Nucleic acid extraction lysis binding solution, nucleic acid extraction kit and extraction method

Abstract

The invention discloses a nucleic acid extraction lysis combination liquid, a nucleic acid extraction kit and an extraction method; the lysis liquid comprises a bioactive agent and a protein solubilizing agent, the bioactive agent can efficiently lyse cell denatured proteins, and the protein solubilizing agent can separate proteins from nucleic acids well, thereby achieving the purpose of effectively releasing nucleic acids; when the lysis combination liquid is used for nucleic acid extraction, it can effectively improve the purity and yield of nucleic acids and ensure the quality of nucleic acid extraction.

Description

Technical Field

[0001] The invention relates to the technical field of nucleic acid extraction, in particular to a nucleic acid extraction lysis combination solution, a nucleic acid extraction kit and an extraction method. Background Art

[0002] With the development of molecular biology technology, research at the genomic level has become a hot topic. In molecular genetics and molecular epidemiology, whether it is genome-wide association analysis (GWAS), candidate SNP locus genotyping, or the establishment of genomic libraries, it is necessary to extract genomic DNA from large quantities of blood samples. The concentration, purity, and integrity of the primary structure of the extracted DNA will affect subsequent research, so efficient and reliable high-throughput DNA extraction methods are urgently needed for basic research in molecular genetics.

[0003] There are many methods for DNA extraction, including the traditional phenol-chloroform method, salting-out method, membrane centrifugal column method, etc. These methods have their own advantages and disadvantages, but due to manual extraction, sample processing is also not suitable for automated operations, which limits clinical applications. Magnetic beads are usually used in traditional extraction methods. The disadvantage of the magnetic bead method is that the purity of the obtained DNA is easily affected by the residual magnetic beads. Factors such as lysis time, sample volume and ethanol concentration may also affect the purity of DNA extraction.

[0004] Nucleic acid lysis is the first and most important step in nucleic acid extraction, and its effect depends on the role of the lysis solution. A lysis solution with excellent performance must not only be able to lyse cells quickly and efficiently, but also achieve the goal of higher nucleic acid extraction purity, while reducing costs while ensuring the quantity and quality of the extracted nucleic acid. Summary of the invention

[0005] The invention provides a nucleic acid extraction lysis combination solution, a whole blood nucleic acid extraction kit and an extraction method. The nucleic acid extraction lysis combination solution can efficiently lyse and separate nucleic acids, thereby reducing costs while ensuring the quantity and quality of the extracted nucleic acids.

[0006] In view of this, the scheme of the present invention is:

[0007] The first aspect of the present invention provides a nucleic acid extraction, lysis and binding solution, comprising a bioactive agent with a concentration of 0.1-2%, a four-arm-polyethylene glycol-amino with a concentration of 1-5%, a high-order salt with a concentration of 2-6M, a first buffer with a concentration of 5-200mM and a surfactant with a concentration of 0.1-1%.

[0008] Furthermore, the bioactive element is selected from at least one of iturin, lichenysin, surfactin and echinocandin.

[0009] Furthermore, the molecular weight of the four-arm-polyethylene glycol-amino group is 1000-5000, preferably 2000-3000; more preferably 3000.

[0010] Further, the high-order salt is selected from at least one of guanidine hydrochloride, guanidine isothiocyanate, potassium thiocyanate, urea, lithium chloride and perchlorate;

[0011] and / or, the first buffer is HEPES, sodium acetate-acetic acid or Tris-HCl buffer;

[0012] And / or, the surfactant is selected from at least one of Triton X-100, NP-40, Tween-20, Tween-80, SDS or SLS.

[0013] The second aspect of the present invention is to provide a nucleic acid extraction kit, comprising the lysis and binding solution described in the first aspect.

[0014] Furthermore, the nucleic acid extraction kit also includes a magnetic bead solution, a washing solution, an eluent and proteinase K.

[0015] Preferably, the washing solution comprises washing solution 1 and washing solution 2; the washing solution 1 contains 1-3M high dissociative salt, 5-200mM second buffer, 1-100mM inorganic salt ions, and 20-6% precipitant; the washing solution 2 contains 5-200mM second buffer, 1-100mM inorganic salt ions, and 30%-80% precipitant.

[0016] Preferably, the eluent contains 5-200 mM of the second buffer solution and 1-100 mM of inorganic salt ions.

[0017] Preferably, the second buffer is selected from at least one of HEPES, sodium acetate-acetic acid or Tris-HCl buffer;

[0018] And / or, the inorganic salt ion is selected from at least one of sodium chloride, potassium chloride, sodium acetate or sodium citrate;

[0019] And / or, the precipitant is selected from ethanol or isopropanol.

