Plasma free nucleic acid co-extraction reagent combination and application thereof

Abstract

The invention discloses a plasma free nucleic acid co-extraction reagent combination and application thereof, and belongs to the technical field of molecular biology. The plasma free nucleic acid co-extraction composition comprises a first lysis solution, a second lysis solution, a binding solution and a magnetic bead dispersion solution, and further comprises an equilibrium solution, a protein cleaning solution, a rinsing solution, a first eluent and / or a second eluent. The kit prepared by utilizing the reagent combination disclosed by the invention is combined with the extraction method disclosed by the invention, so that high-quality and high-purity free nucleic acid can be extracted from a plasma sample, the problem of co-extraction of the free nucleic acid is solved, and the utilization rate of the sample is increased.

Description

[0001] Related patents This application is a divisional application of the Chinese invention patent application with application number 2022112015356, application date September 29, 2022, and invention name “A plasma free nucleic acid co-extraction reagent combination, kit and extraction method”. Technical Field

[0002] The invention belongs to the technical field of molecular biology, and in particular, relates to a plasma free nucleic acid co-extraction reagent combination and application thereof. Background Art

[0003] cfDNA (cell free DNA), also known as circulating DNA, refers to DNA molecules that are free from cells. These DNA molecules mainly come from certain specific release processes of normal cells, cell apoptosis, cell necrosis, and release during the growth and infiltration of cancer cells. Their size is mainly concentrated in the range of 160-170 to hundreds of bp, and their half-life is about several hours.

[0004] The level and type of cfDNA in the blood are associated with a variety of diseases, including tumors. In addition, fetal cfDNA can also be found in maternal blood. cfDNA testing in the blood has been widely used for screening of various cancers, certain autoimmune diseases such as lupus erythematosus, and non-invasive testing of fetal genetic diseases such as Down syndrome.

[0005] Free circulating RNA is a type of RNA that exists in an extracellular free state in the body fluids of animals, plants and humans. Some studies have reported that free circulating RNA-protein complexes can be used as a potential tumor marker; studies have shown that pregnancy-related diseases such as intrauterine growth retardation, fetal aneuploidy and preeclampsia are closely related to free RNA in maternal blood, indicating that free RNA in the blood of pregnant women can be used as a new fetal genetic information material.

[0006] However, the content of free nucleic acids in the blood is low and varies greatly from individual to individual. The content of free nucleic acids in 1 mL of plasma may range from a few ng to dozens of ng. For example, a prenatal screening test requires about 10 mL of blood sample just to extract cfDNA. Ordinary test kits can only extract one of the free nucleic acids, DNA or RNA, and the sample cannot be fully utilized. Summary of the invention

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions: The first aspect of the present invention provides a plasma free nucleic acid co-extraction reagent combination, comprising a first lysate, a second lysate, a binding solution and a magnetic bead dispersion solution, wherein the first lysate contains 2-4% SDS, 80-100mM EDTA, 50-100mM Tris-HCl, and the pH is 6.0-6.8; SDS denatures and precipitates proteins in the sample and depolymerizes nucleoproteins bound to nucleic acids; EDTA is used to inhibit nucleases that may exist in the sample and protect free nucleic acids; Tris-HCl is used to provide a suitable acidic pH environment so that RNA is dissolved in an aqueous phase and DNA is dissolved in an organic phase.

[0008] The second lysis solution is water-saturated phenol, which is used for denaturing and separating proteins and providing an organic phase.

[0009] The binding solution contains 40-60% isopropanol and 1-2M NaCl; wherein the isopropanol is used to absorb water in the solution to precipitate the nucleic acid; and the NaCl is used to provide the sodium ions required for the nucleic acid to bind to the magnetic beads.

[0010] The concentration of the magnetic beads in the magnetic bead dispersion is 50-80 mg / mL, and the magnetic beads are used to adsorb DNA.

[0011] Optionally, the plasma free nucleic acid co-extraction reagent combination further comprises a balancing solution, which is a NaOH solution for adjusting the pH value. Preferably, the concentration of the NaOH solution is 1%.

[0012] Optionally, the plasma free nucleic acid co-extraction reagent combination further includes a protein cleaning solution, which contains 40-60% guanidine hydrochloride and 40-60% anhydrous ethanol, wherein guanidine hydrochloride is used to dissolve protein and anhydrous ethanol is used to clean protein.

[0013] Optionally, the plasma free nucleic acid co-extraction reagent combination further comprises a rinsing solution, which comprises 0.2M Tris-HCl and 60-80% anhydrous ethanol, and is used for rinsing the magnetic beads.

[0014] Optionally, the plasma free nucleic acid co-extraction reagent combination further comprises a first eluent and a second eluent, wherein the first eluent is nuclease-free water, and the second eluent is 5-10 mM Tris-HCl. Preferably, the pH of the Tris-HCl is 8.0.

