Cracking / binding liquid, cleaning liquid, kit and method for nucleic acid extraction

Abstract

The invention relates to a lysis / binding solution for nucleic acid extraction. The lysis / binding solution comprises a protein denaturant, an alcohol-free dehydrating agent, a first surfactant, a buffer solution, ethylenediamine tetraacetic acid and chaotropic salt. The molar concentration range of the protein denaturant is 3.5 mol / L-5. 5mol / L, the mass volume ratio range of the alcohol-free dehydrating agent is 0.1%-1%, the mass volume ratio range of the first surfactant is 0.05%-0.5%, the molar concentration range of the buffer solution is 5mmol / L-150mmol / L, the molar concentration range of the ethylenediamine tetraacetic acid is 5mmol / L-20mmol / L, and the mass volume ratio range of chaotropic salt is 1%-8%.

Description

Lysis / binding solution, cleaning solution, kit and method for nucleic acid extraction Technical Field

[0001] The present disclosure relates to the field of molecular diagnostic technology, and in particular to a lysis / binding solution, a cleaning solution, a kit, and a method for nucleic acid extraction. Background Art

[0002] Molecular diagnostics, using nucleic acids as detection targets, are primarily used in various clinical diagnostic areas, such as tumors, infections, and genetics. Nucleic acid extraction is fundamental to all these processes. Under specific conditions, the surface of adsorption carrier magnetic beads coated with groups such as silicon, amino, or carboxyl groups, or solid-phase silica membrane carriers, enables specific binding of nucleic acids to the beads through electrostatic, hydrophobic, and hydrogen bonding interactions. Several washes are then performed to remove nonspecific impurities and salts, and the nucleic acids are then eluted from the adsorption carrier for purification.

[0003] Summary of the Invention

[0004] In one aspect, a lysis / binding solution for nucleic acid extraction is provided, the lysis / binding solution comprising: a protein denaturant, an alcohol-free dehydrating agent, a first surfactant, a buffer, ethylenediaminetetraacetic acid, and a chaotropic salt. The molar concentration of the protein denaturant ranges from 3.5 mol / L to 5.5 mol / L, the mass volume percentage of the alcohol-free dehydrating agent ranges from 0.1% to 1%, the mass volume percentage of the first surfactant ranges from 0.05% to 0.5%, the molar concentration of the buffer ranges from 5 mmol / L to 150 mmol / L, the molar concentration of the ethylenediaminetetraacetic acid ranges from 5 mmol / L to 20 mmol / L, and the mass volume percentage of the chaotropic salt ranges from 1% to 8%.

[0005] In some embodiments, the anions in the chaotropic salt include: PO4 3- 、SO4 2- 、H2PO4 - HCOO - 、Cl - 、NO3 - CF3COO - 、BF4 - 、ClO4 - and PF6 - At least one of .

[0006] In some embodiments, the cations in the chaotropic salt include: Ca 2+ Mg 2+ 、Li + 、Na + , K + NH4 +, at least one of an imidazolium cation and an N-ethylpyridinium cation.

[0007] In some embodiments, the chaotropic salt includes at least one of ammonium phosphate, ammonium sulfate, ammonium dihydrogen phosphate, ammonium chloride, calcium hexafluorophosphate, potassium hexafluorophosphate, sodium hexafluorophosphate, ammonium hexafluorophosphate, 1-allyl-3-methylimidazolium hexafluorophosphate, N-ethylpyridinium hexafluorophosphate, and N-ethylpyridinium chloride.

[0008] In some embodiments, the alcohol-free dehydrating agent includes at least one of polyoxyethylene lauryl ether, polyvinyl carbazole, polyvinyl alcohol, polyacrylamide, polyacrylic acid, polyoxyethylene sorbitan monolaurate, and sodium lauryl sulfate.

[0009] In some embodiments, the protein denaturant comprises at least one of guanidine isothiocyanate and guanidine hydrochloride.

[0010] In some embodiments, the buffer comprises: at least one of TE buffer and tris(hydroxymethyl)aminomethane; the TE buffer comprises: tris(hydroxymethyl)aminomethane hydrochloride and ethylenediaminetetraacetic acid.

[0011] In some embodiments, the first surfactant includes polyoxyethylene sorbitan monolaurate or polyethylene glycol 4-isooctylphenyl ether.

[0012] In some embodiments, the pH of the lysis / binding solution ranges from 4.4 to 7.4.

[0013] In some embodiments, the lysis / binding solution comprises: guanidine isothiocyanate, sodium lauryl sulfate, polyoxyethylene sorbitan monolaurate, TE buffer, ethylenediaminetetraacetic acid, and N-ethylpyridine hexafluorophosphate. Guanidine isothiocyanate is a protein denaturant, sodium lauryl sulfate is an alcohol-free dehydrating agent, and polyoxyethylene sorbitan monolaurate is a first surfactant. The molar concentration of guanidine isothiocyanate is 5 mol / L, the mass volume ratio of sodium lauryl sulfate is 0.5%, the mass volume ratio of polyoxyethylene sorbitan monolaurate is 0.1%, the molar concentration of TE buffer is 100 mmol / L, the molar concentration of ethylenediaminetetraacetic acid is 10 mmol / L, and the mass volume ratio of N-ethylpyridine hexafluorophosphate is 2%. The pH of the lysis / binding solution is 7.4.

[0014] In some embodiments, the pH of the buffer solution ranges from 7.0 to 7.4.

[0015] In some embodiments, the pH of the buffer is 7.2.

[0016] On the other hand, a cleaning solution for nucleic acid extraction is provided, which includes: sodium chloride and a second surfactant, the molar concentration of the sodium chloride is in the range of 0.05 mol / L to 0.5 mol / L, and the mass volume ratio of the second surfactant is in the range of 0.5% to 5%; the pH value of the cleaning solution is in the range of 6.9 to 7.1.

[0017] In some embodiments, the second surfactant comprises at least one of polyethylene glycol p-isooctylphenyl ether, polyoxyethylene sorbitan monolaurate, and ethylphenyl polyethylene glycol.

[0018] On the other hand, a nucleic acid extraction kit is provided, which includes: a lysis / binding solution for nucleic acid extraction as described in any of the above embodiments, and the nucleic acid extraction kit also includes: a second cleaning solution, which includes the cleaning solution as described in any of the above embodiments.

[0019] In some embodiments, the nucleic acid extraction kit further includes: an erythrocyte lysis solution, wherein the erythrocyte lysis solution includes: sodium chloride, polyoxyethylene sorbitan monolaurate, glucose and tris(hydroxymethyl)aminomethane; the molar concentration range of the sodium chloride is 1 mol / L to 10 mol / L, the mass volume ratio of the polyoxyethylene sorbitan monolaurate is in the range of 3% to 6%, the molar concentration range of the glucose is 200 mmol / L to 500 mmol / L, and the molar concentration range of the tris(hydroxymethyl)aminomethane is 1 mmol / L to 50 mmol / L; the pH value range of the erythrocyte lysis solution is 8.0 to 8.4.

[0020] In some embodiments, the nucleic acid extraction kit further comprises: a proteinase K solution having a concentration range of 10 mg / mL to 30 mg / mL.

[0021] In some embodiments, the nucleic acid extraction kit further comprises: a magnetic bead suspension having a concentration ranging from 10 mg / mL to 40 mg / mL.

[0022] In some embodiments, the nucleic acid extraction kit further includes a first cleaning solution. The first cleaning solution includes guanidine isothiocyanate, tris(hydroxymethyl)aminomethane hydrochloride, polyoxyethylene sorbitan monolaurate, sodium chloride, and isopropyl alcohol; the molar concentration of guanidine isothiocyanate ranges from 0.5 mol / L to 3 mol / L, the molar concentration of tris(hydroxymethyl)aminomethane hydrochloride ranges from 0.5 mmol / L to 10 mmol / L, the mass volume ratio of polyoxyethylene sorbitan monolaurate ranges from 0.01% to 0.1%, the molar concentration of sodium chloride ranges from 0.5 mol / L to 2 mol / L, and the volume ratio of isopropyl alcohol ranges from 20% to 50%; the pH value of the first cleaning solution ranges from 6.5 to 8.0.

[0023] In some embodiments, the nucleic acid extraction kit further comprises: an eluent, and the eluent comprises: a TE buffer.

[0024] On the other hand, a method for nucleic acid extraction is provided, which comprises: A. adding a lysis / binding solution and a proteinase K solution to a sample to be extracted nucleic acid at a temperature of 55°C to 80°C and mixing. B. adding a magnetic bead suspension to the mixed liquid obtained in step A, mixing, placing the mixture on a magnetic stand and letting it stand, removing the supernatant, and obtaining a second precipitate. C. adding a first cleaning solution to the second precipitate, mixing, placing the mixture on a magnetic stand and letting it stand, removing the supernatant, and obtaining a third precipitate. D. adding a second cleaning solution to the third precipitate, mixing, placing the mixture on a magnetic stand and letting it stand, removing the supernatant, and obtaining a fourth precipitate. E. adding an elution solution to the fourth precipitate at a temperature of 40°C to 60°C, mixing, placing the mixture on a magnetic stand and letting it stand, transferring the supernatant, and obtaining the extracted nucleic acid.

[0025] In some embodiments, the volume of the lysis / binding solution is 2 to 4 times the volume of the blood sample, the volume of the proteinase K solution is 0.05 to 0.2 times the volume of the blood sample, the volume of the magnetic bead suspension is 0.075 to 0.2 times the volume of the blood sample, the volume of the first cleaning solution is 2.4 to 4.5 times the volume of the blood sample, and the volume of the second cleaning solution is 2.4 to 4.5 times the volume of the blood sample.

[0026] In some embodiments, before step A, the method further includes: A0, taking a blood sample and placing it in a container, adding red blood cell lysis solution, mixing and centrifuging, removing the supernatant, and obtaining a first precipitate. The sample to be extracted with nucleic acid in step A is the first precipitate. In step A0, the centrifugal speed ranges from 10,000 rpm to 12,000 rpm, and the centrifugal time ranges from 1 minute to 3 minutes. In step A0, the steps of adding red blood cell lysis solution, mixing and centrifuging, and removing the supernatant are repeated at least once. The volume of the red blood cell lysis solution is 3 to 5 times the volume of the blood sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] To more clearly illustrate the technical solutions of the present disclosure, the following briefly introduces the drawings required for use in some embodiments of the present disclosure. Obviously, the drawings described below are only drawings of some embodiments of the present disclosure, and those skilled in the art can also derive other drawings based on these drawings. Furthermore, the drawings described below are schematic diagrams and are not intended to limit the actual dimensions of the products, actual processes of the methods, actual timing of signals, and the like involved in the embodiments of the present disclosure.

