Nucleic acid separation and purification kit and application thereof

Abstract

The invention belongs to the field of molecular biology, and particularly relates to a nucleic acid separation and purification kit and application thereof. The separation and purification reagent in the separation and purification kit provided by the invention does not contain ethanol and isopropanol, is suitable for the totally-enclosed micro-fluidic chip, and can be used for purifying and separating nucleic acid on the totally-enclosed micro-fluidic chip.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to a nucleic acid separation and purification kit and its application. Background Art

[0002] With technological advancements, nucleic acid purification has become essential in the preparatory stages of molecular biology research. Purifying intact, high-quality nucleic acids is a crucial step in experimental procedures such as disease diagnosis, cloning, sequencing, amplification, hybridization, and cDNA analysis. Otherwise, the presence of large amounts of intracellular components (e.g., proteins and polysaccharides) or certain components in the lysate (e.g., organic solvents and metal ions) can hinder the completion of downstream reactions.

[0003] Nucleic acids include deoxyribonucleic acid (DNA) and RNA (RNA). In cells, most nucleic acids exist in a state bound to proteins. The general principles for separating and purifying nucleic acids are: (1) Integrity. To ensure the study of nucleic acid structure and function, a complete primary structure is the most basic requirement, because all genetic information is stored in the primary structure. The primary structure of nucleic acids also determines their higher-order structure and the way they bind to other biological macromolecules. (2) Purity. The separated and purified nucleic acid samples should be free of organic solvents that inhibit enzymes and excessive concentrations of metal ions. Contamination by other biological macromolecules other than nucleic acid components, such as proteins, polysaccharides, and lipid molecules, should be minimized.

[0004] The method of nucleic acid separation and purification mainly includes two steps: the first is lysis, which breaks up the cells to release the nucleic acids. The lysis step is the key link in nucleic acid purification. The lysis process mainly breaks up the cells and releases the nucleic acids; the second is purification, which is to separate the released nucleic acids from other components in the cell (such as proteins, salts and other impurities).

[0005] Currently, the widely used methods for nucleic acid separation and purification include phenol-chloroform extraction and centrifugal column purification. Among them, the phenol-chloroform method uses a large amount of volatile organic solvents, which causes a certain degree of pollution to operators and the environment; the centrifugal column method is complicated to operate and requires repeated centrifugation in a high-speed centrifuge, so it has disadvantages such as difficulty in achieving multi-throughput and automation.

[0006] In recent years, methods using magnetic microspheres for nucleic acid separation and purification have been increasingly used. Magnetic microspheres can be attracted by magnetic rings or magnetic rods, and in conjunction with a robotic arm workstation, a high-throughput, fully automated nucleic acid purification process can be achieved. Traditional methods for nucleic acid separation and purification include CN116694624A, CN108977437A, and CN117230060B. However, the reagents used in traditional methods usually contain a large amount of ethanol and / or isopropanol, which cannot be used in a fully enclosed working environment, thereby seriously affecting the downstream PCR reaction. In addition, ethanol and isopropanol are flammable and explosive dangerous goods, and are subject to strict restrictions in terms of use, storage, transportation, and other aspects.

[0007] In view of this, this application is hereby filed. Summary of the Invention

[0008] Based on this, one or more embodiments of the present application provide a nucleic acid separation and purification kit and its application, including the following technical solutions:

[0009] One or more embodiments of the present application provide a nucleic acid separation and purification kit, wherein the separation and purification reagents in the nucleic acid separation and purification kit include a lysate, a washing solution 1, and a washing solution 2;

[0010] in,

[0011] The lysate comprises 1M-4M guanidine thiocyanate, 40mM-100mM tris(hydroxymethyl)aminomethane hydrochloride, 1wt%-5wt% NP-40, 20wt%-40wt% PEG6000, 1wt%-4wt% Triton X-100, 2mmol / L-4mmol / L EDTA, 1wt%-2wt% catechin, 2wt%-5wt% SDS and 1M-2M NaCl, with a pH value of 3.5-4.5;

[0012] The washing solution 1 comprises 0.5M-1M guanidine thiocyanate, 10mM-50mM tris(hydroxymethyl)aminomethane hydrochloride, 20wt%-40wt% PEG6000 and 200mM-700mM NaCl, with a pH value of 4.8-5.5;

[0013] The washing solution 2 comprises 10 mM-50 mM tris(hydroxymethyl)aminomethane hydrochloride, 0.1 wt%-1 wt% DTT and 5 wt%-30 wt% PEG6000, with a pH value of 4.8-5.5;

[0014] The separation and purification reagent does not include ethanol and isopropanol.