[0015] The second aspect of the present invention provides a plasma free nucleic acid co-extraction kit, comprising any plasma sample cfDNA extraction reagent combination described in the first aspect of the present invention.

[0016] In particular, in a specific embodiment of the present invention, the plasma free nucleic acid co-extraction kit comprises a lysis solution, a binding solution, a magnetic bead dispersion solution, a protein cleaning solution, a rinsing solution and an eluent, wherein the lysis solution contains 2-4% SDS, 50-100mM EDTA, 0.5-1M Tris-HCl, 1-2M NaCl and 10-20% glycerol; the binding solution contains 40-60% isopropanol and 1-2M NaCl; the concentration of magnetic beads in the magnetic bead dispersion solution is 50-80mg / mL; the protein cleaning solution contains 40-60% guanidine hydrochloride and 40-60% anhydrous ethanol; the rinsing solution includes 0.2M Tris-HCl and 60-80% anhydrous ethanol, and the eluent is 5-10mM Tris-HCl.

[0017] The third aspect of the present invention provides a method for co-extraction of plasma free nucleic acids, which is performed using the plasma free nucleic acid co-extraction kit of the second aspect of the present invention, comprising the following steps: S1, adding 0.2-0.4 times the volume of the first lysate to the plasma sample, mixing, adding the second lysate of the same volume as the plasma sample, incubating at 50-80°C, ≥1000 rpm, shaking for 10-20 min; S2, centrifuge at 10000-15000g for 5-15min, aspirate the aqueous supernatant into a new sample tube, named sample tube 1, add 0.2-0.4 times the volume of the plasma sample of the first lysate into the lower organic phase, and then add the balance solution with the same volume as the plasma sample, named sample tube 2; S3, process sample tube 1 and sample tube 2 separately: S31, add 1.5~2.5 times the volume of plasma sample binding solution to sample tube 1, add 0.1~0.2 times the volume of supernatant magnetic beads, mix thoroughly and incubate at room temperature for more than 10 minutes, S32, after sample tube 2 is vigorously shaken and mixed at room temperature, it is allowed to stand at room temperature for 3-10 minutes, centrifuged at 10000-15000g for 8-15 minutes, and the aqueous supernatant is aspirated into a new sample tube, named sample tube 3, and 1.5-2.5 times the volume of the plasma sample binding solution and 0.1-0.2 times the volume of the supernatant magnetic beads are added to sample tube 3, and after being fully mixed, it is incubated at room temperature for more than 10 minutes; S4, place sample tube 1 and sample tube 3 on a magnetic rack, let stand at room temperature for 3-10 min, and discard the supernatant; S5, add 80-150 μL of protein cleaning solution to sample tube 1 and sample tube 3, resuspend the magnetic beads, and let stand at room temperature for 5-15 min; S6, add 180-250 μL of rinsing solution to sample tube 1 and sample tube 3, let stand for more than 10 seconds, discard the supernatant and dry; S7, add 20~30μL of the first elution solution to sample tube 1, add 20~30μL of the second elution solution to sample tube 3, resuspend the magnetic beads by pipetting, let stand at room temperature for 5~15 minutes, place sample tube 1 and sample tube 3 on the magnetic rack, let stand at room temperature for 5~15 minutes, aspirate the supernatant to obtain the cfDNA solution.

[0018] In some embodiments of the present invention, in step S1, a lysis solution with a volume of 0.25 to 0.3 times the volume of the plasma sample is added to the plasma sample.

[0019] In some embodiments of the present invention, in step S1, the mixture is mixed and then incubated in a metal bath at 70° C. for 15 min.

[0020] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial technical effects: By using the reagent combination or kit of the present invention in combination with the extraction method of the present invention, high-quality and high-purity free nucleic acids can be extracted from plasma samples, solving the problem of co-extraction of free nucleic acids and increasing the utilization rate of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The cfDNA quality control result of sample S1 extracted using the reagent combination of the present invention is shown.

[0022] Figure 2 The cfDNA quality control result of sample S2 extracted using the reagent combination of the present invention is shown.

[0023] Figure 3 The cfRNA quality control result of sample S1 extracted using the reagent combination of the present invention is shown.

[0024] Figure 4 The cfRNA quality control result of sample S2 extracted using the reagent combination of the present invention is shown. DETAILED DESCRIPTION

[0025] Unless otherwise indicated, implied from the context, or customary in the prior art, all parts and percentages in this application are based on weight, and the tests and characterization methods used are all synchronized with the filing date of this application. Where applicable, the contents of any patent, patent application or disclosure involved in this application are fully incorporated herein by reference, and their equivalent patent families are also introduced as references, especially the definitions of relevant terms in the art disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0026] Numerical ranges in this application are approximate values, so unless otherwise specified, they may include values ​​outside the range. Numerical ranges include all values ​​from the lower limit to the upper limit increased by 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For a range containing a value less than 1 or containing a fraction greater than 1 (e.g., 1.1, 1.5, etc.), 1 unit is appropriately regarded as 0.0001, 0.001, 0.01 or 0.1. For a range containing a single digit less than 10 (e.g., 1 to 5), 1 unit is generally regarded as 0.1. These are only specific examples of what is intended to be expressed, and all possible combinations of the values ​​between the lowest and highest values ​​listed are considered to be clearly recorded in this application.