[0028] FIG1 is a flow chart of nucleic acid extraction according to some embodiments of the present disclosure;

[0029] FIG2 is a bar graph of nucleic acid extraction efficiency according to some embodiments of the present disclosure;

[0030] FIG3 is a purity diagram of nucleic acid extraction provided according to some embodiments of the present disclosure;

[0031] FIG4 is a gel electrophoresis diagram of nucleic acid extraction provided according to some embodiments of the present disclosure;

[0032] FIG5 is another bar graph of nucleic acid extraction efficiency provided according to some embodiments of the present disclosure;

[0033] FIG6 is a purity diagram of another nucleic acid extraction method provided according to some embodiments of the present disclosure;

[0034] FIG7 is a gel electrophoresis diagram of another nucleic acid extraction method provided according to some embodiments of the present disclosure;

[0035] FIG8 is a graph showing the yield of nucleic acid extraction according to some embodiments of the present disclosure;

[0036] FIG9 is a yield diagram of another nucleic acid extraction method provided according to some embodiments of the present disclosure;

[0037] FIG10 is a yield diagram of another nucleic acid extraction method according to some embodiments of the present disclosure;

[0038] FIG11 is a gel electrophoresis diagram of another nucleic acid extraction method provided according to some embodiments of the present disclosure. DETAILED DESCRIPTION

[0039] The following will be combined with the accompanying drawings to clearly and completely describe the technical solutions in some embodiments of the present disclosure. Obviously, the embodiments described are only some embodiments of the present disclosure, not all embodiments. Based on the embodiments provided by the present disclosure, all other embodiments obtained by ordinary technicians in this field are within the scope of protection of the present disclosure.

[0040] Unless the context requires otherwise, throughout the specification and claims, the term "comprise" and its other forms, such as the third person singular form "comprises" and the present participle form "comprising", are to be interpreted as open and inclusive, that is, "including, but not limited to". In the description of the specification, the terms "one embodiment", "some embodiments", "exemplary embodiments", "example", "specific example" or "some examples" are intended to indicate that the particular features, structures, materials or characteristics associated with the embodiment or example are included in at least one embodiment or example of the present disclosure. The schematic representation of the above terms does not necessarily refer to the same embodiment or example. In addition, the particular features, structures, materials or characteristics may be included in any one or more embodiments or examples in any appropriate manner.

[0041] In the following, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the embodiments of the present disclosure, unless otherwise specified, "plurality" means two or more.

[0042] “At least one of A, B and C” has the same meaning as “at least one of A, B or C” and both include the following combinations of A, B and C: A only, B only, C only, the combination of A and B, the combination of A and C, the combination of B and C, and the combination of A, B and C.

[0043] “A and / or B” includes the following three combinations: A only, B only, and a combination of A and B.

[0044] As used herein, "about," "substantially," or "approximately" includes the stated value and an average value that is within an acceptable range of deviation from the particular value as determined by one of ordinary skill in the art taking into account the measurements in question and the errors associated with the measurement of the particular quantity (i.e., the limitations of the measurement system).

[0045] As used herein, "parallel", "perpendicular", and "equal" include the situations described and situations similar to the situations described, and the range of the similar situations is within an acceptable deviation range, wherein the acceptable deviation range is as determined by a person of ordinary skill in the art taking into account the measurement in question and the errors associated with the measurement of the specific quantity (i.e., the limitations of the measurement system). For example, "parallel" includes absolute parallelism and approximate parallelism, wherein the acceptable deviation range of approximate parallelism can be, for example, a deviation within 5°; "perpendicular" includes absolute perpendicularity and approximate perpendicularity, wherein the acceptable deviation range of approximate perpendicularity can also be, for example, a deviation within 5°. "Equal" includes absolute equality and approximate equality, wherein the acceptable deviation range of approximate equality can be, for example, that the difference between the two equals is less than or equal to 5% of either one.

[0046] As used herein, the term test kit is a box for containing chemical reagents for detecting chemical components, drug residues, virus types, etc. Of course, those skilled in the art will understand that in order to contain chemical reagents, the box may also be other containers such as tubes.

[0047] As used herein, the term "DNA" stands for deoxyribonucleic acid. DNA is the carrier of genetic information found in biological cells, primarily guiding the synthesis of RNA and proteins. DNA is a macromolecular polymer composed of deoxynucleotides, which are composed of phosphate, deoxyribose, and bases. There are four main bases: A (adenine), G (guanine), C (cytosine), and T (thymine).

[0048] As used herein, the term "RNA" is short for ribonucleic acid. RNA is a carrier of genetic information found in biological cells, as well as some viruses and viroids. Its primary function in the body is to guide protein synthesis. RNA is a macromolecular polymer composed of ribonucleotides, which are composed of phosphate, ribose, and bases. There are four main types of bases: A (adenine), G (guanine), C (cytosine), and U (uracil). "Nucleic acid" as used herein includes DNA and / or RNA.

[0049] As used herein, the term "PCR" stands for polymerase chain reaction. PCR is a molecular biology technique used to amplify specific DNA fragments. It can be considered a specialized form of DNA replication in vitro. Its greatest characteristic is its ability to significantly increase the number of DNA fragments from minute amounts.

[0050] As used herein, the term "NGS" stands for high-throughput sequencing technology. Also known as "next-generation" sequencing technology, it is characterized by its ability to sequence hundreds of thousands to millions of DNA molecules simultaneously and its short read length.

[0051] Nucleic acid extraction technology is the foundation of modern molecular diagnostics. Its extraction efficiency and purity have a significant impact on the results of downstream experiments, such as PCR (Polymerase Chain Reaction) and NGS (High-Throughput Sequencing). With the development and popularization of molecular diagnostic technology, especially since the COVID-19 pandemic, the number of samples required for nucleic acid testing has increased significantly. Traditional manual nucleic acid extraction methods can no longer meet actual testing needs. Therefore, manual nucleic acid extraction methods are gradually being replaced by automated nucleic acid extraction methods.

[0052] Currently, the main method for automated nucleic acid extraction includes the magnetic rod method, and the basic steps of the extraction include: 1) lysis of biological samples (such as viruses) in the sample and release of nucleic acids, for example, the sample is a blood sample; 2) binding of nucleic acids and magnetic beads under certain buffer conditions; 3) cleaning of nucleic acids to remove impurities; 4) releasing nucleic acids from the adsorption carrier into the solution, for example, the adsorption carrier is a magnetic bead.

[0053] In step 1), the cell membrane of the biological sample is disrupted and proteins are digested, a process in which Proteinase K is used. The binding solution in step 2) contains isopropanol, a commonly used nucleic acid precipitation reagent. While isopropanol itself does not damage nucleic acids, alcoholic solvents can inhibit enzymes to a certain extent, and isopropanol can inhibit the activity of Proteinase K. Therefore, in conventional nucleic acid extraction processes, the lysate and binding solution components are separated, and the lysis step must be fully completed before the binding solution is added for magnetic bead capture.

[0054] In other words, the biological sample lysis process (step 1) and the nucleic acid-magnetic bead binding process (step 2) are performed separately. Magnetic bar extraction devices typically operate solely by adsorbing, moving, and releasing the magnetic beads. This means that in practice, the process requires manual intervention after the biological sample is lysed and the binding solution is added to the lysis buffer cartridge, preventing a truly fully automated process.

[0055] In addition, the operator's repeated taking and placing of samples in the card slot may contaminate the samples. At the same time, the extraction chamber is closed, the internal gas is not circulating, and the direct introduction of external air into the extractor can easily breed bacteria and cause contamination inside the instrument, thereby reducing the efficiency and quality of nucleic acid extraction.

[0056] Moreover, the reagents used in the automatic extraction instrument are all pre-filled in the test kit slots, and the cleaning liquid components therein often contain ethanol components, which are volatile. The evaporation of ethanol will cause deviations in the component ratios, which is not conducive to the long-term storage of the test kit slots.

[0057] Based on this, an embodiment of the present disclosure provides a lysis / binding solution for nucleic acid extraction, which includes: a protein denaturant, an alcohol-free dehydrating agent, a first surfactant, a buffer, ethylenediaminetetraacetic acid, and a chaotropic salt. The molar concentration of the protein denaturant is in the range of 3.5 mol / L to 5.5 mol / L, the mass volume ratio of the alcohol-free dehydrating agent is in the range of 0.1% to 1%, the mass volume ratio of the first surfactant is in the range of 0.05% to 0.5%, the molar concentration of the buffer is in the range of 5 mmol / L to 150 mmol / L, the molar concentration of the ethylenediaminetetraacetic acid is in the range of 5 mmol / L to 20 mmol / L, and the mass volume ratio of the chaotropic salt is in the range of 1% to 8%.

[0058] This lysis / binding solution is used to release nucleic acids from biological samples (such as viruses) in blood samples and destroy the protein structure of biological samples (such as viruses), promoting the separation of proteins and nucleic acids. In addition, it dehydrates nucleic acid molecules and promotes the effective binding of nucleic acids to adsorption carriers (such as magnetic beads).

[0059] Exemplarily, the protein denaturant includes at least one of guanidine isothiocyanate and guanidine hydrochloride.

[0060] It should be noted that protein denaturants destroy viral protein molecules in the sample. Alcohol-free dehydrating agents primarily prevent DNA from becoming too water-soluble, preventing it from being fully adsorbed by magnetic beads and resulting in loss. The dehydration mechanism of alcohol-free dehydrating agents is to reduce the solubility of DNA in water by competing with its polarity for water molecules.

[0061] Chaotropic salts are salts that disrupt the hydrogen bonds between water molecules, increasing their disorder. Their primary function is to disrupt the cell and nuclear membrane structures within a sample. This occurs primarily by disrupting hydrogen bonds and disulfide bonds within proteins, thereby destroying their secondary structures and significantly weakening the interactions between proteins and DNA. This, in turn, disrupts the membrane structure of biological samples (such as viruses), separates DNA, and disrupts the binding of nucleoproteins to DNA, allowing DNA to adhere to magnetic beads.

[0062] Because no alcohols dehydrating agent content is too high to affect solution solubility, cause no alcohols dehydrating agent to be not molten completely in water, and no alcohols dehydrating agent content is too high to be wrapped in magnetic bead surface, affect the adsorption of magnetic bead to DNA.And the chaotropic salt of appropriate concentration can destroy hydrogen bond between water molecules, strengthens the hydrophobicity of DNA, in conjunction with the no alcohols dehydrating agent of appropriate concentration can make nucleic acid precipitate out and be combined with magnetic bead from aqueous solution.Can reduce like this the usage quantity of no alcohols dehydrating agent, simultaneously chaotropic salt can further coordinate protein denaturant (for example, guanidine isothiocyanate) molecule to destroy the protein molecule of biological sample (such as virus), further reduce protein residual, improve the purity of DNA extraction.And dehydrating agent has certain solubility for polysaccharide and lipid group of cell interior, can reduce impurity interference like this, promote the secondary structure that chaotropic salt destroys protein, no alcohols dehydrating agent further wraps up protein molecule, and takes away protein molecule by solution transfer, reduces the protein impurity in solution.

[0063] The primary function of the first surfactant is to increase the dispersion of the magnetic beads in the aqueous solution, facilitating DNA adsorption. The buffer provides a liquid system for dissolving nucleic acids.

[0064] Ethylenediaminetetraacetic acid is referred to as EDTA, and its molecular formula is C 10 H 16 N2O8. EDTA is a well-characterized complexing agent. It has six coordinating atoms, forming complexes called chelates. EDTA is frequently used in complexometric titrations, typically to determine metal ion content. In biological applications, it is used to eliminate interference from most transition metal ions (such as iron (III), nickel (II), and manganese (II)). Here, EDTA acts as a chelating agent for enzyme catalytic ions (primarily magnesium ions).

[0065] Illustratively, the molar concentration of the protein denaturant is 3.5 mol / L, 4.0 mol / L, 4.5 mol / L, 5.0 mol / L or 5.5 mol / L, etc., which is not limited here.

[0066] Illustratively, the mass volume proportion of the alcohol-free dehydrating agent is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%, etc., which is not limited here.

[0067] It should be noted that "mass-to-volume ratio" is the ratio of the mass of the substance (in grams) to the volume of the liquid (in milliliters). For example, a mass-to-volume ratio of 1% for the non-alcoholic dehydrating agent means that the mass of the non-alcoholic dehydrating agent in 100 milliliters of the lysis / binding solution is 1 gram.

