CN108531472B - Lysis solution for nucleic acid extraction - Google Patents
Lysis solution for nucleic acid extraction Download PDFInfo
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- CN108531472B CN108531472B CN201710268530.8A CN201710268530A CN108531472B CN 108531472 B CN108531472 B CN 108531472B CN 201710268530 A CN201710268530 A CN 201710268530A CN 108531472 B CN108531472 B CN 108531472B
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The invention provides a lysis solution for nucleic acid extraction, which comprises 30-70% of chaotropic salt with high mass concentration, 0.05-20% of anionic surfactant and 0.05-20% of nonionic surfactant. The invention separates and purifies the nucleic acid simply and effectively through the composition and the magnetic beads, does not need the steps of centrifugation, transfer and the like, reduces the loss of the nucleic acid to the maximum extent, can obtain the nucleic acid with high concentration and high purity by optimizing the formula of the composition, can separate and purify the nucleic acid from the body fluid such as blood and the like, can separate and purify virus nucleic acid, and can directly apply the obtained nucleic acid substance to downstream experiments such as Polymerase Chain Reaction (PCR), enzyme digestion reaction, library construction and the like.
Description
Technical Field
The invention relates to the field of nucleic acid extraction, and in particular relates to lysis solution for nucleic acid extraction.
Background
The separation and purification of nucleic acid with high quality is a very critical step in molecular biology experiments. Various methods for isolating and purifying nucleic acids from biological samples have been reported, such as methods for isolating and purifying nucleic acids from samples of blood, plasma, serum, cultured cells, plants, animals, and human tissues.
The separation of nucleic acid mainly refers to the separation of nucleic acid from biological macromolecular substances such as protein, polysaccharide, fat and the like. When separating nucleic acid, the integrity of the primary structure of nucleic acid molecule is ensured, and other molecule pollution is eliminated. The general method for separating and purifying nucleic acid includes several main steps, such as lysis, separation of nucleic acid from other biomacromolecule substances, and purification of nucleic acid.
1. Cracking: the nucleic acid must be released from the cell or other biological material. Cell lysis can be achieved by mechanical action such as ultrasonic lysis, chemical action, enzymatic action, and the like. Extracellular nucleic acids are typically encapsulated in lipid vesicles and also require a lysis process to release them completely.
2. Separation and purification of nucleic acid: nucleic acids are separated and purified by methods such as selective precipitation, chromatography, density gradient centrifugation, affinity adsorption, etc. depending on differences in their physicochemical properties, although some methods require harmful organic solvents, the goal is to obtain high quality nucleic acids. The magnetic bead method adopts nanometer magnetic beads, the surfaces of the magnetic beads are provided with functional groups combined with nucleic acid, and the magnetic beads can perform adsorption reaction with the nucleic acid under certain conditions. The magnetic beads can be aggregated under the action of magnetic force, so that the manual operation process required by centrifugation and the like can be eliminated.
Disclosure of Invention
The present invention provides a lysis solution, particularly a method for separating extracellular nucleic acid from blood, plasma and body fluid samples. In the present invention, the magnetic beads form complexes with nucleic acids in the biological sample in a specific matrix containing chaotropic salts (chaotropic salts) that can destabilize molecular forces (hydrogen bonds) and render hydrophobic proteins more water soluble. Chaotropic salts can disrupt and denature the three-dimensional structure of macromolecules, such as proteins, DNA, RNA; chaotropic salts interfere with intermolecular interactions formed by non-covalent bonds (forces), such as hydrogen bonding, van der waals forces, hydrosulfuric interactions; common chaotropic salts include: guanidine thiocyanate, guanidine hydrochloride, potassium thiocyanate, LiCl and NaClO4And the like. Then, the nucleic acid-magnetic bead complexes are separated from the biological sample by an external magnetic force, and the separated nucleic acids are eluted from the nucleic acid-magnetic bead complexes by a specific salt solution. In order to achieve the above object, the present invention provides the following technical solutions.
In one embodiment, a lysis solution for nucleic acid extraction is provided, comprising 30-70% by mass of a chaotropic salt, 0.05-20% by mass of an anionic surfactant and 0.05-20% by mass of a nonionic surfactant.
In one embodiment, the chaotropic salt is one or more of guanidine hydrochloride, guanidine thiocyanate, guanidine isothiocyanate, potassium thiocyanate, lithium chloride, sodium perchlorate, and the chaotropic salt is present in a concentration of 40 to 65% by mass, preferably 50 to 60% by mass.