[0027] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes, and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, unless explicitly stated, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, adjuvants or compounds. On the contrary, except for those necessary for operating performance, the term "essentially consisting of..." excludes any other components, steps or processes from the scope of any description of the term below. The term "consisting of..." does not include any components, steps or processes that are not specifically described or listed. Unless explicitly stated, the term "or" refers to the listed individual members or any combination thereof.

[0028] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0029] Example The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0031] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0032] The experimental methods not specifically described in the following examples are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0033] Example 1 Plasma sample free nucleic acid extraction reagent combination This embodiment provides a plasma sample free nucleic acid extraction reagent combination, including lysis solution 1, lysis solution 2, balancing solution, binding solution, magnetic beads, protein cleaning solution, rinsing solution, eluent 1, and eluent 2, a total of nine reagents, and the components of each reagent are as follows: 1. Lysis Buffer 1 Composition: 4% SDS, 100 mM EDTA (pH 8.0), 80 mM Tris-HCl (pH 8.0).

[0034] Among them, SDS denatures and precipitates the proteins in the sample and dissociates the nucleoproteins bound to nucleic acids; EDTA is used to inhibit nucleases that may exist in the sample and protect free nucleic acids; Tris-HCl is used to provide a suitable acidic pH environment to separate RNA and DNA into aqueous and organic phases, respectively.

[0035] 2. Lysis Buffer 2 Water-saturated phenol, used to denature and separate proteins while providing an organic phase.

[0036] 3. Balance solution 0.1% sodium hydroxide is used to adjust the pH value of the solution.

[0037] 4. Binding fluid Composition: 50% isopropanol, 1M NaCl.

[0038] Among them, isopropanol is used to absorb water in the solution to precipitate nucleic acids; NaCl is used to provide sodium ions required for nucleic acids to bind to magnetic beads.

[0039] 5. Magnetic beads Monodisperse magnetic microspheres with a particle size of 1-3 μm and a concentration of 80 mg / mL are used to adsorb DNA.

[0040] 6. Protein cleaning solution Components: 50% guanidine hydrochloride, 50% anhydrous ethanol.

[0041] Among them, guanidine hydrochloride is used to dissolve protein; anhydrous ethanol is used to wash protein.

[0042] 7. Rinse solution Components: 0.2M Tris-HCl (pH 8.0), 80% anhydrous ethanol.

[0043] Among them, Tris-HCl is also used to provide a pH environment suitable for DNA stability, and anhydrous ethanol is used to clean proteins.

[0044] 8. Elution 1 Nuclease-free water for eluting RNA.

[0045] 9. Elution 2 Composition: 10 mM Tris-HCl (pH 8.0).

[0046] The above 9 reagents are combined to prepare a plasma sample free nucleic acid extraction kit.

[0047] Example 2 Extraction of free nucleic acids from plasma samples In this example, the reagent combination of Example 1 was used to extract free nucleic acids from two plasma samples (numbered S1 and S2), and the steps were as follows: (1) Add 100 μL of lysis solution 1 to 400 μL of plasma sample. After thorough mixing, add 400 μL of lysis solution 2 and shake vigorously in a metal bath at 70°C and ≥1000 rpm for 15 min.

[0048] (2) Centrifuge at 12000g for 10 min, aspirate the aqueous supernatant into a new sample tube, named Sample Name-1, and add 100 μL of Lysis Solution 1 and 400 μL of Balance Solution to the lower organic phase, named Sample Name-2. (3) Add 800 μL of binding solution and 80 μL of magnetic beads to sample tube 1, mix thoroughly and incubate at room temperature for more than 10 min.

[0049] (4) After vigorous shaking and mixing at room temperature, sample tube 2 was allowed to stand at room temperature for 5 min, and then centrifuged at 12,000 g for 10 min. The aqueous supernatant was aspirated into a new sample tube and named sample name-3.

[0050] (5) Add 800 μL of binding solution and 80 μL of magnetic beads to sample tube 3, mix thoroughly and incubate at room temperature for more than 10 min.

[0051] (6) Place sample tube 1 and sample tube 3 on the magnetic rack and let stand at room temperature for 5 min.

[0052] (7) Aspirate and discard the supernatant.