[0068] Illustratively, the mass volume proportion of the first surfactant is 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 5%, etc., which is not limited here.

[0069] Illustratively, the molar concentration of the buffer is 5mmol / L, 10mmol / L, 15mmol / L, 20mmol / L, 40mmol / L, 55mmol / L, 65mmol / L, 70mmol / L, 75mmol / L, 85mmol / L, 90mmol / L, 100mmol / L, 120mmol / L or 150mmol / L, etc., but is not limited thereto.

[0070] Illustratively, the molar concentration of EDTA is 5 mmol / L, 7 mmol / L, 9 mmol / L, 10 mmol / L, 13 mmol / L, 16 mmol / L or 20 mmol / L, etc., which is not limited here.

[0071] Illustratively, the mass volume ratio of the chaotropic salt is 1%, 2%, 3%, 4%, 5%, 6%, 7% or 8%, etc., which is not limited here.

[0072] Exemplarily, the pH value of the lysis / binding solution is in the range of 4.4 to 7.4, and the pH value of the lysis / binding solution is 4.4, 4.6, 5.2, 5.8, 6.0, 6.3, 6.5, 6.8, 7.2 or 7.4, etc., which are not limited here. Adjusting the pH value of the lysis / binding solution to a weak acidity can enhance the hydrogen bonding ability between the magnetic bead surface and the nucleic acid, thereby allowing the nucleic acid to quickly bind to the magnetic bead surface without affecting the digestion ability of proteinase K, thereby achieving a one-step lysis and binding process.

[0073] Therefore, the embodiments of the present disclosure use an isopropanol-free system reagent to unify the lysis solution and binding solution components, thereby reducing the nucleic acid extraction time, allowing the sample to be lysed and combined with the magnetic beads at the same time, and better matching the nucleic acid automatic extraction instrument to achieve fully automatic extraction.

[0074] In some examples, the anions in the chaotropic salt include: PO4 3- 、SO4 2- 、H2PO4 - HCOO - 、Cl - 、NO3 - CF3COO - 、BF4 - 、ClO4 - and PF6 - Any of the following, the cations in the chaotropic salt include: Ca2+ Mg 2+ 、Li + 、Na + , K + NH4 + , any one of an imidazolium cation and an N-ethylpyridinium cation.

[0075] It should be noted that the structure of the imidazolium cation is shown in Structural Formula I, wherein R1 and R2 include alkyl groups.

[0076] Illustratively, two imidazolium cations are exemplified below.

[0077] Wherein, in structural formula II, R1 is methyl and R2 is ethyl. In structural formula III, R1 is methyl and R2 is propenyl.

[0078] The structural formula of N-ethylpyridinium cation is shown below.

[0079] Exemplary chaotropic salts include ammonium phosphate, ammonium sulfate, ammonium dihydrogen phosphate, ammonium chloride, calcium hexafluorophosphate, potassium hexafluorophosphate, sodium hexafluorophosphate, ammonium hexafluorophosphate, 1-allyl-3-methylimidazolium hexafluorophosphate, N-ethylpyridinium hexafluorophosphate, or N-ethylpyridinium chloride, etc., without limitation.

[0080] The molecular formula of ammonium phosphate is (NH4)3PO4. In aqueous solution, it is PO4 3- and NH4 + exists in the form of .

[0081] The molecular formula of ammonium sulfate is (NH4)2SO4. It is SO4 in aqueous solution. 2- and NH4 + exists in the form of .

[0082] The molecular formula of ammonium dihydrogen phosphate is NH4H2PO4, which is H2PO4 in aqueous solution. - and NH4 + exists in the form of .

[0083] The molecular formula of ammonium chloride is NH4Cl. It is present in aqueous solution as Cl - and NH4 + exists in the form of .

[0084] The molecular formula of calcium hexafluorophosphate is CaF 12 P2, in aqueous solution as PF6 - and Ca 2+ exists in the form of .

[0085] The molecular formula of potassium hexafluorophosphate is KPF6, which is PF6 in aqueous solution.- and K + exists in the form of .

[0086] The molecular formula of sodium hexafluorophosphate is NaPF6, a colorless crystalline powder, which is an inorganic compound. - and Na + exists in the form of .

[0087] The molecular formula of ammonium hexafluorophosphate is NH4PF6, a white crystalline powder, which is an inorganic compound. - and NH4 + exists in the form of .

[0088] The molecular formula of 1-allyl-3-methylimidazolium hexafluorophosphate is C9H 11 PF6N2, in aqueous solution as PF6 - It exists in the form of an imidazolium cation and an imidazolium cation, and the structure of the imidazolium cation is shown in structural formula III.

[0089] The molecular formula of N-ethylpyridine hexafluorophosphate is C7H 10 F6NP, in aqueous solution as PF6 - and N-ethylpyridinium cations.

[0090] The molecular formula of N-ethylpyridinium chloride is C7H 10 ClN, in aqueous solution as Cl - and N-ethylpyridinium cations.

[0091] In some examples, the alcohol-free dehydrating agent includes at least one of polyoxyethylene lauryl ether, polyvinyl carbazole (PVK), polyvinyl alcohol, polyacrylamide, polyacrylic acid, polyoxyethylene sorbitan monolaurate, and sodium lauryl sulfate.

[0092] Since the presence of the non-alcoholic dehydrating agent needs not to affect the solubility of the protein, a polar organic compound is selected as the dehydrating agent so that the non-alcoholic dehydrating agent and the chaotropic salt should be able to maintain a certain degree of mutual solubility in the aqueous solution.

[0093] The molecular formula of polyoxyethylene lauryl ether is C 38 H 76 O 11 , a nonionic surfactant with polar groups in the form of polyoxyethylene separated by polyethylene chains. Its molecular structure consists of single chains of polyethylene glycol (PEG). It is slightly soluble in water and has hydrophobic properties. Polyoxyethylene lauryl ether is a brown, viscous liquid that is readily soluble in water and has emulsifying, wetting, and dispersing properties.

[0094] The molecular formula of polyvinylcarbazole is C 42 H 33N3X2, poly(N-vinylcarbazole) is a colorless, transparent or brown, amorphous thermoplastic resin. The carbazole group imparts high thermal stability, water resistance, and chemical stability to the resin. It is insoluble in aliphatic hydrocarbons, mineral oil, transformer oil, castor oil, carbon tetrachloride, ethanol, ether, dilute acids, and hydrofluoric acid. However, it is readily soluble in concentrated sulfuric acid, concentrated nitric acid, tetrahydrofuran, and chlorinated hydrocarbons.

[0095] The molecular formula of polyvinyl alcohol is (C2H4O)n, where n is a positive integer. The average molecular weight of polyvinyl alcohol is 16,000 to 20,000. Polyvinyl alcohol is an odorless white flaky, flocculent, or powdery solid. It is soluble in water but insoluble in gasoline, kerosene, vegetable oil, benzene, toluene, ethylene dichloride, carbon tetrachloride, acetone, ethyl acetate, methanol, and ethylene glycol. It is slightly soluble in dimethyl sulfoxide.

[0096] The molecular formula of polyacrylamide is (C3H5NO)n, where n is a positive integer. Polyacrylamide is a linear organic high molecular polymer and also a high molecular water treatment flocculant product. It can specifically adsorb suspended particles in water, act as a link and bridge between particles, so that fine particles form relatively large flocs and accelerate the sedimentation rate.

[0097] The molecular formula of polyacrylic acid is (C3H4O2)n, where n is a positive integer. It is used to prepare finishing agents for leather and certain high-end products, and to produce acrylic resin paints. It is a chemical intermediate.

[0098] The molecular formula of polyoxyethylene sorbitan monolaurate is C 26 H 50 O 10 Tween 20, also known as Tween 20, is a surfactant, a type of macromolecule with both hydrophilic and lipophilic parts. It can help plants absorb macromolecules that are insoluble in water and also help water permeate through some lipid-rich biological membranes.

[0099] Sodium dodecyl sulfate is referred to as SDS, and its molecular formula is C 12 H 25 SO4Na is a white or light yellow, slightly viscous substance commonly used in detergents and textiles. It is an anionic surfactant. It is readily soluble in water and has good compatibility with anionic and nonionic surfactants. It exhibits excellent emulsifying, foaming, penetrating, detergency, and dispersing properties.

[0100] In some examples, the buffer comprises at least one of a TE buffer and tris(hydroxymethyl)aminomethane. The TE buffer comprises 10 mmol / L tris(hydroxymethyl)aminomethane hydrochloride and 0.1 mmol / L ethylenediaminetetraacetic acid.

[0101] Exemplarily, the pH value of the buffer solution is in the range of 7.0 to 7.4. For example, the pH value of the buffer solution is 7.0, 7.1, 7.2, 7.3 or 7.4, etc., which is not limited here.

[0102] Tris(hydroxymethyl)aminomethane is referred to as Tris, and its molecular formula is C4H 11 NO3 is a biological buffer used to prepare buffer for gel electrophoresis.

[0103] Tris-HCl is abbreviated as Tris-HCl, and its molecular formula is C4H 11 NO3.HCl.

[0104] For the introduction of ethylenediaminetetraacetic acid, please refer to the above content and will not be repeated here.

[0105] In some examples, the first surfactant includes polyoxyethylene sorbitan monolaurate or polyethylene glycol p-isooctylphenyl ether.

[0106] For the introduction of polyoxyethylene sorbitan monolaurate, please refer to the above content and will not be repeated here.

[0107] Polyethylene glycol 4-isooctylphenyl ether is referred to as Triton X-100 and Triton X-100, with the molecular formula of C 16 H 26 O2.

[0108] The following introduces a nucleic acid extraction kit.

[0109] The embodiments of the present disclosure also provide a nucleic acid extraction kit, which includes: red blood cell lysate, nucleic acid extraction lysis / binding solution, proteinase K solution, magnetic bead suspension, first cleaning solution, second cleaning solution and eluent.

[0110] An erythrocyte lysis solution is used to destroy red blood cells in the blood. In some examples, the erythrocyte lysis solution includes sodium chloride, polyoxyethylene sorbitan monolaurate, glucose, and tris(hydroxymethyl)aminomethane. The molar concentration of sodium chloride ranges from 1 mol / L to 10 mol / L, the mass volume fraction of polyoxyethylene sorbitan monolaurate ranges from 3% to 6%, the molar concentration of glucose ranges from 200 mmol / L to 500 mmol / L, and the molar concentration of tris(hydroxymethyl)aminomethane ranges from 1 mmol / L to 50 mmol / L. The pH of the erythrocyte lysis solution ranges from 8.0 to 8.4.

[0111] Sodium chloride is used to ensure the osmotic pressure between white blood cells and aqueous solution, preventing white blood cells from being osmotically ruptured by aqueous solution during the red blood cell lysis process.

[0112] Illustratively, the molar concentration of sodium chloride is 1 mol / L, 2 mol / L, 3 mol / L, 4 mol / L, 5 mol / L, 6 mol / L, 7 mol / L, 8 mol / L, 9 mol / L or 10 mol / L, etc., which is not limited here.

[0113] Polyoxyethylene sorbitan monolaurate is used to allow membrane fragments, lipid molecules and other organic substances after red blood cell lysis to be eluted by the solution.

[0114] For example, the mass volume percentage of polyoxyethylene sorbitan monolaurate is 3%, 4%, 5% or 6%, etc., which is not limited here.

