CN114480370A - Nucleic acid extraction or purification reagents and methods - Google Patents
Nucleic acid extraction or purification reagents and methods Download PDFInfo
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- CN114480370A CN114480370A CN202111663589.XA CN202111663589A CN114480370A CN 114480370 A CN114480370 A CN 114480370A CN 202111663589 A CN202111663589 A CN 202111663589A CN 114480370 A CN114480370 A CN 114480370A
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- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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Abstract
The present invention relates to nucleic acid extraction or purification reagents and methods. In particular, the invention relates to a lysis binding solution comprising Tris-HCl, guanidine hydrochloride, sodium perchlorate, ammonium chloride, EDTA-Na2Brij30, a polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer and water. The invention also relates to a kit comprising reagents for preparing the lysis binding solution and a method for extracting or purifying nucleic acid using the kit. The invention has the advantages of simple and quick operation, and can especially extract the total nucleic acid of various biological samples.
Description
Technical Field
The invention relates to the field of nucleic acid extraction reagents, in particular to a total nucleic acid extraction reagent of multiple biological sample types.
Background
Nucleic acid is one of the most basic substances of various lives, not only plays a role in storing and transmitting genetic information, but also plays a decisive role in a series of important life phenomena such as growth, heredity, mutation and the like. Since the first separation of nucleic acids from leukocytes by the Swiss physicist Friedrich Miescher in 1869, researchers in various countries around the world have conducted intensive research and exploration on methods for nucleic acid extraction and purification. The traditional methods for extracting nucleic acid include phenol extraction, alkaline lysis, CTAB extraction, boiling and the like. Since the first use of glass fiber for the recovery of DNA fragments from agarose gels in 1979, novel methods for nucleic acid extraction based on solid phase carriers were developed, including adsorption membrane spin column extraction, glass bead adsorption and magnetic bead extraction.
The magnetic bead extraction method is based on the principle similar to that of adsorption membrane centrifugal column extraction, and the magnetic bead surface is modified with specific functional groups capable of adsorbing nucleic acid, so that the purposes of cracking, combining, washing and eluting can be achieved through different solution environments. Meanwhile, the magnetic properties of the magnetic beads are utilized, and directional movement and enrichment can be conveniently realized under the action of an external magnetic field, so that the aim of separating nucleic acid from impurities is fulfilled, the separation and purification of target substances are realized, and the purified nucleic acid is obtained. The technology can separate and obtain high-purity nucleic acid from samples such as animal and plant tissues, body fluids, cultured cells, environment and the like, and the magnetic bead extraction method has many advantages in the aspect of clinical molecular diagnosis: the automation and high-flux operation is easy to realize; the operation is simple, the time consumption is short, and repeated centrifugation and other operations are not needed; toxic reagents such as benzene, chloroform and the like in the traditional method are not used, so that the method is safe and non-toxic; the specific combination of the magnetic beads and the nucleic acid enables the magnetic beads to have very high nucleic acid extraction efficiency, and can meet the project requirement of clinical molecular diagnosis on very high detection sensitivity.
Along with the continuous development of the magnetic bead extraction method, the extraction of nucleic acid based on the magnetic bead method technology becomes diversified, but different magnetic bead modification modes, different nucleic acid adsorption environments and the like have great influence on the efficiency and stability of the magnetic bead method for extracting nucleic acid, which is one of the reasons that the quality of the magnetic bead method nucleic acid extraction products on the market is good and bad although the quantity of the magnetic bead method nucleic acid extraction products is very large. At present, a magnetic bead method nucleic acid extraction or purification reagent which is good for total nucleic acid extraction (genome DNA, virus DNA and virus RNA co-extraction) of various biological sample types is lacked in the market, in addition, most of the nucleic acid extraction or purification reagents in the existing market still adopt four steps of cracking-combining-cleaning-eluting, and after cracking is finished, a binding solution needs to be additionally added to combine magnetic beads and nucleic acid, so that automatic high flux is not easy to realize.
Chinese patent CN112226432 discloses a rapid nucleic acid extraction kit by a magnetic bead method, which is rapid, simple and convenient to operate, can complete an extraction process only within 5 minutes by manual operation, only needs to transfer magnetic beads without adding binding solution after sample lysis, meets the requirement of instrument automation and high flux, but can only be applied to viral nucleic acid in a nasopharyngeal swab sample, and limits further application.