[0015] In some embodiments of the present application, the separation and purification reagent does not include ethanol, isopropanol and histone digestion enzyme.

[0016] In some embodiments of the present application, the histone digestion enzyme comprises proteinase K.

[0017] In some embodiments of the present application, the lysate includes 1.5M-2.5M guanidine thiocyanate, 45mM-55mM tris(hydroxymethyl)aminomethane hydrochloride, 1wt%-2wt% NP-40, 35wt%-40wt% PEG6000, 1.5wt%-2.5% Triton X-100, 2.5mmol / L-3.5mmol / L EDTA, 1.3wt%-1.8wt% catechin, 2wt%-2.5wt% SDS and 1M-2M NaCl, with a pH of 3.5-4.5.

[0018] In some embodiments of the present application, the washing solution 1 comprises 0.5M-0.7M guanidine thiocyanate, 25mM-35mM tris(hydroxymethyl)aminomethane hydrochloride, 25wt%-35wt% PEG6000 and 450mM-550mM NaCl, with a pH value of 4.8-5.5.

[0019] In some embodiments of the present application, the washing solution 2 comprises 35 mM-45 mM tris(hydroxymethyl)aminomethane hydrochloride, 0.3 wt%-0.5 wt% DTT and 5, 8 wt%-12 wt% PEG6000, with a pH value of 4.8-5.5.

[0020] In some embodiments of the present application, the separation and purification reagent further comprises magnetic microspheres;

[0021] Optionally, the composition of the magnetic microspheres includes Fe3O4, or Fe3O4 and SiO2;

[0022] Optionally, the surface modification groups of the magnetic microspheres include hydroxyl groups or carboxyl groups;

[0023] Optionally, the particle size of the magnetic beads ranges from 100 nm to 800 nm.

[0024] In some embodiments of the present application, the magnetic microspheres are suspended in a dispersion medium;

[0025] Optionally, the dispersion medium includes water;

[0026] Optionally, the content of the magnetic microspheres is 2 wt%-3 wt% based on the mass proportion in the suspension system composed of the magnetic microspheres and the dispersion medium.

[0027] In some embodiments of the present application, the separation and purification reagent further comprises an eluent;

[0028] Optionally, the eluent comprises 10 mM-40 mM tris(hydroxymethyl)aminomethane hydrochloride and 2 mmol / L-4 mmol / L EDTA, with a pH value of 7.8-8.2.

[0029] One or more embodiments of the present application further provide a method for preparing a nucleic acid sample, the method comprising the following steps:

[0030] The nucleic acid separation and purification kit is used to separate and purify nucleic acids from the sample to be tested to prepare a nucleic acid sample.

[0031] In some embodiments of the present application, nucleic acid separation and purification is performed in a fully enclosed microfluidic chip.

[0032] In some embodiments of the present application, the sample to be tested includes a blood sample.

[0033] One or more embodiments of the present application further provide a method for detecting a target nucleic acid, the method comprising the following steps:

[0034] preparing a nucleic acid sample using the preparation method; and,

[0035] Detecting the target nucleic acid in the nucleic acid sample.

[0036] The details of one or more embodiments of the present application are set forth in the description below, and other features, objects, and advantages of the application will become apparent from the description and from the claims thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0038] Figure 1 This is the electrophoresis detection diagram corresponding to the separation and purification reagent of Example 1;

[0039] Figure 2 The electrophoresis detection diagrams corresponding to Examples 2 to 4 and Comparative Examples 1 to 3 are shown;

[0040] Figure 3 The electrophoresis detection diagrams corresponding to Examples 5 to 7 and Comparative Examples 4 to 6 are shown;

[0041] Figure 4 The following are test results of Example 1 and a commercial kit. DETAILED DESCRIPTION

[0042] Below in conjunction with accompanying drawing, embodiment and example, the application is described in further detail.It should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms and is not limited to the embodiment and example described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large amount of specific details are given in order to provide a more complete understanding of the application.It should be understood that the application can be implemented without one or more of these details.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing embodiments and examples only and are not intended to limit this application.

[0044] the term

[0045] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0046] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0047] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0048] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0049] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0050] Herein, "preferred", "better", "more preferred" and "suitable" are merely used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0051] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0052] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0053] In this application, the terms "first," "second," "third," "fourth," etc. in "the first aspect," "the second aspect," "the third aspect," "the fourth aspect," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the indicated technical features. Furthermore, "first," "second," "third," "fourth," etc. serve only as non-exhaustive enumeration and description, and should be understood not to constitute a closed-ended limitation on quantity.