[0115] Glucose is used to destroy the cell membranes of red blood cells.

[0116] Illustratively, the molar concentration of glucose is 200 mmol / L, 250 mmol / L, 300 mmol / L, 350 mmol / L, 400 mmol / L, 450 mmol / L or 500 mmol / L, etc., but is not limited thereto.

[0117] The molar concentration of tris(hydroxymethyl)aminomethane is 1 mmol / L, 10 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, 40 mmol / L or 50 mmol / L, etc., but is not limited thereto.

[0118] For blood samples, the red blood cells can be first swelled and lysed using the concentrated salt of the red blood cell lysis solution, and then the white blood cells can be lysed after separation. This can avoid the introduction of large amounts of sugar and protein on the red blood cell surface and contamination of nucleic acids in subsequent steps.

[0119] For the introduction of lysis / binding solution for nucleic acid extraction, please refer to the above content and will not be repeated here.

[0120] Proteinase K is a powerful proteolytic enzyme isolated from Candida albicans. It has high specific activity and is a key reagent for DNA extraction. Proteinase K solution is used to disrupt tissue cells. Exemplarily, the concentration of the proteinase K solution ranges from 10 mg / mL to 30 mg / mL. For example, the concentration of the proteinase K solution is 10 mg / mL, 13 mg / mL, 16 mg / mL, 20 mg / mL, 25 mg / mL, or 30 mg / mL, etc., without limitation.

[0121] The magnetic beads in the magnetic bead suspension are silanol- or carboxyl-modified magnetic beads. Exemplarily, the concentration of the magnetic bead suspension ranges from 10 mg / mL to 40 mg / mL. For example, the concentration of the magnetic bead suspension is 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, or 40 mg / mL, etc., without limitation.

[0122] The first cleaning solution is primarily used to wash the adsorption carrier bound to the nucleic acid to remove protein impurities. Exemplarily, the first cleaning solution includes: guanidine isothiocyanate, tris(hydroxymethyl)aminomethane hydrochloride, polyoxyethylene sorbitan monolaurate, sodium chloride, and isopropyl alcohol; the molar concentration of guanidine isothiocyanate ranges from 0.5 mol / L to 3 mol / L, the molar concentration of tris(hydroxymethyl)aminomethane hydrochloride ranges from 0.5 mmol / L to 10 mmol / L, the mass volume percentage of polyoxyethylene sorbitan monolaurate ranges from 0.01% to 0.1%, the molar concentration of sodium chloride ranges from 0.5 mol / L to 2 mol / L, and the volume percentage of isopropyl alcohol ranges from 20% to 50%. The pH value of the first cleaning solution ranges from 6.5 to 8.0.

[0123] Illustratively, the molar concentration of guanidine isothiocyanate is 0.5 mol / L, 1 mol / L, 1.5 mol / L, 2 mol / L, 2.5 mol / L or 3 mol / L, etc., which is not limited here.

[0124] Illustratively, the molar concentration of tris(hydroxymethyl)aminomethane hydrochloride is 0.5 mmol / L, 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L or 10 mmol / L, etc., but is not limited thereto.

[0125] Polyoxyethylene sorbitan monolaurate is used to increase the dispersion of magnetic beads in aqueous solution. Exemplary, the weight-volume percentage of polyoxyethylene sorbitan monolaurate is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, or 0.1%, etc., without limitation.

[0126] Sodium chloride is used to maintain the ion concentration in the solution, providing ions that are adsorbed between the nucleic acids and the magnetic beads via ionic bridges, thereby preventing them from being washed away by the cleaning solution. For example, the molar concentration of sodium chloride is 0.5 mol / L, 1 mol / L, 1.5 mol / L, or 2 mol / L, etc., although this is not a limitation.

[0127] Isopropanol is used to keep the nucleic acid in a dehydrated state in the solution to prevent it from being eluted by the cleaning solution. For example, the volume percentage of isopropanol is 20%, 30%, 40%, or 50%, etc., and this is not limited here.

[0128] Illustratively, the pH value of the first cleaning solution is 6.5, 6.6, 6.8, 7.0, 7.2, 7.3, 7.5, 7.8 or 8.0, etc., which is not limited here.

[0129] The second cleaning solution includes: sodium chloride and a second surfactant, the molar concentration of sodium chloride is in the range of 0.05 mol / L to 0.5 mol / L, the mass volume ratio of the second surfactant is in the range of 0.5% to 5%; the pH value of the second cleaning solution is in the range of 6.9 to 7.1.

[0130] Exemplarily, the second surfactant includes at least one of polyethylene glycol p-isooctylphenyl ether, polyoxyethylene sorbitan monolaurate, and ethylphenyl polyethylene glycol.

[0131] Ethylphenyl polyethylene glycol is referred to as NP-40, and its molecular formula is C 15 H 24 O·(C2H4O)n, where n is a positive integer. Ethylphenyl polyethylene glycol is a commonly used nonionic, non-denaturing detergent and surfactant.

[0132] The cleaning solution is used to wash the adsorption carrier bound to the nucleic acid to remove non-nucleic acid contaminants and salts, and to maintain the solution in which the nucleic acid is bound to the adsorption carrier.

[0133] Illustratively, the molar concentration of sodium chloride ranges from 0.05 mol / L, 0.1 mol / L, 0.15 mol / L, 0.25 mol / L, 0.3 mol / L, 0.45 mol / L or 0.5 mol / L, etc., which is not limited here.

[0134] Illustratively, the mass volume proportion of the second surfactant is 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5%, 3%, 4.5% or 5%, etc., which is not limited here.

[0135] The cleaning solution contains a sodium chloride solution with a high salt ion concentration and a low pH solution, which can ensure that nucleic acids are not released from the magnetic beads into the solution. At the same time, a certain concentration of the second surfactant can remove impurities on the surface of the magnetic beads, thereby achieving the purpose of purifying nucleic acids.

[0136] Therefore, this example provides an alcohol-free cleaning solution that can improve the long-term storage stability of the performance of the automated extraction kit consumables and reagents.

[0137] The eluent is a solution for eluting the nucleic acid bound to the adsorption carrier. For example, the eluent is TE buffer or nuclease-free water.

[0138] The nucleic acid extraction kit provided in the embodiments of the present disclosure includes the lysis / binding solution provided in the above embodiments. Therefore, the lysis process of the biological sample and the binding process of the nucleic acid and the magnetic beads can be unified, so that the sample can be lysed and bound to the magnetic beads at the same time, thereby reducing the nucleic acid extraction time and better matching the automatic nucleic acid extractor to achieve fully automatic extraction.

[0139] In addition, the nucleic acid extraction kit provided in the embodiment of the present disclosure also includes a red blood cell lysis solution, which uses concentrated salt in the red blood cell lysis solution to swell and lyse the red blood cells, and then lyse the white blood cells after separation. This can avoid the introduction of a large amount of sugar and protein on the surface of the red blood cells and contamination of nucleic acids in subsequent steps.

[0140] In addition, the nucleic acid extraction kit provided in the embodiment of the present disclosure also includes an alcohol-free second cleaning solution, which can improve the long-term storage stability of the consumables and reagents of the automatic extraction kit.

[0141] Based on the nucleic acid extraction kit provided in the above embodiment, the present disclosure further provides a method for nucleic acid extraction, which includes the following steps A0 to E.

[0142] A0. Take a blood sample and place it in a container. Add red blood cell lysis buffer, mix and centrifuge. Remove the supernatant to obtain a first precipitate.

[0143] Exemplarily, the container is a centrifuge tube.

[0144] Exemplarily, the blood sample is bovine blood.

[0145] Illustratively, the volume of the red blood cell lysis solution is 3 to 5 times the volume of the blood sample. For example, the volume of the red blood cell lysis solution is 3, 3.5, 4, 4.5 or 5 times the volume of the blood sample, etc., which is not limited here.

[0146] For example, the blood sample to which the red blood cell lysis solution is added is mixed using a vortex mixer.

[0147] Illustratively, during the centrifugation step, the centrifuge speed ranges from 10,000 rpm to 12,000 rpm, for example, 10,000 rpm, 11,000 rpm, or 12,000 rpm, etc., although this is not intended to be limiting. The centrifugation time ranges from 1 minute to 3 minutes, for example, 1 minute, 2 minutes, or 3 minutes, etc., although this is not intended to be limiting. By appropriately controlling the centrifugation speed and time, red blood cell fragments are retained in the supernatant and removed.

[0148] For example, the steps of adding red blood cell lysis buffer, mixing, centrifuging, and removing the supernatant are repeated at least once. This allows for more complete removal of red blood cells, preventing the introduction of large amounts of sugars and proteins on the red blood cell surface that could subsequently contaminate nucleic acids.

[0149] After the blood sample is lysed through red blood cell lysis to obtain a first precipitate, as shown in Figure 1, the nucleic acid extraction method further includes the following steps: lysing and binding the first precipitate, washing with a first cleaning solution, washing with a second cleaning solution, and eluting. These steps can be combined with an automated nucleic acid extraction instrument to achieve fully automated extraction. The specific operations for each step are as follows.

[0150] The cleavage and binding steps include steps: A and B.

[0151] A. Add lysis / binding solution and proteinase K solution to the first precipitate at a temperature of 55°C to 80°C and mix.

[0152] The first precipitate is also referred to as the sample to be extracted nucleic acid.

[0153] This step is used to release nucleic acids from biological samples (such as viruses) in blood samples, and to destroy the protein structure of biological samples (such as viruses), thereby promoting the separation of proteins and nucleic acids and dehydrating nucleic acid molecules.

[0154] Illustratively, the temperature is 55° C., 60° C., 65° C., 70° C., 75° C., or 80° C., etc., but is not limited thereto.

[0155] Illustratively, the volume of the lysis / binding solution is 2 to 4 times the volume of the blood sample. For example, the volume of the lysis / binding solution is 2, 2.5, 3, 3.5 or 4 times the volume of the blood sample, etc., which is not limited here.

[0156] Exemplarily, the volume of the proteinase K solution is 0.05 to 0.2 times the volume of the blood sample. For example, the volume of the proteinase K solution is 0.05, 0.1, 0.12, 0.15, or 0.2 times the volume of the blood sample, etc., which is not limited here.

[0157] B. Add the magnetic bead suspension to the mixed liquid obtained in step A, mix, place on a magnetic stand and let stand, remove the supernatant to obtain a second precipitate.

[0158] This step is used to effectively bind nucleic acids to adsorption carriers (such as magnetic beads).

[0159] Exemplarily, the volume of the magnetic bead suspension is 0.075 to 0.2 times the volume of the blood sample. For example, the volume of the magnetic bead suspension is 0.075, 0.095, 0.125, 0.15, or 0.2 times the volume of the blood sample, and the like, without limitation.

[0160] Exemplarily, the centrifuge tube is placed on a magnetic stand and allowed to stand for 2 minutes.

[0161] C. Washing with the first washing solution: Add the first washing solution to the second precipitate, mix, place on a magnetic stand and let stand, remove the supernatant, and obtain a third precipitate.

[0162] This step is used to wash the adsorption carrier bound to the nucleic acid to remove protein impurities.

[0163] Exemplarily, the volume of the first cleaning solution is 2.4 to 4.5 times the volume of the blood sample. For example, the volume of the first cleaning solution is 2.4, 2.7, 3.0, 3.3, 4, or 4.5 times the volume of the blood sample, etc., which is not limited here.

[0164] Exemplarily, the centrifuge tube is placed on a magnetic stand and allowed to stand for 2 minutes.