In one embodiment, the anionic surfactant is one or more of Sodium Dodecyl Sulfate (SDS) and Sodium Laurate (SLS), at a mass concentration of 0.1 to 20%, preferably 0.2 to 10%; the nonionic surfactant is one or more of polyoxyethylene type or polyhydric alcohol type nonionic surfactants, and the mass concentration is 0.1-20%, preferably 0.2-10%.
In one embodiment, the nonionic surfactant is one or more of Brij-58, polidocanol, TritonX-100, NP-40, Tween-20, Tween-80, polyoxyethylene ether.
In one embodiment, the lysis solution comprises guanidine hydrochloride at a mass concentration of 52%, Triton X-100 at a mass concentration of 10% and sodium laurate at a mass concentration of 20%; or guanidinium isothiocyanate with mass concentration of 36%, Brij-58 with mass concentration of 10% and sodium laurate with mass concentration of 10%; or guanidinium isothiocyanate with mass concentration of 40%, Brij58 with mass concentration of 1% and sodium laurate with mass concentration of 1%; or 40% of guanidinium isothiocyanate, 20% of TWEEN-20 and 0.2% of sodium laurate; or guanidinium isothiocyanate with a mass concentration of 40%, polidocanol with a mass concentration of 1%, and SDS with a mass concentration of 0.1%.
The invention also provides a nucleic acid extraction kit, which comprises the lysate.
Further, the invention also provides an extracellular nucleic acid separation and purification method, which comprises the following steps: 1) releasing extracellular nucleic acid by the lysis solution; 2) adsorbing extracellular nucleic acids with a hydroxylated solid phase support; and 3) eluting the extracellular nucleic acid by eluent.
The nucleic acid isolated according to the invention is preferably a deoxyribonucleic acid (DNA). The magnetic beads used according to the invention are preferably silica-coated magnetic beads. In the present invention, first, a biological sample is mixed with the composition of the present invention and magnetic beads; the nucleic acids in the mixture are adsorbed to magnetic beads, which are then separated from the mixture by magnetic force. Finally, the nucleic acid on the magnetic beads is eluted with an eluent. In addition, the nucleic acid extraction kit of the present invention further comprises other components required for separating and purifying nucleic acid, including: magnetic bead suspension, wash solution and eluent, etc.
The invention provides a simple and effective method for separating and purifying nucleic acid in a biological sample, the nucleic acid is simply and effectively separated and purified by a composition and magnetic beads, steps such as centrifugation, transfer and the like are not needed, the loss of the nucleic acid is reduced to the maximum extent, the high-concentration and high-purity nucleic acid can be obtained by optimizing the formula of the composition, the nucleic acid can be separated and purified from body fluid such as blood and the like, the virus nucleic acid can also be separated and purified, and the obtained nucleic acid substance can be directly applied to downstream experiments such as Polymerase Chain Reaction (PCR), enzyme digestion reaction, library construction and the like, so the invention provides a simple and effective method for separating and purifying the nucleic acid with wider application range.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described below with reference to the following embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Example 1 extraction of nucleic acids from human plasma
10ml of blood is collected from a cancer patient and is evenly mixed in a Streck blood collection tube for 8 to 10 times in a reversed way, so that the blood is fully mixed with the protective agent in the blood collection tube. Separating plasma according to plasma separation step, centrifuging at 4 deg.C and 2000g for 10min, transferring supernatant into new tube, centrifuging at 4 deg.C and 16000g for 10min, transferring supernatant into new tube, and storing the obtained plasma specimen in refrigerator at-70 deg.C for a long period.
The nucleic acid extraction of the experimental group was performed according to the following steps: to 1ml of plasma was added 10. mu.L of proteinase K solution, followed by addition of 1.5 sample volumes of lysis buffer (50% guanidinium isothiocyanate, 15% NP-40, 2% SDS), and after mixing, the mixture was allowed to stand at room temperature for 5 min. 3/4 sample volumes of isopropanol and 30-50. mu.L of magnetic bead suspension (Thermo Fisher, 37002D) were added. Mix by inversion several times. The tube was placed on a vertical mixer and mixed for 10min at room temperature. Placing the centrifuge tube in a magnetic frame, performing magnetic separation, and removing the supernatant by a liquid transfer device. Then, 1mL of a washing solution (30% guanidine hydrochloride, 50% isopropyl alcohol) was added thereto, the magnetic beads were resuspended, the mixture was transferred to a 1.5mL centrifuge tube, and the tube was allowed to stand on a magnetic frame for 2min for magnetic separation, and the supernatant was discarded. The washing step was repeated 2 times. Then 30. mu.L of eluent (10mM Tris HCl pH8.0) was added, and after mixing with the magnetic beads, the mixture was incubated at 70 ℃ for 10 min. Placing the centrifuge tube in a magnetic frame, performing magnetic separation, carefully sucking the supernatant into a new centrifuge tube, and storing the obtained nucleic acid at-20 deg.C for a long time at-80 deg.C. The plasma was extracted simultaneously with a commercial kit (Thermo Fisher, 37011D) according to the instructions, and used as a control group.