[0054] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0055] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0056] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0057] In this application, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0058] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0059] In traditional nucleic acid separation and purification methods, the reagents used usually contain a large amount of ethanol and / or isopropanol. The residual alcohol can be volatilized by opening the lid and standing still in the workstation. However, in a fully enclosed microfluidic chip, there will be residues because the lid cannot be opened, which seriously affects the downstream PCR reaction. In addition, fully enclosed microfluidic chips are mostly used in POCT (point-of-care testing) scenarios. Most of the reagents used require less manual operation and are actually stored and transported at room temperature. Therefore, the operating procedures and reagent formulas of the existing conventional magnetic bead method cannot be directly applied to fully enclosed microfluidic chips. In addition, the traditional method also involves the use of enzyme activity reagents such as proteinase K for digesting histones, but the storage of such reagents usually requires low temperature and cannot be mixed with other reagents in advance, that is, it is difficult to prefabricate. This will affect the overall storage conditions of the purification kit and increase the complexity of the operation. In view of the shortcomings of traditional technology, this application scheme is proposed.

[0060] In a first aspect of the embodiments of the present application, a nucleic acid separation and purification kit is provided, wherein the separation and purification reagents in the nucleic acid separation and purification kit include a lysate, a washing solution 1, and a washing solution 2;

[0061] in,

[0062] The lysate comprises 1M-4M guanidine thiocyanate, 40mM-100mM tris(hydroxymethyl)aminomethane hydrochloride, 1wt%-5wt% NP-40, 20wt%-40wt% PEG6000, 1wt%-4wt% Triton X-100, 2mmol / L-4mmol / L EDTA, 1wt%-2wt% catechin, 2wt%-5wt% SDS and 1M-2M NaCl, with a pH value of 3.5-4.5;

[0063] The washing solution 1 comprises 0.5M-1M guanidine thiocyanate, 10mM-50mM tris(hydroxymethyl)aminomethane hydrochloride, 20wt%-40wt% PEG6000 and 200mM-700mM NaCl, with a pH value of 4.8-5.5;

[0064] The washing solution 2 comprises 10 mM-50 mM tris(hydroxymethyl)aminomethane hydrochloride, 0.1 wt%-1 wt% DTT and 5 wt%-30 wt% PEG6000, with a pH value of 4.8-5.5;

[0065] The separation and purification reagent does not include ethanol and isopropanol.

[0066] The embodiments of the present application provide a nucleic acid separation and purification kit suitable for a fully enclosed microfluidic chip and its application. The separation and purification reagents in the kit can quickly purify and separate nucleic acids on a fully enclosed microfluidic chip, with a recovery rate and purity that are no less than existing purification reagents, and do not contain ethanol and isopropanol (or further do not contain proteinase K), significantly improving downstream PCR reaction performance.

[0067] In the lysate of the embodiment of the present application, the concentration of guanidine thiocyanate is, for example, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4M, the concentration of tris (hydroxymethyl)aminomethane hydrochloride is, for example, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100mM, the concentration of NP-40 is, for example, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%, the concentration of PEG6000 is, for example, 20wt%, 25wt%, 30wt%, 35wt%, 40wt%, and the concentration of Triton is, for example, 40wt%. The concentration of X-100 is, for example, 1wt%, 1.5wt%, 2wt%, 2.5wt%, 3wt%, 3.5wt%, and 4wt%; the concentration of EDTA is, for example, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, and 4 mmol / L; the concentration of catechin is, for example, 1wt%, 1.2wt%, 1.4wt%, 1.6wt%, 1.8wt%, and 2wt%; the concentration of SDS is, for example, 2wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, and 5wt%; the concentration of NaCl is, for example, 1, 1.2, 1.4, 1.6, 1.8, and 2M; and the pH value is, for example, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, and 4.5.

[0068] In the washing solution 1 of the embodiment of the present application, the concentration of guanidine thiocyanate is, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1 M, the concentration of tris (hydroxymethyl)aminomethane hydrochloride is, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50 mM, the concentration of PEG6000 is, for example, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, the concentration of NaCl is, for example, 200, 250, 3000, 350, 400, 450, 500, 550, 600, 650, 700 mM, and the pH value is, for example, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5.

[0069] In the washing solution 2 of the embodiment of the present application, the concentration of tris(hydroxymethyl)aminomethane hydrochloride is, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50 mM, the concentration of DTT is, for example, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, the concentration of PEG6000 is, for example, 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, and the pH value is, for example, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5.