[0165] D. Washing with the second washing solution: Add the second washing solution to the third precipitate, mix, place on a magnetic stand and let stand, remove the supernatant, and obtain a fourth precipitate.

[0166] This step is used to wash the adsorption carrier bound to the nucleic acid to remove non-nucleic acid contaminants and salts, and to maintain a solution in which the nucleic acid is bound to the adsorption carrier.

[0167] Exemplarily, the volume of the second cleaning solution is 2.4 to 4.5 times the volume of the blood sample. For example, the volume of the second cleaning solution is 2.4, 2.7, 3.0, 3.3, 4, or 4.5 times the volume of the blood sample, etc., which is not limited here.

[0168] Exemplarily, the centrifuge tube is placed on a magnetic stand and allowed to stand for 2 minutes.

[0169] E. Elution: Add eluent to the fourth precipitate at 40°C to 60°C, mix, place on a magnetic stand and let stand, transfer the supernatant to obtain the extracted nucleic acid.

[0170] This step is used to elute the nucleic acid bound to the adsorption carrier.

[0171] Illustratively, the temperature is 40° C., 45° C., 48° C., 52° C., 55° C., or 60° C., etc., but is not limited thereto.

[0172] Exemplarily, the centrifuge tube is placed on a magnetic stand and allowed to stand for 2 minutes.

[0173] The nucleic acid extraction method provided by the embodiments of the present disclosure includes the lysis / binding solution provided by the above embodiments. Therefore, during the nucleic acid extraction process, after adding the blood sample, the biological sample lysis process and the nucleic acid-magnetic bead binding process can be unified, so that the sample can be lysed and bound to the magnetic beads at the same time, thereby reducing the nucleic acid extraction time and better matching the nucleic acid automatic extractor to achieve fully automatic extraction.

[0174] Moreover, before matching the automatic nucleic acid extractor to realize nucleic acid extraction, the step of red blood cell lysis is also included, that is, the red blood cells are first swelled and lysed using the concentrated salt of the red blood cell lysis solution, and then the white blood cells are lysed after separation. This can avoid the introduction of a large amount of sugar and protein on the surface of the red blood cells and contamination of nucleic acids in subsequent steps.

[0175] Based on the nucleic acid extraction kit and nucleic acid extraction method provided in the above embodiments, the following specific embodiments are provided.

[0176] Example 1

[0177] The nucleic acid properties used to compare the nucleic acid extracted by the nucleic acid extraction kit provided in the embodiment of the present disclosure with the nucleic acid extracted by commercially available nucleic acid extraction kits include the concentration and purity of the nucleic acid.

[0178] The reagent components of the nucleic acid extraction kit provided in the embodiments of the present disclosure are as follows.

[0179] (1) Red blood cell lysis solution: 5 mmol / L sodium chloride (NaCl), 5% polyoxyethylene sorbitan monolaurate (Tween 20) by mass volume, 320 mmol / L glucose, 10 mmol / L tris (hydroxymethyl)aminomethane (Tris), the pH value of tris (hydroxymethyl)aminomethane (Tris) is 8.2.

[0180] (2) Lysis / binding solution: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by mass volume, 100 mmol / L TE buffer, pH value of TE buffer is 7.2, 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate (SDS) by mass volume, and 3% ammonium phosphate by mass volume.

[0181] That is, the protein denaturant used in Example 1 is guanidine isothiocyanate, the alcohol-free dehydrating agent is sodium dodecyl sulfate (SDS), the first surfactant is polyoxyethylene sorbitan monolaurate (Tween 20), the buffer is TE buffer with a pH value of 7.2, and the chaotropic salt is ammonium phosphate.

[0182] (3) Proteinase K solution: concentration is 20 mg / mL.

[0183] (4) The first cleaning solution: 1 mol / L guanidine thiocyanate, 1 mmol / L tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 0.05% polyoxyethylene sorbitan monolaurate (Tween 20) by mass volume, 1 mol / L sodium chloride (NaCl), 40% isopropanol by volume, and the pH value of the first cleaning solution is 7.5.

[0184] (5) Second cleaning solution: 0.5 mol / L sodium chloride (NaCl), 0.5% by mass volume of polyethylene glycol p-isooctylphenyl ether (Triton 100), the pH value of the second cleaning solution is 7.0.

[0185] (6) Eluent: TE buffer.

[0186] In this embodiment, whole blood of a cow is used as a blood sample. The specific operation process of nucleic acid extraction is as follows, including the steps of removing red blood cells from the blood sample and extracting nucleic acid.

[0187] The step of removing red blood cells from the blood sample includes: S1 to S4.

[0188] S1. Take 250 μL of blood sample and place it in a 2 mL centrifuge tube. Add 800 μL of red blood cell lysis buffer and shake thoroughly for 10 seconds to obtain a red, transparent, and clear mixture.

[0189] The volume of cell lysate is 3.2 times that of blood sample.

[0190] S2. The mixture was centrifuged at a speed of 12,000 revolutions per minute (rpm) for 1 minute, and the supernatant was removed to obtain a white precipitate.

[0191] S3. Add 800 μl of red blood cell lysis buffer to the centrifuge tube in step S2, shake thoroughly to mix, and resuspend the precipitate to obtain a mixed solution.

[0192] S4. Centrifuge the mixed solution obtained in step S3 at a centrifugal speed of 12,000 revolutions per minute (rpm) for 1 minute, remove the supernatant, and obtain a first precipitate.

[0193] The nucleic acid extraction step also includes: S5 to S10.

[0194] S5. Add 600 μl of lysis / binding solution to the first precipitate at 65° C., mix well using a vortex mixer, and let stand for 10 minutes.

[0195] S6. Add 20 μl of the magnetic bead suspension to the mixed liquid obtained in step S5, mix well using a vortex mixer, let it stand for 10 minutes, and then mix it using a vortex mixer for 3 seconds every 2 minutes.

[0196] The volume of the magnetic bead suspension is 0.08 times the volume of the blood sample.

[0197] S7. Place the mixed solution obtained in step S6 on a magnetic rack and let it stand for 2 minutes, remove the supernatant, and obtain a second precipitate.

[0198] Before placing the mixed solution obtained in step S6 on the magnetic rack, the mixed solution obtained in step S6 may be subjected to low-speed centrifugation to accelerate the subsequent deposition of magnetic beads.

[0199] S8. Add 600 μL of the first washing solution to the second precipitate and mix manually. Then, place the centrifuge tube on a magnetic stand and let it stand for 2 minutes. Remove the supernatant to obtain a third precipitate.

[0200] The volume of the first washing solution is 2.4 times the volume of the blood sample.

[0201] For example, manual mixing includes tapping the outside of the centrifuge tube with your fingers or inverting the centrifuge tube upside down.

[0202] S9. Add 600 μL of the second wash solution to the third precipitate and mix manually. Then, place the centrifuge tube on a magnetic plate and let it adsorb for 2 minutes. Remove the supernatant to obtain the fourth precipitate, and air dry it at room temperature until no liquid is visible on the surface of the magnetic beads.

[0203] The volume of the second washing solution is 2.4 times the volume of the blood sample.

[0204] S10. Add 100 μL of elution buffer and shake for elution at 56°C for 10 minutes. Then, place the centrifuge tube on a magnetic stand for 2 minutes. Transfer the supernatant to a new centrifuge tube to obtain the extracted nucleic acid.

[0205] The concentration of the nucleic acid extracted in the above example was tested using a Qubit 4.0 nucleic acid / protein fluorescence quantifier, and the purity of the nucleic acid was tested using a micro-spectrophotometer (nanodrop).

[0206] It should be noted that when using a micro-spectrophotometer (nanodrop), A260 / A280 is used to assess nucleic acid purity, and a larger value indicates a higher nucleic acid purity.

[0207] Compare nucleic acid concentration and purity test results to the performance of commercially available reagents.

[0208] Table 1 is a comparison of the performance of nucleic acids extracted in Example 1 and those extracted by commercially available kits, Figure 2 is a bar graph of nucleic acid extraction efficiency, and Figure 3 is a purity graph of nucleic acid extraction. Judging from the data results, the reagent system provided by this scheme is slightly better than the commercially available reagents in terms of DNA extraction yield and purity. Figure 4 is a gel electrophoresis diagram of nucleic acid extraction, and the ordinate represents the gel electrophoresis migration rate (bp is base pair, the abbreviation of base pair). It can be seen from Figure 4 that the DNA band distribution between Example 1 and the commercially available reagents is not much different. It should be noted that the DNA band distribution of the nucleic acid sample extracted by the commercially available nucleic acid extraction kit shows that the nucleic acid did not degrade during the experiment. The nucleic acid sample extracted in Example 1 is compared with the nucleic acid sample extracted by the commercially available nucleic acid extraction kit, and the DNA band distribution is not much different, indicating that the nucleic acid sample extracted by the nucleic acid extraction kit provided in Example 1 of the present disclosure did not degrade during the experiment and can be used for downstream experiments.

[0209] Example 1 shows that the nucleic acid extraction kit provided by the embodiments of the present disclosure can allow the sample to be lysed and combined with magnetic beads at the same time, and the performance of the extracted nucleic acid is better than that of the nucleic acid extracted by commercially available reagents.

[0210] Table 1 Comparison of the performance of nucleic acids extracted from Example 1 and nucleic acids extracted using commercially available kits

[0211] It should be noted that, as shown in Table 1, the three data points in each table represent data obtained from three experiments repeated under the same experimental conditions. For example, the data for DNA recovery concentration (ng / μL) of a commercially available reagent, "106 / 112 / 111," indicates that nucleic acid was extracted three times using the commercially available reagent. The nucleic acid concentration of each extraction was measured using a Qubit 4.0 Nucleic Acid / Protein Fluorometer. The DNA recovery concentrations in these three experiments were 106 ng / μL, 112 ng / μL, and 111 ng / μL, respectively. The same applies to the following data.

[0212] Example 2

[0213] The reagent components of this embodiment are similar to those of Example 1. The reagent components are pre-filled into the reagent slot holes in the automatic nucleic acid extraction instrument. The reagents corresponding to the respective slot holes are shown in Table 2 below.

[0214] Among them, the proteinase K solution needs to be refrigerated and stored at a temperature of 2°C to 8°C, and is added to well 2 before the reaction to ensure that the proteinase K has sufficient activity.

[0215] Table 2 Correspondence between reagent components and reagent slot positions in the automatic nucleic acid extraction instrument

[0216] It should be noted that the lysis in step 1 corresponds to the lysis in step S5 in Example 1. Before step 1, a manual operation step of removing red blood cells from the blood sample is also included, and the specific operation steps are the same as steps S1 to S4 in Example 1.

[0217] After obtaining the first precipitate using steps S1-S4, it is necessary to transfer the first precipitate to reagent slot position 2 of the automated nucleic acid extraction instrument. Specifically, the first precipitate is mixed with 200 μL of phosphate-buffered saline (PBS) buffer and the mixed solution is transferred to reagent slot position 2 of the automated nucleic acid extraction instrument. The instrument is then turned on, and the parameters are set as described in Table 1. After the settings are complete, the automated extraction process is initiated. After the automated nucleic acid extraction process is complete, 80 μL of the nucleic acid eluate from reagent slot position 5 is transferred to a new centrifuge tube for use in the next step of testing.