[0020] Preferably, the magnetic beads are superparamagnetic silicon oxide nanospheres.

[0021] The third aspect of the present invention is to provide a method for extracting nucleic acid, comprising the steps of:

[0022] The lysis binding solution described in the first aspect is mixed with the sample, proteinase K and magnetic bead solution, and lysed at 50-80° C. for 5-20 min, and the magnetic bead-nucleic acid complex is collected;

[0023] Wash the magnetic bead-nucleic acid complex with a washing solution, and magnetically absorb and collect the magnetic bead-nucleic acid complex;

[0024] Add elution buffer to the magnetic bead-nucleic acid complex, elute at 50-80°C for 2-5 minutes, and collect the nucleic acid.

[0025] Compared with the prior art, the present invention includes but is not limited to the following beneficial effects:

[0026] The bioactive element in the nucleic acid lysis binding solution of the present invention can efficiently lyse cells and release nucleic acids, and the protein solubilizing agent enables the protein to be more effectively separated from the nucleic acid. When used for nucleic acid extraction using the magnetic bead method, the purity and yield of nucleic acids can be effectively improved, thereby ensuring the quality of nucleic acid extraction. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below in conjunction with the preferred embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] In one embodiment, a nucleic acid extraction, lysis and binding solution is proposed, comprising a bioactive agent with a mass concentration of 0.1-2%, a four-arm-polyethylene glycol-amino (4-arm-NH2-PEG) with a mass concentration of 1-5%, a high-order salt with a concentration of 2-6M, a first buffer with a concentration of 5-200mM and a surfactant with a mass concentration of 0.1-1%.

[0029] In the above embodiments, the bioactive element in the nucleic acid lysis binding solution can efficiently lyse cells and release nucleic acids. The four-arm-polyethylene glycol-amino group serves as a protein solubilizer, which enables the protein to be more effectively separated from the nucleic acid, and can effectively improve the purity and yield of the nucleic acid, thereby ensuring the quality of nucleic acid extraction.

[0030] In a preferred embodiment, the bioactive element is selected from at least one of iturin, lichenysin, surfactin and echinocandin. The bioactive element is a cyclic lipopeptide surfactant that can efficiently lyse cells and increase the yield of nucleic acids;

[0031] In a preferred embodiment, the molecular weight of the four-arm-polyethylene glycol-amino group is 1000-5000, preferably 2000-3000, and more preferably 3000. The concentration of the four-arm-polyethylene glycol-amino group is preferably 1-3%; preferably 2-3%; more preferably 2%;

[0032] In a preferred embodiment, the bioactive element is preferably iturin or echinocandin, and the concentration is preferably 0.5-1%; more preferably 0.5%;

[0033] In the above embodiment, the nucleic acid lysis binding solution is suitable for lysis and effective release of nucleic acids in whole blood, plasma or serum. Personnel in the art can extract free nucleic acids by magnetic bead method to obtain high-quality and high-purity nucleic acids. The above nucleic acid lysis solution can be made into a kit to match an automated extraction instrument to improve the convenience of operation, and can be applied to downstream PCR, sequencing, hybridization, etc. without interfering with the results.

[0034] The magnetic bead method is used to verify the effect of the nucleic acid lysis binding solution on the above technology. Unless otherwise specified, the operations used in the following experimental examples are commonly used methods in the field, and the reagents used are commercially available.

[0035] Example 1 (different concentrations of bioactive element 1)

[0036] (1) Take 200 μl of EDTA anticoagulated blood and place it in a centrifuge tube. Add 265 μl of lysis binding solution, 40 μl of proteinase K and 20 μl of magnetic bead solution to the centrifuge tube. Heat and mix at 70°C for 10 min. Place the centrifuge tube on a magnetic rack and perform magnetic separation for 20 seconds. Discard the supernatant.