[0070] In some embodiments, the separation and purification reagents do not include ethanol, isopropanol, and histone digestion enzymes.

[0071] The present application does not particularly limit the type of histone digestion enzyme, including but not limited to proteinase K.

[0072] Optionally, the lysate comprises 1.5M-2.5M guanidine thiocyanate, 45mM-55mM tris(hydroxymethyl)aminomethane hydrochloride, 1wt%-2wt% NP-40, 35wt%-40wt% PEG6000, 1.5wt%-2.5% Triton X-100, 2.5mmol / L-3.5mmol / L EDTA, 1.3wt%-1.8wt% catechin, 2wt%-2.5wt% SDS and 1M-2M NaCl, with a pH of 3.5-4.5.

[0073] Optionally, the washing solution 1 comprises 0.5M-0.7M guanidine thiocyanate, 25mM-35mM tris(hydroxymethyl)aminomethane hydrochloride, 25wt%-35wt% PEG6000 and 450mM-550mM NaCl, with a pH value of 4.8-5.5.

[0074] Optionally, the washing solution 2 comprises 35 mM-45 mM tris(hydroxymethyl)aminomethane hydrochloride, 0.3 wt%-0.5 wt% DTT and 5, 8 wt%-12 wt% PEG6000, with a pH value of 4.8-5.5.

[0075] In some embodiments, the separation and purification reagent further comprises magnetic microspheres;

[0076] Optionally, the composition of the magnetic microspheres includes Fe3O4, or Fe3O4 and SiO2;

[0077] Optionally, the surface modification groups of the magnetic microspheres include hydroxyl groups or carboxyl groups;

[0078] Optionally, the particle size range of the magnetic beads is 100 nm-800 nm, for example, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 nm.

[0079] In some embodiments, the magnetic microspheres are suspended in a dispersion medium. The present application does not particularly limit the dispersion medium, and includes but is not limited to water. Optionally, the content of the magnetic microspheres is 2 wt% to 3 wt%, for example, 2 wt%, 2.2 wt%, 2.4 wt%, 2.6 wt%, 2.8 wt%, or 3 wt%, based on the mass percentage of the magnetic microspheres and the dispersion medium in the suspension system.

[0080] In some embodiments, the separation and purification reagent also includes an eluent; optionally, the eluent includes 10mM-40mM (for example, 10, 15, 20, 25, 30, 35, 40mM) tris(hydroxymethylaminomethane) hydrochloride and 2mmol / L-4mmol / L (for example, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4mmol / L) EDTA, and the pH value is 7.8-8.2 (for example, 7.8, 7.9, 8, 8.1, 8.2).

[0081] A second aspect of the present invention provides a method for preparing a nucleic acid sample, the method comprising the following steps:

[0082] The nucleic acid separation and purification kit is used to separate and purify nucleic acids from the sample to be tested to prepare a nucleic acid sample.

[0083] In some embodiments of the present application, separation and purification of nucleic acids are performed in a fully enclosed microfluidic chip.

[0084] The present application does not particularly limit the type of sample to be tested, which may be a biological sample, such as a sample taken from a mammal, including but not limited to a blood sample, which may be a pig blood sample.

[0085] In a third aspect of the embodiments of the present application, a method for detecting a target nucleic acid is provided, the method comprising the following steps:

[0086] preparing a nucleic acid sample using the preparation method; and,

[0087] Detecting the target nucleic acid in the nucleic acid sample.

[0088] The present application does not specifically limit the type of target nucleic acid, including but not limited to the GAPDH gene.

[0089] In the examples of the present application, the lysis solution mainly ruptures the outer structure of cells, bacteria or viruses, denatures histones, and releases nucleic acids, while providing a suitable environment for the magnetic beads to adsorb nucleic acids.

[0090] In the embodiment of the present application, the washing solution 1 is mainly used to wash away cell debris, settled proteins, etc.

[0091] In the embodiment of the present application, the washing solution 2 is mainly used to further clean impurities other than nucleic acids. The elution solution is mainly used to elute the nucleic acids from the magnetic microspheres, dissolve them in them, form a nucleic acid solution, and transfer them to the downstream for PCR reaction.

[0092] In the examples of the present application, the main function of the magnetic microspheres is to adsorb free nucleic acids.