Chinese patent CN106244583 discloses a magnetic bead method for extracting nucleic acid, which can be used for nucleic acid of biological samples such as whole blood, cell cultures, bacterial cultures, swabs, tissues, saliva and the like, but the invention is only suitable for extracting genomic DNA of various sample types, and can not be used for extracting virus nucleic acid, and after the sample is cracked, a binding solution needs to be additionally added for binding nucleic acid and magnetic beads, so that the method is not easy to automate high-throughput operation.
In summary, although the prior art cannot provide a nucleic acid extraction reagent for total nucleic acid extraction by magnetic bead method, which can process multiple types of biological samples, it is not necessary to add a binding solution to combine nucleic acid and magnetic bead after the completion of sample lysis, and the reagent is suitable for an automated high-throughput instrument.
Disclosure of Invention
The invention aims to provide a nucleic acid extraction or purification reagent, which is simple and quick to operate and can be used for extracting total nucleic acid (genomic DNA, virus DNA and virus RNA) of various biological samples.
In order to achieve the above object of the present invention, the present invention provides in a first aspect a lysis conjugate comprising: Tris-HCl, guanidine hydrochloride, sodium perchlorate, ammonium chloride, EDTA-Na2Brij30, a polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer and water.
In some preferred embodiments, the lysis conjugate comprises: 20-600mM Tris-HCl, 1-5M guanidine hydrochloride, 1-10M sodium perchlorate, 100mM ammonium chloride,1-50mM EDTA-Na20.1-20% Brij30, 0.1-20% polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer and the balance of water, and the pH of the cleavage binding solution is 5.0-8.5.
In other preferred embodiments, the lysis binding liquid comprises: 50-500mM Tris-HCl, 2-4M guanidine hydrochloride, 1-5M sodium perchlorate, 100mM ammonium chloride, 5-50mM EDTA-Na20.5-10% Brij30, 0.1-10% polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer and the balance water, and the pH of the cleavage binding solution is 6.0-8.0.
In other preferred embodiments, the lysis binding liquid comprises: 100-200mM Tris-HCl, 2-3M guanidine hydrochloride, 2-5M sodium perchlorate, 100-200mM ammonium chloride, 10-40mM EDTA-Na21-5% Brij30, 0.2-2% polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer and balance water, and the pH of the cleavage binding solution is 6.5;
in other preferred embodiments, the lysis binding solution comprises 100mM Tris-HCl, 3M guanidine hydrochloride, 3M sodium perchlorate, 100mM ammonium chloride, 10mM EDTA-Na22% Brij30, 0.25% of a 4400 molecular weight polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer and the balance of water, wherein the pH of the cracking binding solution is 6.5.
In the cracking binding solution, Tris-HCl can provide a buffer environment with stable pH, maintain the charge state of a phosphate group of nucleic acid and protect DNA base.
Guanidine hydrochloride is a chaotropic agent for destroying the three-dimensional structure of protein, can quickly destroy cell membranes or viruses, releases nucleic acid, and denatures the protein, so that the nucleic acid can get rid of the winding of the protein, and simultaneously, the guanidine hydrochloride is a strong inhibitor of nuclease, can reduce the activity of the nuclease, and inhibits the activity of the nuclease.
Sodium perchlorate is also a protein denaturant, similar to guanidine hydrochloride, and simultaneously provides a large amount of univalent cations (sodium ions) to establish a salt bridge between the phosphate of nucleic acid and the silicon hydroxyl surface of magnetic beads.
EDTA-Na2Is a divalent ion chelating agentBeing able to chelate Mg2+Or Ca2+Divalent cations, which inhibit the activity of metal-dependent enzymes such as DNase and RNase, can inhibit the degradation of the cleaved free exposed nucleic acids by nucleases, thereby reducing the degradation of nucleic acids.
The polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer exists as a nucleic acid protective agent to prevent the degradation of the nucleic acid protective agent by nuclease, and can be combined with trace nucleic acid when the pH value is lower than 7.0 (such as 6.5), and can be separated from the nucleic acid when the pH value is higher than 7.5 (such as 8.0), so that the downstream nucleic acid detection is not influenced. Surprisingly, the addition of low concentrations (0.1-1%) of polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymers greatly enhances the enrichment of low copy nucleic acids. Thus, in some preferred embodiments, the polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer has a molecular weight of 2000-20000. Preferably, the molecular weight of the polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer is 4000-10000.