[0037] The components added to the lysis and binding solution are 4 M guanidine hydrochloride, 20 mM Tris-HCl buffer, and 0.5% Tween-20. The bioactive element added is iturin, and the concentrations are 0%, 0.1%, 0.5% and 1% respectively;

[0038] (2) Continue to add 1000 μl of washing solution 1 to the centrifuge tube, mix for 1 min, place the centrifuge tube on a magnetic rack, perform magnetic separation for 20 s, and discard the supernatant;

[0039] The washing solution 1 comprises 2M guanidine hydrochloride, 100 mM Tris-HCl and 50% ethanol solution;

[0040] (3) Continue to add 500 μl of washing solution 2 to the centrifuge tube, mix for 1 min, place the centrifuge tube on a magnetic rack, perform magnetic separation for 20 s, discard the supernatant, and repeat the washing once;

[0041] The washing solution 2 comprises 200 mM Tris-HCl and 80% ethanol solution;

[0042] (4) Dry until all the ethanol on the surface of the magnetic beads evaporates, add the eluent, heat and mix at 70°C for 5 min, place the centrifuge tube on a magnetic rack, perform magnetic separation for 20 s, and collect the supernatant, which is the nucleic acid;

[0043] The elution buffer consisted of 20 mM Tris-HCl and 10 mM EDTA.

[0044] After the extraction was completed, the NanoDrop One spectrophotometer was zeroed with the eluent, the interface DNA was selected, and 2 μL of nucleic acid was extracted from whole blood for detection. The test results are shown in Table 1.

[0045] Table 1:

[0046] iturin concentration ND (ng / μl) 260 / 280 260 / 230 0 112.7 1.64 1.46 0.1% 155.8 1.71 1.52 0.5% 258.2 1.76 1.67 1% 204.6 1.73 1.57 2% 188.7 1.70 1.48

[0047] Example 2 (Different concentrations of bioactive element 2)

[0048] (1) Take 200 μl of EDTA anticoagulated blood and place it in a centrifuge tube. Add 265 μl of lysis binding solution, 40 μl of proteinase K and 20 μl of magnetic bead solution to the centrifuge tube. Heat and mix at 70°C for 10 min. Place the centrifuge tube on a magnetic rack and perform magnetic separation for 20 s. Discard the supernatant.

[0049] The components added to the lysis and binding solution are 4 M guanidine hydrochloride, 20 mM Tris-HCl buffer, and 0.5% Tween-20. The bioactive substance added is echinocandin, and the concentrations are 0%, 0.1%, 0.5% and 1% respectively;

[0050] (2) Continue to add 1000 μl of washing solution 1 to the centrifuge tube, mix for 1 min, place the centrifuge tube on a magnetic rack, perform magnetic separation for 20 s, and discard the supernatant;

[0051] The washing solution 1 comprises 2M guanidine hydrochloride, 100 mM Tris-HCl and 50% ethanol solution;

[0052] (3) Continue to add 500 μl of washing solution 2 to the centrifuge tube, mix for 1 min, place the centrifuge tube on a magnetic rack, perform magnetic separation for 20 s, discard the supernatant, and repeat the washing once;

[0053] The washing solution 2 comprises 200 mM Tris-HCl and 80% ethanol solution;

[0054] (4) Dry until all the ethanol on the surface of the magnetic beads evaporates, add the eluent, heat and mix at 70°C for 5 min, place the centrifuge tube on a magnetic rack, perform magnetic separation for 20 s, and collect the supernatant, which is the nucleic acid;

[0055] The composition of the elution solution is 20 mM Tris-HCl and 10 mM EDTA;

[0056] After the extraction was completed, the NanoDrop One spectrophotometer was zeroed with the eluent, the interface DNA was selected, and 2 μL of nucleic acid was extracted from whole blood for detection. The test results are shown in Table 2.

[0057] Table 2:

[0058] Example 3 (Protein solubilizing agent, such as four-arm-polyethylene glycol-amino)

[0059] (1) Take 200 μl of EDTA anticoagulated blood and place it in a centrifuge tube. Add 265 μl of lysis binding solution, 40 μl of proteinase K and 20 μl of magnetic bead solution to the centrifuge tube. Heat and mix at 70°C for 10 min. Place the centrifuge tube on a magnetic rack and perform magnetic separation for 20 s. Discard the supernatant.

[0060] The components added to the lysis and binding solution are 2% four-arm-polyethylene glycol-amino, 4 M guanidine hydrochloride, 20mM Tris-HCl buffer, and 0.5% Tween-20. In addition, the molecular weights of four-arm-polyethylene glycol-amino are 1000, 2000, 3000, and 5000 respectively;

[0061] (2) Continue to add 1000 μl of washing solution 1 to the centrifuge tube, mix for 1 min, place the centrifuge tube on a magnetic rack, perform magnetic separation for 20 s, and discard the supernatant;

[0062] The washing solution 1 comprises 2M guanidine hydrochloride, 100 mM Tris-HCl and 50% ethanol solution;