[0093] The method for separating and purifying nucleic acids using the reagent kit of the embodiment of the present application can be carried out in a fully enclosed microfluidic device. The four reagents in the reagent kit are pre-infused in four different reagent chambers, which are connected by multiple liquid pipelines, and a reaction chamber is provided at a specific position, and a magnet and a mixing device are provided at the lower part of the reaction chamber. The reagents can flow into the reaction chamber through the pipeline under the push of the pump. There are at least five reagent chambers, which can be made of hard or flexible resilient polymer materials. The pump can be driven by a motor rotating to push the piston rod, a motor driving a button to squeeze the flexible reagent chamber, etc. The mixing device can be an ultrasonic transducer or a vibration mixer, etc., and can be equipped with a heating function.

[0094] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0095] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0096] The following implementation plans are all carried out with reference to the following steps:

[0097] 1. Fill the lysis solution (800 μL), cleaning solution 1 (850 μL), cleaning solution 2 (850 μL), and eluent (400 μL) into reagent chamber 1, reagent chamber 2, reagent chamber 3, and reagent chamber 4, respectively, and seal these reagent chambers;

[0098] 2. Add 100 μL of blood sample to the reagent chamber (reagent chamber 0) without reagent, and seal the reagent chamber;

[0099] 3. Use the pump to push 100 μL of lysate into reagent compartment 0 to mix the lysate with the sample;

[0100] 4. The mixture of lysate and sample is pushed into the reaction chamber to dissolve the magnetic beads pre-prepared in the reaction chamber. The magnetic beads and the mixture are thoroughly mixed using a mixing device. The mixing process lasts for 5 minutes to allow the cells in the sample to be fully lysed and the nucleic acids to be released and adsorbed onto the magnetic beads.

[0101] 5. Stop the mixing device and move the magnet close to the reaction chamber to fix the magnetic beads adsorbed with nucleic acid in the reaction chamber;

[0102] 6. Push 200 μL of cleaning solution 1 into the reaction chamber and push the waste liquid in the original reaction chamber back to the reagent chamber 0;

[0103] 7. Move the magnet away from the reaction chamber and release the magnetic beads. Turn on the mixing device and mix for 5 seconds each time, pause for 2 seconds, and repeat 3 times. Use cleaning solution 1 to thoroughly clean the reaction chamber and magnetic beads to remove cell debris, protein and other impurities;

[0104] 8. Stop the mixing device and move the magnet close to the reaction chamber to fix the magnetic beads adsorbed with nucleic acid in the reaction chamber;

[0105] 9. Push 200 μL of cleaning solution 2 into the reaction chamber and push the waste liquid in the original reaction chamber back to the reagent chamber 2;

[0106] 10. Move the magnet away from the reaction chamber and release the magnetic beads. Turn on the mixing device and mix for 5 seconds each time, pause for 2 seconds, and repeat 3 times. Rinse the reaction chamber and magnetic beads again with cleaning solution 2 to remove residual guanidine salts, proteins and other impurities;

[0107] 11. Stop the mixing device and move the magnet close to the reaction chamber to fix the magnetic beads adsorbed with nucleic acid in the reaction chamber;

[0108] 12. Push 200 μL of eluent into the reaction chamber and push the waste liquid in the original reaction chamber back to the reagent chamber 3;

[0109] 13. Remove the magnet from the reaction chamber to release the magnetic beads. Turn on the heating function to 60°C. Turn on the mixing device and mix for 1 second each time, then pause for 10 seconds, repeating three times to fully dissolve the nucleic acids adsorbed on the magnetic beads in the eluent.

[0110] 14. Stop the mixing device and move the magnet close to the reaction chamber to fix the magnetic beads in the reaction chamber and transfer the nucleic acid liquid out for subsequent experimental operations or low-temperature storage.

[0111] Example 1

[0112] This embodiment provides a nucleic acid separation and purification kit, comprising:

[0113] The main components of the magnetic bead suspension are Fe3O4 and SiO2, and the surface is modified with hydroxyl groups, namely silicon hydroxyl magnetic beads, with a particle size of 400nm and a solid content of 2.5wt%. The dispersion medium of the magnetic bead suspension is water.

[0114] The lysis buffer consists of 2 M guanidine thiocyanate, 50 mM tris-HCl (pH 4.0), 1.5 wt% NP-40, 40 wt% polyethylene glycol 6000 (PEG6000), 2 wt% polyethylene glycol tert-octylphenyl ether (Triton X-100), 3 mmol / L ethylenediaminetetraacetic acid (EDTA), 1.5 wt% catechin, 2 wt% sodium dodecyl sulfate (SDS), 2 M sodium chloride (NaCl), and water.