Brij30 (CAS: 9002-92-0) is a relatively mild non-ionic surfactant (or detergent) that solubilizes lipids, promotes hydrogen bonding and cationic bridge formation, and is often used as an additive to stabilize proteins, particularly membrane proteins, in their native conformation. The present invention in the examples attempted to replace with other conventional non-ionic surfactants such as triton X-100 or tween 20, impressively Brij30 had better cell lysis and nucleic acid quality and recovery in blood samples than the two surfactants, resulting in clear eluates.
In a second aspect, the present invention provides a nucleic acid extraction or purification kit comprising reagents for formulating a lysis conjugate according to the first aspect of the invention.
In some preferred embodiments, the kit further comprises a reagent for preparing a suspension of magnetic beads, a reagent for preparing a wash solution, and a reagent for preparing an eluent.
In other preferred embodiments, the magnetic bead suspension comprises 5 to 20% by mass volume of superparamagnetic silicon particles having a particle size of 0.1 to 10 μm, the pH of the magnetic bead suspension is 4.0 to 8.0, the dispersion comprises 10 to 50% by volume of PEG300 and the balance water; preferably, the magnetic bead suspension comprises 10-15% by mass volume of superparamagnetic silicon particles with a particle size of 0.5-5 μm, the pH of the magnetic bead suspension is 5.0-6.0, and the dispersion comprises 20% by volume of PEG300 and the balance of water; more preferably, the magnetic bead suspension comprises 15% by volume of superparamagnetic silicon particles having a particle size of 0.5 to 5 μm, the pH of the magnetic bead suspension is 5.0, and the dispersion comprises 20% by volume of PEG300 and the balance water. The water is preferably nuclease-free water. The amount of superparamagnetic silicon particles in the suspension of magnetic beads is in mass volume percent, i.e. the mass of superparamagnetic silicon particles is a percentage of the volume of the suspension of magnetic beads.
In other preferred embodiments, the first universal wash comprises 10-100mM Tris-HCl, 10-200mM lithium chloride, 1-20mM EDTA-Na21-5M guanidine hydrochloride and 50-80% absolute ethyl alcohol; preferably, the first general-purpose washing solution is 10-50mM Tris-HCl, 50-100mM lithium chloride and 2-10mM EDTA-Na22-4M guanidine hydrochloride and 50% absolute ethanol; more preferably, the first general-purpose washing solution contains 50mM Tris-HCl, 100mM lithium chloride and 2mM EDTA-Na23M guanidine hydrochloride and 50% absolute ethanol.
In other preferred embodiments, the second universal cleaning solution comprises: 5-100mM Tris-HCl, 5-100mM sodium chloride and 40-60% PEG 300; preferably, the second general purpose wash comprises 10-20mM Tris-HCl, 10-25mM sodium chloride and 50% PEG 300; more preferably, the second general wash comprises 10mM Tris-HCl, 20mM sodium chloride and 50% PEG 300.
In other preferred embodiments, the eluent comprises 10mM Tris-HCl and a pH of 8.5.
In a fourth aspect, the present invention provides a method of extracting or purifying nucleic acid from a biological sample, said method using a lysis binding solution according to the first aspect of the invention.
Preferably, the method for extracting or purifying nucleic acid from a biological sample provided by the fourth aspect of the present invention is carried out by using the kit of the second aspect of the present invention.
In some preferred embodiments, the method comprises the steps of:
(1) sample preparation: adding a biological sample into a centrifuge tube;
(2) sample lysis: adding the lysis binding solution into the biological sample obtained in the step (1), and heating at the temperature of 50-80 ℃ for 3-20min to obtain sample lysate;
(3) nucleic acid adsorption: adding the magnetic bead suspension into the sample lysis solution obtained in the step (2), uniformly mixing and standing, and removing liquid after the magnetic beads are completely adsorbed to obtain adsorbed magnetic beads;
(4) first cleaning: adding the first general cleaning solution into the adsorbed magnetic beads obtained in the step (3), uniformly mixing, standing until the magnetic beads are completely adsorbed, and discarding the liquid to obtain first cleaning magnetic beads;
(5) and (3) second cleaning: adding the second universal cleaning solution into the first cleaning magnetic beads obtained in the step (4), uniformly mixing, standing until the magnetic beads are completely adsorbed, and discarding the liquid to obtain second cleaning magnetic beads;
(6) sample elution: and (5) adding the eluent into the second washed magnetic beads obtained in the step (5), uniformly mixing, incubating and eluting at 50-80 ℃ for 5-10min, and collecting the nucleic acid solution obtained after elution.