[0063] (3) Continue to add 500 μl of washing solution 2 to the centrifuge tube, mix for 1 min, place the centrifuge tube on a magnetic rack, perform magnetic separation for 20 s, discard the supernatant, and repeat the washing once;

[0064] The washing solution 2 comprises 200 mM Tris-HCl and 80% ethanol solution;

[0065] (4) Dry until all the ethanol on the surface of the magnetic beads evaporates, add the eluent, heat and mix at 50-80°C for 5 min, place the centrifuge tube on a magnetic rack, perform magnetic separation for 20 s, and collect the supernatant, which is the nucleic acid;

[0066] The composition of the elution solution is 20 mM Tris-HCl and 10 mM EDTA;

[0067] After the extraction was completed, the NanoDrop One spectrophotometer was zeroed with the eluent, the interface DNA was selected, and 2 μL of nucleic acid was extracted from whole blood for detection. The test results are shown in Table 3.

[0068] Table 3:

[0069] Example 4 (Biologically active substances and protein solubilizers)

[0070] (1) Take 200 μl of EDTA anticoagulated blood and place it in a centrifuge tube. Add 265 μl of lysis binding solution, 40 μl of proteinase K and 20 μl of magnetic bead solution to the centrifuge tube. Heat and mix at 70°C for 10 min. Place the centrifuge tube on a magnetic rack and perform magnetic separation for 20 s. Discard the supernatant.

[0071] The components added to the lysis and binding solution are 0.5% iturin, 2% four-arm-polyethylene glycol-amino, 4 M guanidine hydrochloride, 20 mM Tris-HCl buffer, and 0.5% Tween-20. In addition, the molecular weight of four-arm-polyethylene glycol-amino is 3000;

[0072] (2) Continue to add 1000 μl of washing solution 1 to the centrifuge tube, mix for 1 min, place the centrifuge tube on a magnetic rack, perform magnetic separation for 20 s, and discard the supernatant;

[0073] The washing solution 1 comprises 2M guanidine hydrochloride, 100 mM Tris-HCl and 50% ethanol solution;

[0074] (3) Continue to add 500 μl of washing solution 2 to the centrifuge tube, mix for 1 min, place the centrifuge tube on a magnetic rack, perform magnetic separation for 20 s, discard the supernatant, and repeat the washing once;

[0075] The washing solution 2 comprises 200 mM Tris-HCl and 80% ethanol solution;

[0076] (4) Dry until all the ethanol on the surface of the magnetic beads evaporates, add the eluent, heat and mix at 50-80°C for 5 min, place the centrifuge tube on a magnetic rack, perform magnetic separation for 20 s, and collect the supernatant, which is the nucleic acid;

[0077] The composition of the elution solution is 20 mM Tris-HCl and 10 mM EDTA;

[0078] The above nucleic acid extraction was performed in parallel for 3 groups. In addition, no iturin or four-arm-polyethylene glycol-amino was added to the lysis and binding solution as a control group for comparative extraction.

[0079] After the extraction was completed, the NanoDrop One spectrophotometer was zeroed with the eluent, the interface DNA was selected, and 2 μL of nucleic acid was extracted from whole blood for detection. The test results are shown in Table 4.

[0080] Table 4:

[0081] Example 5 (Comparison with commercially available kits)

[0082] Four whole blood samples were taken, 400 μl of each sample was extracted using a commercial kit (Meiji) and this method for comparison, and each method extracted 200 μl of the same sample. The Meiji extraction method was operated according to the instructions of D6310-01. This method was performed as follows;

[0083] (1) Take 200 μl of EDTA anticoagulated blood and place it in a centrifuge tube. Add 265 μl of lysis binding solution, 40 μl of proteinase K and 20 μl of magnetic bead solution to the centrifuge tube. Heat and mix at 70°C for 10 min. Place the centrifuge tube on a magnetic rack and perform magnetic separation for 20 s. Discard the supernatant.

[0084] The components added to the lysis and binding solution are 0.5% iturin, 2% four-arm-polyethylene glycol-amino, 4 M guanidine hydrochloride, 20 mM Tris-HCl buffer, and 0.5% Tween-20. In addition, the molecular weight of four-arm-polyethylene glycol-amino is 3K;

[0085] (2) Continue to add 1000 μl of washing solution 1 to the centrifuge tube, mix for 1 min, place the centrifuge tube on a magnetic rack, perform magnetic separation for 20 s, and discard the supernatant;

[0086] The washing solution 1 comprises 2M guanidine hydrochloride, 100 mM Tris-HCl and 50% ethanol solution;

