acid scavenging reagent according to claim 1 for scavenging exogenous nucleic acid contamination of magnetic beads.

3. Method for scavenging nucleic acid of magnetic beads, characterized in that, scavenging is carried out using the nucleic acid scavenging reagent according to claim 1.

4. The method according to claim 3, characterized in that, it comprises the following steps: adding Solution A to the magnetic beads, mixing and washing, and then removing the waste liquid; adding Solution B, mixing and washing, and obtaining clean magnetic beads after removing the waste liquid.

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