In other preferred embodiments, the volume usage of the biological sample is 200 μ L in case the biological sample is a liquid biological sample selected from the group consisting of blood, plasma, serum and saliva, and 200-400 μ L in case the biological sample is a various swab eluate; in the step (2), adding 1-2 times of the lysis binding solution by volume of the biological sample; in the step (3), 5-30 μ L of the magnetic bead suspension is added; in the step (4), adding 200-; in the step (5), adding 200-; in step (6), 40-100. mu.L of the eluent is added.
In other preferred embodiments, in step (1), in the case where the biological sample is a liquid biological sample selected from the group consisting of blood, plasma, serum, and saliva, the volume usage of the biological sample is 200 μ L; in the case where the biological sample is a variety of swab elutions, the volume usage of the biological sample is 300 μ L; in the step (2), 2 times of the volume of the cracking binding solution is added, and the mixture is heated for 5-10min at 50-80 ℃; in the step (3), 10-20 μ L of the magnetic bead suspension is added; in the step (4), 500-; in the step (5), 500-; in step (6), elution was incubated at 60 ℃ for 5 min.
In some more specific embodiments, the method of extracting or purifying total nucleic acids (co-extraction or purification of genomic DNA, RNA, viral DNA, viral RNA can be achieved) from blood, plasma, serum, saliva and swab eluate samples using the kit of the second aspect of the invention comprises the steps of:
(1) sample preparation: the samples were mixed well, and 200. mu.L of each body fluid (blood, plasma, serum, saliva) or 200. mu.L of each swab eluate sample were taken into a centrifuge tube for detection.
(2) Sample lysis: adding 1-2 times of the volume of the lysis binding solution into the sample detected in the step (1), and heating at 50-80 ℃ for 3-20min to obtain sample lysis solution;
(3) nucleic acid adsorption: adding 5-30 mu L of magnetic bead suspension into the sample lysate in the step (2), uniformly mixing for 1min, placing on a magnetic frame, standing, and discarding liquid after the magnetic beads are completely adsorbed to obtain adsorbed magnetic beads;
(4) first cleaning: adding 200-;
(5) and (3) second cleaning: adding 200-;
(6) sample elution: and (3) adding 40-100 mu L of eluent into the washed magnetic beads obtained in the step (5), shaking, uniformly mixing, heating at 50-80 ℃ for 5-10min, placing on a magnetic frame, after the magnetic beads are adsorbed, collecting the nucleic acid solution obtained after elution, and placing at-80 ℃ for storage.
Compared with the prior art, the invention has the following beneficial technical effects:
(1) provides a reagent for extracting or purifying total nucleic acid of a plurality of types of samples, and does not need to select different nucleic acid extracting or purifying kits for different types of samples.
(2) Can be matched with a self-made nucleic acid extractor to realize rapid high-flux nucleic acid extraction or purification.
(3) Genomic DNA, viral DNA and viral RNA in a sample can be analyzed and detected simultaneously.
(4) According to the scheme provided by the invention, the second cleaning solution does not contain volatile ethanol or isopropanol, so that the volatilization and drying control of alcohol is not required, the flow time is saved, and the pollution of aerosol is reduced.
(5) The scheme of the invention does not contain proteinase K, so the operation is more convenient and the cost is lower.
Drawings
FIG. 1 is a graph showing the amplification curve of OFR1ab fluorescent quantitative PCR of Covid-19 RNA extraction products from 20 replicate swab samples in example 4 of the present invention.
Detailed Description
The present application is further described below with reference to examples.
In the following description, different "one embodiment" or "an embodiment" may not necessarily refer to the same embodiment, in order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art. Various embodiments may be replaced or combined, and other embodiments may be obtained according to the embodiments without creative efforts for those skilled in the art.
Definition of
Before the present teachings are described in detail, it is to be understood that this disclosure is not limited to particular compositions or process steps, as these may vary. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
When ranges of values are provided herein, the ranges are intended to include the starting and ending values, as well as the values or ranges of values therebetween, unless expressly stated otherwise. For example, "from 0.2 to 0.5" means 0.2, 0.3, 0.4, 0.5; ranges therebetween such as 0.2-0.3, 0.3-0.4, 0.2-0.4; increments therein such as 0.25, 0.35, 0.220, 0.325, 0.49; and increments therein ranging from 0.26-0.39 and the like.