[0115] The components of the washing solution 1 are 0.5 M guanidine thiocyanate, 30 mM tris-HCl (pH=5.0), 30 wt % polyethylene glycol 6000 (PEG6000), and 500 mM sodium chloride (NaCl).

[0116] The components of washing solution 2 are 40 mM tris-HCl (pH=5.0), 0.4 wt% DL-dithiothreitol (DTT), and 10% polyethylene glycol 6000 (PEG6000).

[0117] The eluent consisted of 30 mM Tris-HCl (pH=8.0) and 3 mmol / L ethylenediaminetetraacetic acid (EDTA).

[0118] Example 2 to Example 4

[0119] Examples 2 to 4 respectively provide a nucleic acid separation and purification kit. The difference from Example 1 lies in the formula of the lysate, as shown in Table 1. The formulas of the other three reagents are the same as those in Example 1.

[0120] Table 1

[0121]

[0122] Comparative Examples 1 to 3

[0123] Comparative Examples 1 to 3 respectively provide a nucleic acid separation and purification kit. The formula of the lysate is shown in Table 2, and the formulas of the other three reagents are the same as in Example 1.

[0124] Table 2

[0125]

[0126] According to the above operating steps, the separation and purification kit of Example 1 was used to separate and purify nucleic acids from three blood samples collected from pigs. The 260 / 280 ratio of the nucleic acid solution was detected using nanodrop to calculate the purity and concentration of the nucleic acid. The results are shown in the following table and Figure 1 As shown, the 260 / 280 values ​​of the three blood samples were all above 1.8, the concentration was above 10 ng / μL, and the electrophoresis detection bands were clear and bright without tailing.

[0127] Table 3

[0128]

[0129]

[0130] For the same blood sample (H1), separation and purification were carried out according to the above-mentioned operation steps using the separation and purification reagents of Examples 2 to 4 and Comparative Examples 1 to 3. The results are shown in the following table and Figure 2 As shown:

[0131] The nucleic acid purity 260 / 280 corresponding to the three examples was greater than 1.7, but only Example 2 was greater than 1.8. For the same blood sample, the nucleic acid concentrations of the nucleic acid solutions obtained using different separation and purification reagents were slightly different, indicating that the components of the separation and purification reagents did affect the nucleic acid concentration, but all were within an acceptable range.

[0132] Compared with the examples, the nucleic acid purity corresponding to the three comparative examples was significantly reduced, and the electrophoresis bands were also significantly inferior to the three examples, which indicates that when the reagent components exceeded the preset range, the purification effect was significantly reduced.

[0133] Table 4

[0134] Separation and purification reagents ng / μL A260 / A280 Example 2 11.7 1.82 Example 3 12.9 1.72 Example 4 17.5 1.78 Comparative Example 1 1.7 1.3 Comparative Example 2 2.3 1.62 Comparative Example 3 1.6 1.28

[0135] Example 5 to Example 7

[0136] Examples 5 to 7 respectively provide a nucleic acid separation and purification kit. The changes compared to Example 1 are the formulas of washing solution 1 and washing solution 2, as shown in the table below. The formulas of the other two reagents are the same as those in Example 1.

[0137] Table 5

[0138]

[0139] Comparative Examples 4 to 6

[0140] Comparative Examples 4 to 6 respectively provide a set of nucleic acid separation and purification kits. The difference from Example 1 lies in the formulas of washing solution 1 and washing solution 2, as shown in the table below. The formulas of the other two reagents are the same as in Example 1.

[0141] Table 6

[0142]

[0143] For the same blood sample (H1), the above-mentioned operation steps were followed, and the separation and purification reagents of Examples 5 to 7 and Comparative Examples 4 to 6 were used. The results are shown in the following table and Figure 3 :

[0144] The nucleic acid purity 260 / 280 corresponding to the three examples was greater than 1.8. For the same blood sample, the nucleic acid solutions obtained using different separation and purification reagents had slightly different nucleic acid concentrations, indicating that the reagent components did affect the nucleic acid concentration, but all were within an acceptable range.

[0145] Compared with the examples, the nucleic acid purity corresponding to the three comparative examples was significantly reduced, and the electrophoresis bands were also significantly inferior to the three examples, which indicates that when the reagent components exceeded the preset range, the purification effect was significantly reduced.

[0146] Table 7

[0147] Separation and purification kit ng / μL A260 / A280 Example 5 17.7 1.82 Example 6 14.9 1.86 Example 7 13.5 1.92 Comparative Example 4 2.7 1.10 Comparative Example 5 4.3 1.66 Comparative Example 6 3.3 1.48

[0148] Example 8 to Example 15

[0149] Examples 8 to 15 all provide a nucleic acid separation and purification kit, which differs from Example 1 in the formulation of each reagent, as shown in the table below.