[0053] (8) Remove sample tube 1 and sample tube 3 from the magnetic rack, add 100 μL of protein cleaning solution, resuspend the magnetic beads, and let stand at room temperature for 10 min.

[0054] (9) Place sample tube 1 and sample tube 3 on the magnetic rack and let stand at room temperature for 5 min.

[0055] (10) Aspirate and discard the supernatant.

[0056] (11) Place sample tube 1 and sample tube 3 on a magnetic rack, add 100 μL of rinse solution, let stand for more than 10 seconds, and discard the supernatant.

[0057] (12) Repeat step (10) to ensure that the supernatant is completely discarded.

[0058] (13) Place sample tubes 1 and 3 on the magnetic rack and dry them for 2 minutes with the lid open. (13) Remove sample tube 1 and sample tube 3 from the magnetic rack, add 20 μL of eluent 1 to sample tube 1 and 20 μL of eluent 2 to sample tube 3, pipette to resuspend the magnetic beads, and let stand at room temperature for 5 min. (14) Place sample tubes 1 and 3 on a magnetic rack and let them stand at room temperature for 5 min.

[0059] (15) Aspirate the supernatant. The supernatant in tube Sample Name-1 is the extracted cfRNA, named Sample Name-R. The supernatant in tube Sample Name-3 is the extracted cfDNA, named Sample Name-D. They can be used for subsequent experiments or stored at -80°C.

[0060] (16) Sample quantification and quality inspection: The extracted cfDNA was quantified using the Qubit™ dsDNA HS kit and Qubit 2.0 instrument, the sample purity was determined using Nanodrop1000, and the DNA fragment distribution was quality checked using the Agilent High Sensitivity DNA Kit and Agilentbioanalyzer 2100 instrument.

[0061] The extracted cfRNA was quantified using the Qubit™ RNA HS Assay Kit and the Qubit 2.0 instrument, the sample purity was determined using the Nanodrop 1000, and the RNA fragment distribution was quality checked using the Agilent RNA 6000 Pico Kit and the Agilent bioanalyzer 2100 instrument.

[0062] Table 1 Sample quality inspection results

[0063] The fragment distribution of cfDNA extracted from sample S1 and sample S2 are as follows: Figure 1 and Figure 2As shown in Figure 2, the fragment distribution of cfRNA extracted from samples S1 and S2 is shown in Figure 2. Figure 3 and Figure 4 shown.

[0064] Depend on Figure 1~Figure 4 It can be seen that by using the reagent combination of Example 1, very high-quality free cfDNA and cfRNA can be obtained.

[0065] Example 3 Optimization of plasma sample cfDNA extraction kit The inventors optimized the components or contents in the reagent combination of Example 1. It was proved that the reagent combination of Example 1 achieved unexpected technical effects in extracting plasma free nucleic acids. To further prove this conclusion, the inventors listed the results of extracting free nucleic acids from 400 μL plasma sample S1 after the relevant ratios were changed in Table 2.

[0066] Table 2 Effects of changes in reagent combination ratio or proportion on free nucleic acid extraction

[0067] It can be seen from Table 2 that any change in the components of any reagent in the reagent combination in Example 1 will lead to a significant decrease in the total amount of cfDNA finally extracted. It can be seen that the reagents in the reagent combination of Example 1 cooperate and interact with each other to achieve an amazing technical effect of extracting free nucleic acids from plasma samples.

[0068] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A plasma free nucleic acid co-extraction reagent combination, characterized in that: The reagent combination includes a first lysate, a second lysate, a binding solution and a magnetic bead dispersion solution, wherein the first lysate components are: 2-4% SDS, 80-100mM EDTA, 50-100mM Tris-HCl, pH 6.0-6.8; the second lysate is water-saturated phenol, the binding solution components are: 40-60% isopropanol and 1-2M NaCl; the concentration of magnetic beads in the magnetic bead dispersion solution is 50-80mg / mL.

2. The plasma free nucleic acid co-extraction reagent combination according to claim 1, characterized in that: The method further comprises a balancing solution, which is a NaOH solution.

3. The plasma free nucleic acid co-extraction reagent combination according to claim 1, characterized in that: The invention also comprises a protein cleaning solution, wherein the components of the protein cleaning solution are: 40-60% guanidine hydrochloride and 40-60% anhydrous ethanol.

4. The plasma free nucleic acid co-extraction reagent combination according to claim 1, characterized in that: The invention also comprises a rinsing solution, wherein the rinsing solution comprises: 0.2M Tris-HCl and 60-80% anhydrous ethanol.

5. The plasma free nucleic acid co-extraction reagent combination according to claim 1, characterized in that: The system also includes a first eluent and a second eluent, wherein the first eluent is nuclease-free water and the second eluent is 5-10 mM Tris-HCl.

6. Use of the reagent combination according to any one of claims 1 to 5 in preparing a kit for extracting free nucleic acids from plasma.

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