The term "sample" or "specimen" as used herein will be understood to mean any such sample: the term "sample" or "specimen" may include a sample that may contain a nucleic acid substance of interest, or may include blood, plasma, serum, saliva, cerebrospinal fluid, swab eluate, or a combination of one or more thereof.
It is understood that there is an implied "about" before the temperatures, masses, weights, volume ratios, concentrations, times, etc. discussed in this disclosure such that slight and insubstantial deviations are within the scope of the teachings herein. Generally, the term "about" refers to insubstantial changes in the amounts of the components of the composition, which do not have any significant effect on the effectiveness or stability of the composition. Also, the use of "including," "containing," and "including" is not intended to be limiting. It is to be understood that both the foregoing general description and the detailed description are exemplary and explanatory only and are not restrictive of the present teachings. To the extent that any material incorporated by reference does not conform to the teachings of the present disclosure, that description controls.
Unless specifically stated otherwise, embodiments in the specification that are described as "comprising" various components are also considered to "consist of" or "consist essentially of" the components; embodiments in the specification that are described as "consisting of" various components are also considered to be "comprising" or "consisting essentially of" the components.
"nucleic acid" refers to a polymeric compound comprising two or more covalently bonded nucleosides or nucleoside analogs having a nitrogen-containing heterocyclic base or base analog, wherein the nucleosides are linked together by phosphodiester or other linkages to form a polynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNA polymers or oligonucleotides and analogs thereof. The nucleic acid "backbone" may be composed of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid linkages. Nucleic acids may include modified bases to alter the function or behavior of the nucleic acid, such as the addition of 3' -terminal dideoxynucleotides to prevent additional nucleotides from being added to the nucleic acid. Synthetic methods for preparing nucleic acids in vitro are well known in the art, although nucleic acids can be purified from natural sources using conventional techniques.
In this application, "extraction," "isolation," or "purification" refers to the removal of one or more components of a sample or the separation from other sample components. The sample components comprise target nucleic acids, often in a generally aqueous solution phase, which may also comprise cell fragments, proteins, carbohydrates, lipids, salt ions, metal ions, and other nucleic acids. "extraction", "isolation" or "purification" does not imply any degree of purification. Typically, at least 70% or at least 80% or at least 90% of the target nucleic acid from the other sample components is isolated or purified.
Examples
For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the materials are commercially available, unless otherwise specified.
Preparation example 1 nucleic acid extraction or purification kit:
the preparation example provides a nucleic acid extraction or purification kit, which comprises a reagent for preparing a lysis binding solution, a reagent for preparing a magnetic bead suspension, a reagent for preparing a first general-purpose cleaning solution, a reagent for preparing a second general-purpose cleaning solution and a reagent for preparing an eluent.
1) The lysis binding solution consists of: 100mM Tris-HCl, 3M guanidinium hydrochloride, 3M sodium perchlorate, 100mM ammonium chloride, 10mM EDTA-Na22% of Brij30, 0.25% of a polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer (molecular weight 4400), and the balance of water, and the pH of the cleavage-bound solution was 6.5.
2) The magnetic bead suspension comprises 15 mass percent of superparamagnetic silicon particles with the particle size of 0.5-5 mu m, the pH value of the magnetic bead suspension is 5.0, and the dispersion liquid is 20 mass percent of PEG 300-free nuclease water.
3) The first universal cleaning solution consists of the following components: 50mM Tris-HCl, 100mM lithium chloride, 2mM EDTA-Na23M guanidine hydrochloride, 50% absolute ethanol.
4) The second general cleaning solution consists of the following components: 10mM Tris-HCl, 20mM sodium chloride, 50% PEG 300.
5) The eluent comprises the following components: an aqueous solution (pH8.5) containing 10mM Tris-HCl.
Comparative preparation example 1:
the cleavage conjugate solution contained no polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer, and the remaining components were the same as those in preparation example 1.
Comparative preparation example 2:
the Brij30 in the cleavage conjugate solution was replaced with an equal volume of Tween 20, and the remaining components were identical to those in preparation example 1.