[0218] The performance of the manually extracted nucleic acid sample in Example 1 was compared with that of the automatically extracted nucleic acid sample in Example 2. Table 3 shows a comparison of the effects of manual and automatic extraction methods on nucleic acid performance. Figure 5 is a bar chart of nucleic acid extraction efficiency, and Figure 6 is a chart of nucleic acid extraction purity. Tables 3, 5, and 6 show that the automatic extraction method is inferior to manual extraction in terms of extraction yield and purity. Figure 7 shows a gel electrophoresis image of the nucleic acid extraction, showing that the DNA band distribution is not significantly different, indicating that the manually and automatically extracted nucleic acid samples did not degrade during the experiment.

[0219] Table 3 Comparison of the effects of manual and automatic extraction methods on nucleic acid performance

[0220] As shown in Example 2, the nucleic acid extraction lysis / binding solution and the kit containing the lysis / binding solution provided in the embodiments of the present disclosure can be used with an automated nucleic acid extraction instrument to achieve fully automated extraction. Although automated extraction methods are inferior to manual extraction methods in terms of extraction yield and purity, they do not affect the performance of the extracted nucleic acids.

[0221] Example 3

[0222] This example is used to verify the effects of different types of anions in chaotropic salt on the concentration and purity of extracted nucleic acids.

[0223] When a chaotropic salt dissociates into an ionic salt solution in an aqueous solution, hydrogen bonds in the water aggregate around the salt ions, driving the aggregation of water molecules. This disrupts the arrangement of hydrogen bonds in the aqueous solution, thereby strengthening the internal interactions of ionic organic matter (such as DNA) and causing coagulation. Different ionic salts interfere with water-soluble hydrogen bonds to varying degrees. Based on the Hofmeister sequence, this example selected different anions and cations for comparison. By comparing their effects on other components in the lysate, such as proteinase K and proteins, a set of ion pairs was selected that had the best effect on DNA precipitation while not significantly interfering with protein digestion.

[0224] It should be noted that the Hofmeister sequence refers to the change in the aggregation state of polymers, which can be achieved by simply adding specific ions, where different ions have different abilities to precipitate polymers.

[0225] Example 3 uses an automatic nucleic acid extractor to extract nucleic acid, and the operation process can refer to Example 2.

[0226] To verify the effects of different types of anions in chaotropic salt on the concentration and purity of extracted nucleic acids, the following examples are provided.

[0227] In Example 3.1, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate (SDS) by weight by volume, and 3% ammonium phosphate by weight by volume. The pH of the lysis / binding solution is 7.2.

[0228] In Example 3.2, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate (SDS) by weight by volume, and 3% ammonium sulfate by weight by volume. The pH of the lysis / binding solution is 7.2.

[0229] In Example 3.3, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate (SDS) by weight by volume, and 3% ammonium dihydrogen phosphate by weight by volume. The pH of the lysis / binding solution is 7.2.

[0230] In Example 3.4, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate (SDS) by weight by volume, and 3% ammonium chloride by weight by volume. The pH of the lysis / binding solution is 7.2.

[0231] In Example 3.5, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate (SDS) by weight by volume, and 3% ammonium nitrate by weight by volume. The pH of the lysis / binding solution is 7.2.

[0232] In Example 3.6, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate (SDS) by weight by volume, and 3% ammonium hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 7.2.

[0233] That is, for Examples 3.1 to 3.6, the chaotropic salt components of the lysis / binding solution added to the reagent cartridge well 2 are different. Specifically, the cations of the chaotropic salts are all ammonium ions (NH4 + ), the anions of the chaotropic salt are different, and the specific chaotropic salt components used are ammonium phosphate, ammonium sulfate, ammonium dihydrogen phosphate, ammonium chloride, ammonium nitrate and ammonium hexafluorophosphate.

[0234] Table 4 Comparison of the effects of different anions in chaotropic salts on nucleic acid extraction performance

[0235] Table 4 compares the effects of different anions in chaotropic salts on nucleic acid extraction performance. Figure 8 shows the nucleic acid extraction yield. The test results show that among the six different anionic chaotropic salt compositions, the lysis / binding solution containing ammonium hexafluorophosphate has the best concentration and purity of nucleic acids extracted.

[0236] Example 4

[0237] This example is used to verify the effects of different types of cations in chaotropic salt on the concentration and purity of extracted nucleic acids.

[0238] Example 4 uses an automatic nucleic acid extractor to extract nucleic acid, and the operation process can refer to Example 2.

[0239] To demonstrate the effects of different cations in chaotropic salts on the concentration and purity of extracted nucleic acids, the following examples are provided.

[0240] In Example 4.1, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate (SDS) by weight by volume, and 3% calcium hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 7.2.

[0241] In Example 4.2, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate (SDS) by weight by volume, and 3% magnesium hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 7.2.

[0242] In Example 4.3, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate (SDS) by weight by volume, and 3% potassium hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 7.2.

[0243] In Example 4.4, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecylsulfonate (SDS) by weight by volume, and 3% sodium hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 7.2.

[0244] In Example 4.5, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate (SDS) by weight by volume, and 3% ammonium hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 7.2.

[0245] In Example 4.6, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecylsulfonate (SDS) by weight by volume, and 3% N-ethylpyridinium hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 7.2.

[0246] That is, for Examples 4.1 to 4.6, the chaotropic salt components of the lysis / binding solution added to the reagent cartridge well 2 are different. Specifically, the anion of the chaotropic salt is hexafluorophosphate (PF6 - ), the cations of the chaotropic salts are different, and the specific chaotropic salt components used are calcium hexafluorophosphate, magnesium hexafluorophosphate, potassium hexafluorophosphate, sodium hexafluorophosphate, ammonium hexafluorophosphate and N-ethylpyridine hexafluorophosphate.

[0247] Table 5 Comparison of the effects of different cations in chaotropic salts on nucleic acid extraction performance

[0248] Table 5 compares the effects of different cations in chaotropic salts on nucleic acid extraction performance, and Figure 9 shows the yield of nucleic acid extraction. From the test results, different cations have different effects on the extraction performance. Among them, the concentration and purity of nucleic acids extracted by the lysis / binding solution containing N-ethylpyridinium cationic chaotropic salt are the best.

[0249] Example 5

[0250] This example is used to verify the effect of the concentration of the chaotropic salt on the concentration and purity of the extracted nucleic acid.

[0251] In this embodiment, an automatic nucleic acid extractor is used to extract nucleic acid. The operation process can refer to Example 2.

[0252] As can be seen from Examples 3 and 4, the concentration and purity of nucleic acids extracted from the lysis / binding solution containing N-ethylpyridine hexafluorophosphate as the chaotropic salt component are better. Therefore, N-ethylpyridine hexafluorophosphate is used as the chaotropic salt component in the lysis / binding solution.

[0253] To verify the effect of the concentration of chaotropic salt on the concentration and purity of extracted nucleic acids, the following example is provided.

[0254] In Example 5.1, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight / volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), and 0.5% sodium dodecyl sulfate (SDS) by weight / volume. The pH of the lysis / binding solution is 7.2.

[0255] In Example 5.2, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecylsulfonate (SDS) by weight by volume, and 0.5% N-ethylpyridinium hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 7.2.

[0256] In Example 5.3, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecylsulfonate (SDS) by weight by volume, and 1% N-ethylpyridine hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 7.2.

[0257] In Example 5.4, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecylsulfonate (SDS) by weight by volume, and 1.5% N-ethylpyridinium hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 7.2.

[0258] In Example 5.5, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecylsulfonate (SDS) by weight by volume, and 2% N-ethylpyridinium hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 7.2.

[0259] In Example 5.6, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecylsulfonate (SDS) by weight by volume, and 2.5% N-ethylpyridinium hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 7.2.

[0260] In Example 5.7, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecylsulfonate (SDS) by weight by volume, and 3% N-ethylpyridine hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 7.2.

[0261] In Example 5.8, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecylsulfonate (SDS) by weight by volume, and 3.5% N-ethylpyridinium hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 7.2.

[0262] In Example 5.9, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecylsulfonate (SDS) by weight by volume, and 4% N-ethylpyridinium hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 7.2.

[0263] That is to say, for Examples 5.1 to 5.9, the mass volume ratio of the chaotropic salt (N-ethylpyridine hexafluorophosphate) in the lysis / binding solution added to the reagent card slot hole 2 is different, and the mass volume ratios of the chaotropic salt used are 0%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, and 4%, respectively.

[0264] Table 6 Comparison of the effects of different chaotropic salt concentrations on nucleic acid extraction performance

[0265] Table 6 compares the effects of different chaotropic salt concentrations on nucleic acid extraction performance, and Figure 10 is a graph showing the yield of nucleic acid extraction. It can be seen from Table 6 and Figure 10 that when the mass volume ratio of chaotropic salt in the lysis / binding solution is 2%, the extracted nucleic acid performance is relatively good.

[0266] Example 6

[0267] This example is used to verify the effects of different pH values ​​of the lysis / binding solution on the concentration and purity of the extracted nucleic acid.

[0268] In this embodiment, an automatic nucleic acid extractor is used to extract nucleic acid. The operation process can refer to Example 2.

[0269] In order to verify the effect of different pH values ​​of the lysis / binding solution on the concentration and purity of the extracted nucleic acid, the following examples are provided.

[0270] In Example 6.1, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecylsulfonate (SDS) by weight by volume, and 2% N-ethylpyridinium hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 4.4.

[0271] In Example 6.2, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecylsulfonate (SDS) by weight by volume, and 2% N-ethylpyridinium hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 5.4.

[0272] In Example 6.3, the lysis / binding solution comprises: 5 mol / L guanidine isothiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecylsulfonate (SDS) by weight by volume, and 2% N-ethylpyridinium hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 6.4.

[0273] In Example 6.4, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecylsulfonate (SDS) by weight by volume, and 2% N-ethylpyridinium hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 7.4.

[0274] In Example 6.5, the lysis / binding solution comprises: 5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight by volume, 100 mmol / L TE buffer (pH 7.2), 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecylsulfonate (SDS) by weight by volume, and 2% N-ethylpyridinium hexafluorophosphate by weight by volume. The pH of the lysis / binding solution is 8.4.

[0275] That is, for Examples 6.1 to 6.5, the pH values ​​of the lysis / binding solution added to the reagent cartridge well 2 are different, and the pH values ​​of the lysis / binding solution are 4.4, 5.4, 6.4, 7.4, and 8.4, respectively.

[0276] Table 7 Comparison of the effects of different pH values ​​of lysis / binding solution on nucleic acid extraction performance

[0277] Table 7 compares the effects of different pH values ​​of the lysis / binding solution on nucleic acid extraction performance. As can be seen from the table, when the pH value of the lysis / binding solution is 7.4, the concentration and purity of the extracted nucleic acids are better. It should be noted that when verifying the effects of different pH values ​​of the lysis / binding solution on nucleic acid extraction performance, the pH difference between the lysis / binding solution and the binding solution was designed to be 1. Due to deviations in reagent preparation, the nucleic acid performance test results when the pH value of the lysis / binding solution is 7.2 are similar to those when the pH value of the lysis / binding solution is 7.4. In other words, when the pH value of the lysis / binding solution is around 7.4, the concentration and purity of the extracted nucleic acids are better.

[0278] Based on the data of Example 3 on the effects of different types of anions in chaotropic salt on the concentration and purity of extracted nucleic acids, Example 4 on the effects of different types of cations in chaotropic salt on the concentration and purity of extracted nucleic acids, Example 5 on the effects of the concentration of chaotropic salt on the concentration and purity of extracted nucleic acids, and Example 6 on the effects of different pH values ​​of the lysis / binding solution on the concentration and purity of the extracted nucleic acids, the following examples of preferred lysis / binding solutions are provided.