[0087] (3) Continue to add 500 μl of washing solution 2 to the centrifuge tube, mix for 1 min, place the centrifuge tube on a magnetic rack, perform magnetic separation for 20 s, discard the supernatant, and repeat the washing once;

[0088] The washing solution 2 comprises 200 mM Tris-HCl and 80% ethanol solution;

[0089] (4) Dry until all the ethanol on the surface of the magnetic beads evaporates, add the elution solution, heat and mix at 50-80℃ for 5 minutes, place the centrifuge tube on the magnetic rack, magnetically separate for 20 seconds, and collect the supernatant, which is the nucleic acid. (Use 50 μl of elution solution for elution);

[0090] The composition of the eluent is 20 mM Tris-HCl and 10 mM EDTA. The commercial kit was compared with this method. After the extraction was completed, the eluent was used to zero the NanoDrop One spectrophotometer, the interface DNA was selected, and 2 μl of nucleic acid was extracted from whole blood for detection. The detection results are shown in Table 5.

[0091] Table 5:

[0092]

[0093] discuss:

[0094] It is not difficult to see from the above embodiments that when only bioactive elements are used in embodiments 1 and 2, the amount and purity of nucleic acid extraction can be increased relative to no addition, but the improvement is limited; and when only protein solubilizer four-arm-polyethylene glycol-amino is used in embodiment 3, the amount and purity of nucleic acid extraction can be increased relative to no addition, but the improvement is also limited; when bioactive elements and four-arm-polyethylene glycol-amino are used simultaneously in embodiment 4, the quality of nucleic acid extraction is significantly improved relative to the control group, and is improved relative to embodiments 1-3, which can explain that bioactive elements and four-arm-polyethylene glycol-amino play a synergistic role in cell lysis and nucleic acid release extraction, and effectively improve the extraction quality of nucleic acid. In addition, the results of A260 / 280 and A260 / 230 and the results of concentration comparison in embodiment 5 show that the quality of nucleic acid extraction is better than this commercial kit.

[0095] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A nucleic acid extraction, lysis and binding solution, characterized in that: The invention is composed of the following components: a bioactive substance with a concentration of 0.1-2% w / v, a four-arm-polyethylene glycol-amino with a concentration of 1-5% w / v, a high-order salt with a concentration of 2-6M, a first buffer with a concentration of 5-200mM and a surfactant with a concentration of 0.1-1% w / v; The bioactive element is selected from at least one of iturin, lichenin and echinocandin; the molecular weight of the four-arm-polyethylene glycol-amino is 2000-3000; the high-order salt is selected from at least one of guanidine hydrochloride, urea, lithium chloride and perchlorate; the first buffer is HEPES, sodium acetate-acetic acid or Tris-HCl buffer; the surfactant is selected from at least one of NP-40, Tween-20, Tween-80, SDS or SLS.

2. A nucleic acid extraction kit, characterized in that: The invention comprises the nucleic acid extraction, lysis and binding solution according to claim 1.

3. The nucleic acid extraction kit according to claim 2, characterized in that It also includes magnetic bead solution, washing solution, elution solution and proteinase K.

4. The nucleic acid extraction kit according to claim 3, characterized in that The washing solution comprises washing solution 1 and washing solution 2; the washing solution 1 contains 1-3M high-order salt, 5-200mM second buffer, 1-100mM inorganic salt ions and 6-20% precipitant; the washing solution 2 contains 5-200mM second buffer, 1-100mM inorganic salt ions and 30%-80% precipitant.

5. The nucleic acid extraction kit according to claim 4, characterized in that The eluent contains 5-200 mM of the second buffer and 1-100 mM of inorganic salt ions.

6. The nucleic acid extraction kit according to claim 4 or 5, characterized in that: The second buffer is selected from at least one of HEPES, sodium acetate-acetic acid or Tris-HCl buffer; And / or, the inorganic salt ion is selected from at least one of sodium chloride, potassium chloride, sodium acetate or sodium citrate; And / or, the precipitant is selected from ethanol or isopropanol.

7. A method for extracting nucleic acid, characterized in that the steps include: The nucleic acid extraction, lysis and binding solution of claim 1 is mixed with the sample, proteinase K and magnetic bead solution, and the mixture is lysed at 50 to 80° C. for 5 to 20 minutes, and the magnetic bead-nucleic acid complex is collected; Wash the magnetic bead-nucleic acid complex with a washing solution, and magnetically absorb and collect the magnetic bead-nucleic acid complex; Add elution solution to the magnetic bead-nucleic acid complex, elute at 50-80°C for 2-5 minutes, and collect the nucleic acid.

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