Comparative preparation example 3:
the Brij30 in the cleavage conjugate solution was replaced with an equal volume of Triton X-100, and the remaining components were identical to those of preparation example 1.
Example 1: blood genomic DNA extraction
In this example, the nucleic acid extraction or purification kits of preparation example 1, comparative preparation example 2 and comparative preparation example 3 were used to extract genomic DNA from different blood samples, and the specific steps were as follows:
(1) sample preparation: mixing the samples evenly, and adding 200 mu L of blood sample into a 2mL centrifuge tube;
(2) sample lysis: adding 2 times of the volume of the lysis binding solution into the sample obtained in the step (1), and heating at 60 ℃ for 10min to obtain a sample lysis solution;
(3) nucleic acid adsorption: adding 15 mu L of magnetic bead suspension into the sample lysate in the step (2), uniformly mixing for 1min, placing on a magnetic frame, standing, and discarding liquid after the magnetic beads are completely adsorbed to obtain adsorbed magnetic beads;
(4) first cleaning: adding 500 mu L of first general cleaning liquid into the adsorbed magnetic beads obtained in the step (3), vibrating and uniformly mixing for 1min, placing on a magnetic frame, standing until the magnetic beads are completely adsorbed, and discarding all liquid to obtain first cleaning magnetic beads;
(5) and (3) second cleaning: adding 500 mu L of second universal cleaning liquid into the first cleaning magnetic beads obtained in the step (4), vibrating and uniformly mixing for 1min, placing on a magnetic frame, standing until the magnetic beads are completely adsorbed, and discarding all liquid to obtain second cleaning magnetic beads;
(6) sample elution: and (3) adding 100 mu L of eluent into the second cleaned magnetic beads obtained in the step (5), shaking, uniformly mixing, heating at 60 ℃ for 5min, placing on a magnetic frame, after the magnetic beads are adsorbed, collecting the nucleic acid solution obtained after elution, and placing at-80 ℃ for storage.
The 6 human blood samples extracted as above were compared and then the nucleic acid concentration and purity were measured, and the results of nucleic acid extraction of preparation example 1 and the comparative preparation example were compared, and the results of the measurements are shown in table 1 below. As can be seen from the results of table 1, the extraction or purification effect of the nucleic acid extraction or purification kit of the present invention is significantly superior to that of the comparative preparation example. Furthermore, the present inventors observed that the DNA eluates extracted in comparative preparation example 2 and comparative preparation example 3 were not clear in color, showing pale yellow or pale red, while the DNA eluates of preparation example 1 were clear and colorless.
TABLE 1 genomic DNA extraction test results of blood samples of example 1
Example 2: genomic DNA extraction of saliva samples and swab eluate samples
The extraction of genomic DNA from different saliva samples and sample eluate samples was carried out using the nucleic acid extraction or purification kit and the comparison kit (QIAamp DNA mini kit, Qiagen, Cat #51304) of preparation example 1, in substantially the same procedure as in example 1, except that in the sample preparation of step (1), the subject saliva sample was taken at 200. mu.L, and in the elution step in step 6, the eluate volume was 50. mu.L. In addition, the swab eluate was freshly collected, and after oral cells were scraped off by a flocked swab, the eluate was resuspended in 600. mu.L of physiological saline, and 200. mu.L of both the kit of preparation example 1 of the present invention and the control kit was used for nucleic acid extraction. In the elution step in step 6, the volume of the eluate was 50. mu.L.
The results of the nucleic acid concentration and purity test were performed by comparing the 6 human saliva samples extracted as described above with the 6 swab eluates, and the results of the nucleic acid extraction test performed on the comparative kit were compared, and are shown in table 2 below. As can be seen from Table 2, the extraction effect of the nucleic acid extraction or purification kit of the present invention is significantly superior to that of the comparative imported kit.