[0279] Exemplary components of the lysis / binding solution include: guanidine isothiocyanate at a molar concentration of 5 mol / L, sodium lauryl sulfate at a weight volume percentage of 0.5%, polyoxyethylene sorbitan monolaurate at a weight volume percentage of 0.1%, TE buffer at a molar concentration of 100 mmol / L, ethylenediaminetetraacetic acid at a molar concentration of 10 mmol / L, and N-ethylpyridine hexafluorophosphate at a weight volume percentage of 2%. The pH of the lysis / binding solution is 7.4.

[0280] Exemplary components of the lysis / binding solution include: guanidine isothiocyanate at a molar concentration of 5 mol / L, sodium lauryl sulfate at a weight volume percentage of 0.5%, polyoxyethylene sorbitan monolaurate at a weight volume percentage of 0.1%, TE buffer at a molar concentration of 100 mmol / L, ethylenediaminetetraacetic acid at a molar concentration of 10 mmol / L, and N-ethylpyridine hexafluorophosphate at a weight volume percentage of 2%. The pH of the lysis / binding solution is 7.2.

[0281] Example 7

[0282] This example is used to verify the effects of using a lysis / binding solution without isopropanol and a binding solution containing isopropanol on the concentration and purity of the extracted nucleic acid.

[0283] The lysis / binding solution in this example comprises: 5 mol / L guanidine isothiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by volume, 100 mmol / L TE buffer, 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecylsulfonate (SDS) by volume, and 2% N-ethylpyridinium hexafluorophosphate by volume. The pH of the lysis / binding solution is 7.2.

[0284] The other components of the kit are the same as those in Example 1, and the nucleic acid extraction steps are the same as those in Example 1.

[0285] The comparative example of Example 7 (referred to as comparative example 1) is a reagent containing isopropyl alcohol in the binding solution.

[0286] The reagent components of the kit of Comparative Example 1 are as follows.

[0287] (1) Red blood cell lysis solution: 5 mmol / L sodium chloride (NaCl), 5% polyoxyethylene sorbitan monolaurate (Tween 20), 320 mmol / L glucose, 10 mmol / L tris(hydroxymethyl)aminomethane (Tris), pH 8.2.

[0288] (2) Lysis buffer: 3.5 mol / L guanidine thiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by mass volume, 100 mmol / L TE buffer, pH value of TE buffer is 7.5, 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecylsulfonate (SDS) by mass volume, and 1% polyethylene glycol 4-isooctylphenyl ether (Triton 100) by mass volume.

[0289] (3) Proteinase K solution includes: proteinase K at a concentration of 10 mg / mL, 50 mmol / L tris(hydroxymethyl)aminomethane (Tris), pH 8.0, 5 mmol / L calcium chloride (CaCl2), and 20% by volume of glycerol.

[0290] (4) Binding solution: 0.5 mol / L sodium chloride (NaCl), 45% isopropanol by volume, 15% PEG800 by mass volume, 100 mmol / L tris (hydroxymethyl)aminomethane (Tris), pH value of Tris is 8.0, 5 mmol / L ethylenediaminetetraacetic acid (EDTA).

[0291] Among them, PEG 800 (Polyethylene Glycol 800) is polyethylene glycol with an average molecular weight of 800.

[0292] (5) The first cleaning solution: 1 mol / L guanidine thiocyanate, 1 mmol / L tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 0.05% polyoxyethylene sorbitan monolaurate (Tween 20) by mass volume, 1 mol / L sodium chloride (NaCl), 40% isopropanol by volume, and the pH value of the first cleaning solution is 7.5.

[0293] (6) Second cleaning solution: 0.5 mol / L sodium chloride (NaCl), 0.5% by mass volume of polyethylene glycol p-isooctylphenyl ether (Triton 100), the pH value of the second cleaning solution is 7.0.

[0294] (7) Eluent: TE buffer.

[0295] Comparative Example 1 uses whole blood from a cow as a blood sample, and the specific operation process for nucleic acid extraction is as follows, including the steps of removing red blood cells from the blood sample and extracting nucleic acid.

[0296] The steps of removing red blood cells from the blood sample are the same as those in Example 1, specifically including steps R1 to R4.

[0297] R1. Take 250 μL of blood sample and place it in a 2 mL centrifuge tube. Add 800 μL of red blood cell lysis buffer and shake thoroughly for 10 seconds to obtain a red, transparent, and clear mixture.

[0298] The volume of cell lysate is 3.2 times that of blood sample.

[0299] R2. Centrifuge the mixture at a speed of 12,000 revolutions per minute (rpm) for 1 minute, remove the supernatant, and obtain a white precipitate.

[0300] R3. Add 800 μl of red blood cell lysis buffer to the centrifuge tube in step R2, shake thoroughly to mix, and resuspend the precipitate to obtain a mixed solution.

[0301] R4. Centrifuge the mixed solution obtained in step R3 at a centrifugal speed of 12,000 revolutions per minute (rpm) for 1 minute, remove the supernatant, and obtain a first precipitate.

[0302] The steps of nucleic acid extraction also include: R5 to R12.

[0303] R5. Add 400 μl of lysis buffer to the first precipitate at 65°C, mix well using a vortex mixer, and let stand for 30 minutes.

[0304] R6. Add 400 μl of isopropanol to the mixture in step R5 and mix well using a vortex mixer.

[0305] R7. Add 20 μl of the magnetic bead suspension to the mixed liquid obtained in step R6, mix well using a vortex mixer, let it stand for 5 minutes, and then mix it using a vortex mixer for 3 seconds every 2 minutes.

[0306] R8. Add 600 μl of binding solution to the mixture obtained in step R7, mix well using a vortex mixer, let it stand for 10 minutes, and then mix it using a vortex mixer for 3 seconds every 2 minutes.

[0307] R9. Place the mixed solution obtained in step R8 on a magnetic rack and let it stand for 2 minutes. Remove the supernatant to obtain a second precipitate.

[0308] Before placing the mixed solution obtained in step R8 on the magnetic rack, the mixed solution obtained in step R8 can also be subjected to low-speed centrifugation to accelerate the deposition of magnetic beads in subsequent steps.

[0309] R10. Add 600 μL of the first wash solution to the second precipitate and mix manually. Then, place the centrifuge tube on a magnetic stand and let it adsorb for 2 minutes. Remove the supernatant to obtain the third precipitate.

[0310] R11. Add 600 μL of the second wash solution to the third precipitate and mix manually. Then, place the centrifuge tube on a magnetic plate and let it adsorb for 2 minutes. Remove the supernatant to obtain the fourth precipitate, and air dry it at room temperature until no liquid is visible on the surface of the magnetic beads.

[0311] R12. Add 100 μL of elution buffer and shake for 10 minutes at 56°C. Then, place the centrifuge tube on a magnetic stand for 2 minutes. Transfer the supernatant to a new centrifuge tube to extract the nucleic acid.

[0312] Table 8 Comparison of the effects of the lysis / binding solution without isopropanol and the binding solution with isopropanol on the nucleic acid extraction performance

[0313] Table 8 compares the effects of extraction methods with and without isopropanol in the lysis / binding solution on nucleic acid extraction performance. The test results show that the performance of nucleic acids extracted using the lysis / binding solution without isopropanol in Example 7 and the extraction method with isopropanol in the binding solution in Comparative Example 17 shows little difference in concentration, and the purity of nucleic acids extracted using the lysis / binding solution without isopropanol is improved. Figure 11 shows gel electrophoresis images of nucleic acid extraction. The figure shows little difference in DNA band distribution, indicating that the nucleic acid samples extracted using the lysis / binding solution without isopropanol in Example 7 and the extraction method using isopropanol in the binding solution in Comparative Example 1 did not degrade during the experiment.

[0314] In order to compare the effect of the second cleaning solution containing ethanol on the nucleic acid extraction performance, the following comparative example 2 is provided.

[0315] Comparative Example 2 is compared with Example 7.

[0316] The reagent components of the kit of Comparative Example 2 are as follows.

[0317] (1) Red blood cell lysis solution: 5 mmol / L sodium chloride (NaCl), 5% polyoxyethylene sorbitan monolaurate (Tween 20), 320 mmol / L glucose, 10 mmol / L tris(hydroxymethyl)aminomethane (Tris), pH 8.2.

[0318] (2) Lysis / binding solution: 5 mol / L guanidine isothiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by mass volume, 100 mmol / L TE buffer, pH value of TE buffer is 7.2, 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecylsulfonate (SDS) by mass volume, and 2% N-ethylpyridine hexafluorophosphate by mass volume.

[0319] (3) Proteinase K solution includes: Proteinase K solution with a concentration of 10 mg / mL, 50 mmol / L tris(hydroxymethyl)aminomethane (Tris), pH value of 8.0, 5 mmol / L calcium chloride (CaCl2), and 20% by volume of glycerol.

[0320] (4) The first cleaning solution: 1 mol / L guanidine thiocyanate, 1 mmol / L tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 0.05% polyoxyethylene sorbitan monolaurate (Tween 20) by mass volume, 1 mol / L sodium chloride (NaCl), 40% isopropanol by volume, and the pH value of the first cleaning solution is 7.5.

[0321] (5) Second cleaning solution: TE buffer and 80% ethanol solution by volume.

[0322] (6) Eluent: TE buffer.

[0323] Table 9 Comparison of the effects of the extraction method with and without isopropanol in the second cleaning solution on the nucleic acid extraction performance

[0324] Table 9 compares the effects of the extraction methods in which the second cleaning solution does not contain isopropanol and the second cleaning solution contains isopropanol on the nucleic acid extraction performance. The test results show that the extraction methods in which the second cleaning solution does not contain isopropanol in Example 7 and the second cleaning solution contains isopropanol in Comparative Example 2 have little effect on the performance of the extracted nucleic acid.

[0325] Therefore, under the condition of ensuring the performance of the extracted nucleic acid, using a second cleaning solution that does not contain isopropanol can improve the performance of the reagent in long-term storage.

[0326] In order to compare the effects of a lysis / binding solution containing a chaotropic salt and a lysis / binding solution not containing a chaotropic salt on nucleic acid extraction performance, the following Comparative Example 3 is provided.

[0327] Comparative Example 3 is compared with Example 7.

[0328] The components of the lysis / binding solution in Comparative Example 3 include: 5 mol / L guanidine isothiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by mass volume, 100 mmol / L TE buffer, the pH value of the TE buffer is 7.2, 10 mmol / L ethylenediaminetetraacetic acid (EDTA), and 0.5% sodium dodecyl sulfate (SDS) by mass volume.

[0329] The other components of the kit are the same as those in Example 1 (it is understood that the other components of the kit are also the same as those in Example 7), and the nucleic acid extraction steps are the same as those in Example 1.

[0330] Table 10 Comparison of the effects of the lysis / binding solution without chaotropic salt and the lysis / binding solution with chaotropic salt on the nucleic acid extraction performance

[0331] Table 10 compares the effects of the lysis / binding solution without chaotropic salt and the lysis / binding solution with chaotropic salt on nucleic acid extraction performance. The test results show that the concentration and purity of nucleic acids extracted by the lysis / binding solution with chaotropic salt are higher than those of the lysis / binding solution without chaotropic salt in ratio 3.

[0332] Therefore, Comparative Example 3 proves that chaotropic salt is helpful for the extraction of nucleic acid.