[0150] Table 8

[0151]

[0152]

[0153] Comparative Examples 7 to 17

[0154] Comparative Examples 7 to 17 all provide nucleic acid separation and purification kits, which are comparative examples of Example 1, specifically:

[0155] The only difference between Comparative Example 7 and Example 1 is that the concentration of Tris-HCl in the lysate is 30 mM, and the other reagents are the same as those in Example 1.

[0156] The only difference between Comparative Example 8 and Example 1 is that the concentration of PEG6000 in the lysate is 15 wt %, and the other reagents are the same as those in Example 1.

[0157] The only difference between Comparative Example 9 and Example 1 is that the concentration of PEG6000 in the lysate is 50 wt %, and the other reagents are the same as those in Example 1.

[0158] The only difference between Comparative Example 10 and Example 1 is that the concentration of guanidine isothiocyanate in the lysate is 500 mM, and the other reagents are the same as those in Example 1.

[0159] The only difference between Comparative Example 11 and Example 1 is that the concentration of guanidine isothiocyanate in the lysate is 5 M, and the other reagents are the same as those in Example 1.

[0160] The only difference between Comparative Example 12 and Example 1 is that the concentration of EDTA in the lysate is 4 mmol / L, and the other reagents are the same as those in Example 1.

[0161] The only difference between Comparative Example 13 and Example 1 is that the concentration of SDS in the lysate is 1.5 wt %, and the other reagents are the same as those in Example 1.

[0162] The only difference between Comparative Example 14 and Example 1 is that the concentration of guanidine isothiocyanate in washing solution 1 is 2 M, and the other reagents are the same as those in Example 1.

[0163] The only difference between Comparative Example 15 and Example 1 is that the concentration of PEG6000 in the washing solution 1 is 50 wt %, and the other reagents are the same as those in Example 1.

[0164] The only difference between Comparative Example 16 and Example 1 is that the concentration of Tris-HCl in the washing solution 1 is 60 mM, and the other reagents are the same as those in Example 1.

[0165] The only difference between Comparative Example 17 and Example 1 is that the concentration of PEG6000 in the washing solution 2 is 40 wt %, and the other reagents are the same as those in Example 1.

[0166] The only difference between Comparative Example 18 and Example 1 is that Tris-HCl in washing solution 2 is replaced by water, and the other reagents are the same as those in Example 1.

[0167] Table 9

[0168]

[0169]

[0170] The kit of Example 1 of the present application was compared with a commercial kit (the commercial kit also contains four reagents: lysate, cleaning solution 1, cleaning solution 2 and eluent, wherein isopropanol is added to cleaning solution 1 and ethanol is added to cleaning solution 2). The operating procedures of the kit of Example 1 are as described above. After being placed in a fully enclosed microfluidic chip, the nucleic acids of the four blood samples were purified at the same time, and the obtained nucleic acid products were subjected to QPCR experiments. The samples used were all pig blood, and the target region of the primer probe used for QPCR was located on the pig housekeeping gene GAPDH. The specific primer probe sequences are as follows:

[0171] Table 10

[0172] pig-GAPDH-F (SEQ ID NO. 1) TTGATGCCCTCCCTCGTCC pig-GAPDH-R (SEQ ID NO. 2) CCGTTCTCCGCCTTGACT pig-GAPDH-CY5 (SEQ ID NO. 3) 5`CY5-CCACCCACGGCAAGTTCCAC-3`BHQ

[0173] The QPCR reaction system is as follows:

[0174] Table 11

[0175] name Single serving volume (μL) DEPC water 28.9 Air-Dryable qPCR Mix 12.5 pig-GAPDH-F (50 μM) 0.2 pig-GAPDH-R (50 μM) 0.2 pig-GAPDH-CY5 (50 μM) 0.2 DNA Elution Buffer 8 Total volume of the system (μL) 50

[0176] The experimental results are shown in the following table and Figure 4 :

[0177] Table 12

[0178]

[0179] Using the same sample and the same fully automated microfluidic experimental process, the final QPCR result corresponding to Example 1 was significantly better than that of the commercial kit.

[0180] Conclusion: Conventional commercial magnetic bead-based nucleic acid isolation kits cannot be directly adapted to our fully enclosed microfluidic chip. However, the purification reagents proposed in the examples of this application can realize the nucleic acid separation and purification process in a fully enclosed and fully automatic microfluidic chip, ensuring the accuracy and reliability of subsequent QPCR experiments.