TABLE 2 example 2 detection results of genomic DNA extraction of saliva samples and swab samples
Example 3: co-extraction of viral DNA and RNA from multiple sample types
In this example, the nucleic acid extraction or purification kit of preparation example 1 and the reagent kit of comparative preparation example 1 were used to perform co-extraction of viral DNA and RNA of multiple sample types, the DNA virus was selected from porcine epidemic diarrhea virus (PEDV quality control), and the RNA virus was selected from Covid-19 pseudovirus quality control. DNA virus or RNA virus with the same titer is respectively added into eluent of blood, plasma, saliva and swab, and virus with the concentration of about 5000 copies is added into every 200 mu L, and the specific steps are as follows:
(1) sample preparation: mixing samples of different types, respectively taking 200 mu L of blood, plasma, saliva and swab eluent, and adding the eluent into a 2mL centrifuge tube (added with a virus quality control substance);
(2) sample lysis: adding 2 times of the volume of the lysis binding solution into the sample obtained in the step (1), and heating at 60 ℃ for 10min to obtain a sample lysis solution;
(3) nucleic acid adsorption: adding 10 mu L of magnetic bead suspension into the sample lysate in the step (2), uniformly mixing for 1min, placing on a magnetic frame, standing, and discarding liquid after the magnetic beads are completely adsorbed to obtain adsorbed magnetic beads;
(4) first cleaning: adding 500 mu L of first general cleaning liquid into the magnetic beads obtained in the step (3), shaking and uniformly mixing for 1min, placing on a magnetic frame, standing until the magnetic beads are completely adsorbed, and discarding all liquid to obtain first cleaning magnetic beads;
(5) and (3) second cleaning: adding 500 mu L of second universal cleaning liquid into the first cleaning magnetic beads obtained in the step (4), vibrating and uniformly mixing for 1min, placing on a magnetic frame, standing until the magnetic beads are completely adsorbed, and discarding all liquid to obtain second cleaning magnetic beads;
(6) sample elution: and (5) adding 60 mu L of eluent into the second cleaning magnetic beads obtained in the step (5), shaking, uniformly mixing, heating at 60 ℃ for 5min, placing on a magnetic frame, after the magnetic beads are adsorbed, collecting the nucleic acid solution obtained after elution, and placing at-80 ℃ for storage.
The nucleic acid concentration was detected by performing fluorescence quantitative PCR on viral DNA and RNA in the extracted 4 blood, 4 plasma, 4 saliva samples and 4 swab eluates, and the detection results were shown in Table 3 below by comparing Ct values with the nucleic acid extraction results of the contrast reagent using the PEDV fluorescence quantitative PCR kit Covid-19 fluorescence quantitative PCR kit (OFR1ab gene) from Shenzhen Biotechnology, Inc. and Guangzhou Danan Gen Ltd. As can be seen from Table 3, the extraction effect of the nucleic acid extraction or purification kit of the present invention is significantly superior to that of comparative preparation example 1, which can be attributed to the addition of a polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer, which improves the enrichment capacity for trace viral nucleic acids.
TABLE 3 example 3 viral DNA and RNA extraction test results for multiple sample types
Example 4: repeatability of viral RNA extraction from swab samples
In this example, a Covid-19 pseudovirus quality control was added to freshly prepared 20 swab specimen resuspensions (physiological saline) using the nucleic acid extraction or purification kit of preparation example 1. Adding virus with the concentration of about 5000 copies into every 200 mu L, and specifically extracting the virus by the following steps:
(1) sample preparation: mixing the sample swab resuspension solution sample, and adding 300 mu L of swab eluent into 2mL centrifuge tubes (to which the virus quality control substances are added) respectively;
(2) sample lysis: adding 2 times of the volume of the lysis binding solution into the sample obtained in the step (1), and heating at 60 ℃ for 5min to obtain a sample lysate;
(3) nucleic acid adsorption: adding 10 mu L of magnetic bead suspension into the sample lysate in the step (2), uniformly mixing for 1min, placing on a magnetic frame, standing, and discarding liquid after the magnetic beads are completely adsorbed to obtain adsorbed magnetic beads;
(4) first cleaning: adding 500 mu L of first general cleaning liquid into the adsorbed magnetic beads obtained in the step (3), vibrating and uniformly mixing for 1min, placing on a magnetic frame, standing until the magnetic beads are completely adsorbed, and discarding all liquid to obtain first cleaning magnetic beads;
(5) and (3) second cleaning: adding 500 mu L of second universal cleaning liquid into the first cleaning magnetic beads obtained in the step (4), vibrating and uniformly mixing for 1min, placing on a magnetic frame, standing until the magnetic beads are completely adsorbed, and discarding all liquid to obtain second cleaning magnetic beads;
(6) sample elution: and (3) adding 60 mu L of eluent into the second cleaning magnetic beads obtained in the step (5), shaking, uniformly mixing, heating at 60 ℃ for 5min, placing on a magnetic frame, after the magnetic beads are adsorbed, collecting the nucleic acid solution obtained after elution, and placing at-80 ℃ for storage.