[0333] Example 8

[0334] This example aims to verify the effects of different types of chaotropic salts on the concentration and purity of extracted nucleic acids and to compare the performance of nucleic acids extracted with commercially available reagents.

[0335] The reagent components of the lysis / binding solution provided in this example are: 5 mol / L guanidine isothiocyanate, 0.1% polyoxyethylene sorbitan monolaurate (Tween 20) by weight volume, 100 mmol / L TE buffer, the pH value of the TE buffer is 7.2, 10 mmol / L ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate (SDS) by weight volume, and 2% by weight volume of chaotropic salt.

[0336] The difference is that the chaotropic salt components in the lysis / binding solution are different, and the chaotropic salt components are: sodium hexafluorophosphate, ammonium hexafluorophosphate, 1-allyl-3-methylimidazole hexafluorophosphate, N-ethylpyridinium hexafluorophosphate, 1-allyl-3-methylimidazole chloride, N-ethylpyridinium chloride, 1-allyl-3-methylimidazole phosphate and N-ethylpyridinium phosphate.

[0337] The other components of the kit are the same as those in Example 1, and the nucleic acid extraction steps are the same as those in Example 1.

[0338] Table 11 Effects of different types of chaotropic salts on the performance of extracted nucleic acids and comparison with the performance of nucleic acids extracted using commercially available reagents

[0339] Table 11 shows the effects of different types of chaotropic salts on the performance of the extracted nucleic acids, and compares the performance of nucleic acids extracted with commercially available reagents. From the test results, when the chaotropic salts in the lysis / binding solution are sodium hexafluorophosphate, ammonium hexafluorophosphate, 1-allyl-3-methylimidazolium hexafluorophosphate, N-ethylpyridinium hexafluorophosphate, and N-ethylpyridinium chloride, the performance of the extracted nucleic acids is not much different from that of the nucleic acids extracted with commercially available reagents.

[0340] Therefore, unifying the components of the lysis buffer and the binding buffer and selecting appropriate chaotropic salts not only reduces the nucleic acid extraction time, but also allows the sample to bind to the magnetic beads while being lysed, and can better match the nucleic acid automatic extractor to achieve fully automatic extraction without affecting the performance of the extracted nucleic acid.

[0341] The above description is merely a specific embodiment of the present disclosure, but the scope of protection of the present disclosure is not limited thereto. Any changes or substitutions that a person skilled in the art can conceive within the technical scope disclosed in the present disclosure should be included within the scope of protection of the present disclosure. Therefore, the scope of protection of the present disclosure should be based on the scope of protection of the claims.

Claims

1. A lysis / binding solution for nucleic acid extraction, comprising: A protein denaturant, an alcohol-free dehydrating agent, a first surfactant, a buffer, ethylenediaminetetraacetic acid, and a chaotropic salt; The molar concentration range of the protein denaturant is 3.5mol / L to 5.5mol / L, the mass volume ratio of the alcohol-free dehydrating agent is 0.1% to 1%, the mass volume ratio of the first surfactant is 0.05% to 0.5%, the molar concentration range of the buffer solution is 5mmol / L to 150mmol / L, the molar concentration range of the ethylenediaminetetraacetic acid is 5mmol / L to 20mmol / L, and the mass volume ratio of the chaotropic salt is 1% to 8%.

2. The lysis / binding solution for nucleic acid extraction according to claim 1, wherein: The anions in the chaotropic salt include: PO4 3- 、SO4 2- 、H2PO4 - HCOO - , Cl - 、NO3 - CF3COO - 、BF4 - 、ClO4 - and PF6 - At least one of .

3. The lysis / binding solution for nucleic acid extraction according to claim 1 or 2, wherein: The cations in the chaotropic salt include: Ca 2+ Mg 2+ , Li + 、Na + , K + NH4 + At least one of an imidazolium cation and an N-ethylpyridinium cation.

4. The lysis / binding solution for nucleic acid extraction according to any one of claims 1 to 3, wherein: The chaotropic salt comprises at least one of ammonium phosphate, ammonium sulfate, ammonium dihydrogen phosphate, ammonium chloride, calcium hexafluorophosphate, potassium hexafluorophosphate, sodium hexafluorophosphate, ammonium hexafluorophosphate, 1-allyl-3-methylimidazolium hexafluorophosphate, N-ethylpyridine hexafluorophosphate and N-ethylpyridine chloride.

5. The lysis / binding solution for nucleic acid extraction according to any one of claims 1 to 4, wherein: The alcohol-free dehydrating agent comprises: At least one of polyoxyethylene lauryl ether, polyvinyl carbazole, polyvinyl alcohol, polyacrylamide, polyacrylic acid, polyoxyethylene sorbitan monolaurate and sodium lauryl sulfate.

6. The lysis / binding solution for nucleic acid extraction according to any one of claims 1 to 5, wherein: The protein denaturant comprises at least one of guanidine isothiocyanate and guanidine hydrochloride.

7. The lysis / binding solution for nucleic acid extraction according to any one of claims 1 to 6, wherein: The buffer comprises: at least one of TE buffer and tris(hydroxymethyl)aminomethane; the TE buffer comprises: tris(hydroxymethyl)aminomethane hydrochloride and ethylenediaminetetraacetic acid.

8. The lysis / binding solution for nucleic acid extraction according to any one of claims 1 to 7, wherein: The first surfactant includes: polyoxyethylene sorbitan monolaurate or polyethylene glycol p-isooctylphenyl ether. 9 . The lysis / binding solution for nucleic acid extraction according to claim 1 , wherein the pH value of the lysis / binding solution is in the range of 4.4 to 7.

4.

10. The lysis / binding solution for nucleic acid extraction according to any one of claims 1 to 9, comprising: Guanidine isothiocyanate, sodium dodecyl sulfate, polyoxyethylene sorbitan monolaurate, TE buffer, ethylenediaminetetraacetic acid and N-ethylpyridine hexafluorophosphate; wherein guanidine isothiocyanate is a protein denaturant, sodium dodecyl sulfate is an alcohol-free dehydrating agent, and polyoxyethylene sorbitan monolaurate is a first surfactant; The molar concentration of guanidine isothiocyanate is 5 mol / L, the mass volume proportion of sodium dodecyl sulfate is 0.5%, the mass volume proportion of polyoxyethylene sorbitan monolaurate is 0.1%, the molar concentration of TE buffer is 100 mmol / L, the molar concentration of ethylenediaminetetraacetic acid is 10 mmol / L, and the mass volume proportion of N-ethylpyridine hexafluorophosphate is 2%; the pH value of the lysis / binding solution is 7.

4.

11. The lysis / binding solution for nucleic acid extraction according to any one of claims 1 to 10, wherein: The pH value range of the buffer solution is 7.0-7.

4.

12. The lysis / binding solution for nucleic acid extraction according to any one of claims 1 to 11, wherein: The pH value of the buffer solution is 7.

2.

13. A cleaning solution for nucleic acid extraction, comprising: Sodium chloride and a second surfactant, wherein the molar concentration of the sodium chloride is in the range of 0.05 mol / L to 0.5 mol / L, and the mass volume ratio of the second surfactant is in the range of 0.5% to 5%; and the pH value of the cleaning solution is in the range of 6.9 to 7.

1.

14. The cleaning solution for nucleic acid extraction according to claim 13, wherein The second surfactant includes at least one of polyethylene glycol p-isooctylphenyl ether, polyoxyethylene sorbitan monolaurate and ethylphenyl polyethylene glycol.

15. A nucleic acid extraction kit, comprising: The lysis / binding solution for nucleic acid extraction according to any one of claims 1 to 12; The second cleaning liquid comprises: the cleaning liquid as claimed in claim 13 or 14.

16. The nucleic acid extraction kit according to claim 15, further comprising: Red blood cell lysis buffer; The red blood cell lysing solution comprises: sodium chloride, polyoxyethylene sorbitan monolaurate, glucose and tris(hydroxymethyl)aminomethane; The molar concentration of the sodium chloride is in the range of 1 mol / L to 10 mol / L, the mass volume ratio of the polyoxyethylene sorbitan monolaurate is in the range of 3% to 6%, the molar concentration of the glucose is in the range of 200 mmol / L to 500 mmol / L, and the molar concentration of the tris(hydroxymethyl)aminomethane is in the range of 1 mmol / L to 50 mmol / L; The pH value of the red blood cell lysis solution ranges from 8.0 to 8.

4.

17. The nucleic acid extraction kit according to claim 15 or 16, further comprising: Proteinase K solution, concentration range is 10mg / mL~30mg / mL.

18. The nucleic acid extraction kit according to any one of claims 15 to 17, further comprising: Magnetic bead suspension, concentration range is 10mg / mL~40mg / mL.

19. The nucleic acid extraction kit according to any one of claims 15 to 18, further comprising: The first cleaning solution comprises: guanidine isothiocyanate, tris(hydroxymethyl)aminomethane hydrochloride, polyoxyethylene sorbitan monolaurate, sodium chloride and isopropanol; the molar concentration range of the guanidine isothiocyanate is 0.5 mol / L to 3 mol / L, the molar concentration range of the tris(hydroxymethyl)aminomethane hydrochloride is 0.5 mmol / L to 10 mmol / L, the mass volume ratio range of the polyoxyethylene sorbitan monolaurate is 0.01% to 0.1%, and the molar concentration range of the sodium chloride is 0.5mol / L~2mol / L, the volume proportion range of the isopropanol is 20%~50%; the pH value range of the first cleaning solution is 6.5~8.

0.

20. The nucleic acid extraction kit according to any one of claims 15 to 19, further comprising: The elution solution includes: TE buffer.

21. A method for extracting nucleic acid, comprising: A. Add lysis / binding solution and proteinase K solution to the sample to be extracted nucleic acid at a temperature of 55°C to 80°C and mix; B. Add the magnetic bead suspension to the mixed liquid obtained in step A, mix, place on a magnetic stand and let stand, remove the supernatant, and obtain a second precipitate; C. Add the first cleaning solution to the second precipitate, mix, place on a magnetic stand and let stand, remove the supernatant, and obtain a third precipitate; D. Add the second cleaning solution to the third precipitate, mix, place on a magnetic stand and let stand, remove the supernatant, and obtain a fourth precipitate; E. At a temperature of 40°C to 60°C, add the eluent to the fourth precipitate, mix, place on a magnetic stand to stand, transfer the supernatant, and obtain the extracted nucleic acid.

22. The method for extracting nucleic acid according to claim 21, wherein: The volume of the lysis / binding solution is 2 to 4 times the volume of the blood sample, the volume of the proteinase K solution is 0.05 to 0.2 times the volume of the blood sample, the volume of the magnetic bead suspension is 0.075 to 0.2 times the volume of the blood sample, the volume of the first cleaning solution is 2.4 to 4.5 times the volume of the blood sample, and the volume of the second cleaning solution is 2.4 to 4.5 times the volume of the blood sample.

23. The method for extracting nucleic acid according to claim 21 or 22, wherein: Before step A, the method further includes: A0, taking a blood sample and placing it in a container, adding a red blood cell lysing solution, mixing and centrifuging, removing the supernatant, and obtaining a first precipitate; wherein the sample to be extracted nucleic acid in step A is the first precipitate; Wherein, in step A0, the centrifugal speed ranges from 10000 rpm to 12000 rpm, and the centrifugal time ranges from 1 minute to 3 minutes; in step A0, the steps of adding red blood cell lysate, mixing, centrifuging, and removing the supernatant are repeated at least once; The volume of the red blood cell lysis solution is 3 to 5 times the volume of the blood sample.

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