[0181] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the description in this specification.

[0182] The above-described embodiments only express several implementation methods of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that, for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of the present application shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A nucleic acid separation and purification kit, characterized in that: The separation and purification reagents in the nucleic acid separation and purification kit include lysis solution, washing solution 1 and washing solution 2; in, The lysate comprises 1M-4M guanidine thiocyanate, 40mM-100mM tris(hydroxymethyl)aminomethane hydrochloride, 1wt%-5wt% NP-40, 20wt%-40wt% PEG6000, 1wt%-4wt% Triton X-100, 2mmol / L-4mmol / L EDTA, 1wt%-2wt% catechin, 2wt%-5wt% SDS and 1M-2M NaCl, with a pH value of 3.5-4.5; The washing solution 1 comprises 0.5M-1M guanidine thiocyanate, 10mM-50mM tris(hydroxymethyl)aminomethane hydrochloride, 20wt%-40wt% PEG6000 and 200mM-700mM NaCl, with a pH value of 4.8-5.5; The washing solution 2 comprises 10 mM-50 mM tris(hydroxymethyl)aminomethane hydrochloride, 0.1 wt%-1 wt% DTT and 5 wt%-30 wt% PEG6000, with a pH value of 4.8-5.5; The separation and purification reagent does not include ethanol and isopropanol; optionally, the separation and purification reagent does not include ethanol, isopropanol and histone digestion enzyme.

2. The nucleic acid separation and purification kit according to claim 1, wherein The lysate comprises 1.5M-2.5M guanidine thiocyanate, 45mM-55mM tris(hydroxymethyl)aminomethane hydrochloride, 1wt%-2wt% NP-40, 35wt%-40wt% PEG6000, 1.5wt%-2.5% Triton X-100, 2.5mmol / L-3.5mmol / L EDTA, 1.3wt%-1.8wt% catechin, 2wt%-2.5wt% SDS and 1M-2M NaCl, with a pH value of 3.5-4.

5.

3. The nucleic acid separation and purification kit according to claim 1, wherein The washing solution 1 comprises 0.5M-0.7M guanidine isothiocyanate, 25mM-35mM tris(hydroxymethyl)aminomethane hydrochloride, 25wt%-35wt% PEG6000 and 450mM-550mM NaCl, with a pH value of 4.8-5.

5.

4. The nucleic acid separation and purification kit according to claim 1, wherein The washing solution 2 includes 35mM-45mM tris(hydroxymethyl)aminomethane hydrochloride, 0.3wt%-0.5wt% DTT and 5, 8wt%-12wt% PEG6000, with a pH value of 4.8-5.

5.

5. The nucleic acid separation and purification kit according to any one of claims 1 to 4, characterized in that The separation and purification reagent also includes magnetic microspheres; Optionally, the composition of the magnetic microspheres includes Fe3O4, or Fe3O4 and SiO2; Optionally, the surface modification groups of the magnetic microspheres are hydroxyl groups or carboxyl groups; Optionally, the particle size of the magnetic microspheres ranges from 100 nm to 800 nm.

6. The nucleic acid separation and purification kit according to claim 5, characterized in that The magnetic microspheres are suspended in a dispersion medium; Optionally, the dispersion medium includes water; Optionally, the content of the magnetic microspheres is 2 wt%-3 wt% based on the mass proportion in the suspension system composed of the magnetic microspheres and the dispersion medium.

7. The nucleic acid separation and purification kit according to any one of claims 1 to 4 and 6, characterized in that The separation and purification reagent also includes an eluent; Optionally, the eluent comprises 10 mM-40 mM tris(hydroxymethyl)aminomethane hydrochloride and 2 mmol / L-4 mmol / L EDTA, with a pH value of 7.8-8.

2.

8. A method for preparing a nucleic acid sample, characterized in that: The preparation method comprises the following steps: Using the nucleic acid separation and purification kit according to any one of claims 1 to 7 to separate and purify nucleic acid from a sample to be tested to prepare a nucleic acid sample; Optionally, the separation and purification of nucleic acids is performed in a fully enclosed microfluidic chip.

9. The method for preparing a nucleic acid sample according to claim 8, wherein: The sample to be tested includes a blood sample.

10. A method for detecting a target nucleic acid, characterized in that: The detection method comprises the following steps: preparing a nucleic acid sample using the preparation method according to claim 8 or 9; and Detecting the target nucleic acid in the nucleic acid sample.

Images