The extracted nucleic acids were quantified using a qubit3.0 fluorimeter (Thermo Fisher) and the DNA concentrations determined were: the experimental group was 0.43 ng/. mu.l, and the control group was 0.31 ng/. mu.l. Extracting nucleic acids from different biological samples (such as blood, urine, interstitial fluid, other body fluids, etc.), wherein the above buffers (including lysis solution, washing solution, etc.) can be adjusted properly, including adjusting concentration, pH, solvent addition amount, etc.
Example 2 efficiency of lysate extraction
The efficiency of lysis to release nucleic acids depends mainly on the concentration of chaotropic salts, the type and amount of surfactants. Preparation of lysate buffers of various formulations 1ml of plasma DNA was extracted according to the nucleic acid extraction method of example 1, the DNA was bound to magnetic beads (Thermo Fisher, 37002D), then the DNA was eluted with an eluent (10mM Tris HCl pH8.5), and the concentration of the obtained DNA was measured by Qubit, and the results are shown in Table 1 below.
TABLE 1 extraction efficiency of different lysates
As can be seen from Table 1, the use of a combination of two different surfactants as the lysate component provides a better extraction than the use of one surfactant, with a significant difference (P < 0.001).
Example 3 extraction of nucleic acids from blood
To 200. mu.l of EDTA anticoagulated blood was added 600. mu.l of erythrocyte lysate (RT122, Tiangen), mixed by inversion, and left at room temperature for 5min, during which mixed by inversion was repeated several times. Centrifuging at 10000rpm for 1min, discarding the supernatant to leave leukocyte precipitate, adding 200 μ l PBS, and shaking to mix thoroughly. Then 20. mu.l proteinase K solution was added to the cell suspension and mixed well. Then, 300. mu.l of a lysis solution (40% guanidine isothiocyanate, 10% Triton X-100, 5% SDS) was added thereto, and after mixing, the mixture was allowed to stand at room temperature for 5 min. Then, the nucleic acid extraction was performed according to the nucleic acid extraction procedure in example 1. Finally, the nucleic acid was eluted with 100. mu.l of an eluent. The extracted nucleic acid is stored at-20 deg.C. The product is stored at-80 ℃ for a long time. The concentration of nucleic acid determined by NanoDrop ND-1000(NanoDrop) was 52.5 ng/. mu.l, and the purity was OD260/280 ═ 1.82.
It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Claims (3)
1. A lysis solution for nucleic acid extraction comprises guanidine hydrochloride with the mass concentration of 52%, Triton X-100 with the mass concentration of 10% and sodium laurate with the mass concentration of 20%.
2. A nucleic acid extraction kit comprising the lysate of claim 1.
3. An extracellular nucleic acid separation and purification method, which comprises the following steps:
1) releasing extracellular nucleic acid from the lysate of claim 1;
2) adsorbing extracellular nucleic acids with a hydroxylated solid phase support; and
3) extracellular nucleic acids were eluted by eluent.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109722431B (en) * | 2019-01-21 | 2024-01-23 | 上海科华生物工程股份有限公司 | Non-alcohol virus nucleic acid extraction kit based on magnetic bead method |
| CN109750030A (en) * | 2019-01-21 | 2019-05-14 | 上海科华生物工程股份有限公司 | A kind of quick, nucleic acid extraction kit for not heating based on paramagnetic particle method |
| CN109628313A (en) * | 2019-01-24 | 2019-04-16 | 长春万成生物电子工程有限公司 | A kind of E. coli lysate and its preparation method and application |
| CN110129312B (en) * | 2019-04-03 | 2021-02-05 | 深圳市芯思微生物科技有限公司 | Lysate and nucleic acid extraction kit |
| CN110747255B (en) * | 2019-11-27 | 2023-08-22 | 郑州安图生物工程股份有限公司 | Pretreatment reagent and method for whole blood sample for nucleic acid detection |
| CN113073096B (en) * | 2021-03-31 | 2023-02-03 | 北京源微生物科技有限公司 | Method and reagent for separating and enriching pathogenic microorganisms in blood and extracting nucleic acid |
| CN113881663A (en) * | 2021-10-25 | 2022-01-04 | 苏州缔因安生物科技有限公司 | Nucleic acid extraction method based on ultrasonic waves |
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