The nucleic acid concentration of the viral RNA in the 20 swab eluates extracted above was detected by fluorescence quantitative PCR, and the Ct value and the curve were compared, and the results are shown in FIG. 1. As can be seen from FIG. 1, the results of 20 times of repeated verification are very good, and the CV value of the Ct value is 1.8%, which shows that the method has very good stability and sensitivity for extracting the new coronavirus RNA.
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
It should be noted that the above embodiments can be freely combined as necessary. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A lysis binding fluid, wherein said lysis binding fluid comprises: Tris-HCl, guanidine hydrochloride, sodium perchlorate, ammonium chloride, EDTA-Na2Brij30, a polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer and water.
2. The lysis conjugate of claim 1, wherein:
the cleavage binding liquid comprises: 20-600mM Tris-HCl, 1-5M guanidine hydrochloride, 1-10M sodium perchlorate, 100mM ammonium chloride, 1-50mM EDTA-Na2、0.1-20％Brij30, 0.1-20% of polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer and the balance of water, and the pH of the cracking binding solution is 5.0-8.5;
preferably, the cleavage binding liquid comprises: 50-500mM Tris-HCl, 2-4M guanidine hydrochloride, 1-5M sodium perchlorate, 100mM ammonium chloride, 5-50mM EDTA-Na20.5-10% Brij30, 0.1-10% polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer and balance water, and the pH of the split binding solution is 6.0-8.0;
more preferably, the lysis binding solution comprises: 100-200mM Tris-HCl, 2-3M guanidine hydrochloride, 2-5M sodium perchlorate, 100-200mM ammonium chloride, 10-40mM EDTA-Na21-5% Brij30, 0.2-2% polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer and balance water, and the pH of the cleavage binding solution is 6.5;
it is further preferred that the lysis binding solution comprises 100mM Tris-HCl, 3M guanidine hydrochloride, 3M sodium perchlorate, 100mM ammonium chloride, 10mM EDTA-Na22% Brij30, 0.25% of a 4400 molecular weight polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer and the balance of water, wherein the pH of the cracking binding solution is 6.5.
3. Lysis binding fluid according to claim 1 or 2, characterized in that:
the molecular weight of the polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer is 2000-20000;
preferably, the molecular weight of the polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer is 4000-10000.
4. A nucleic acid extraction or purification kit comprising reagents for formulating the lysis binding solution of any one of claims 1 to 3.
5. The kit of claim 4, further comprising reagents for preparing a suspension of magnetic beads, reagents for preparing a wash solution, and reagents for preparing an eluent.
6. The kit according to claim 4 or 5, characterized in that:
the magnetic bead suspension comprises 5-20% by mass volume of superparamagnetic silicon particles with the particle size of 0.1-10 mu m, the pH of the magnetic bead suspension is 4.0-8.0, and the dispersion comprises 10-50% by volume of PEG300 and the balance of water; preferably, the magnetic bead suspension comprises 10-15% by mass volume of superparamagnetic silicon particles with a particle size of 0.5-5 μm, the pH of the magnetic bead suspension is 5.0-6.0, and the dispersion comprises 20% by volume of PEG300 and the balance of water; more preferably, the magnetic bead suspension comprises 15% by volume of superparamagnetic silicon particles with a particle size of 0.5-5 μm, and a dispersion with a pH of 5.0, wherein the dispersion comprises 20% by volume of PEG300 and the balance of water;
the first universal cleaning solution comprises 10-100mM Tris-HCl, 10-200mM lithium chloride and 1-20mM EDTA-Na21-5M guanidine hydrochloride and 50-80% absolute ethyl alcohol; preferably, the first general-purpose washing solution is 10-50mM Tris-HCl, 50-100mM lithium chloride and 2-10mM EDTA-Na22-4M guanidine hydrochloride and 50% absolute ethanol; more preferably, the first general-purpose washing solution contains 50mM Tris-HCl, 100mM lithium chloride and 2mM EDTA-Na23M guanidine hydrochloride and 50% absolute ethanol;
the second general cleaning liquid includes: 5-100mM Tris-HCl, 5-100mM sodium chloride and 40-60% PEG 300; preferably, the second general purpose wash comprises 10-20mM Tris-HCl, 10-25mM sodium chloride and 50% PEG 300; more preferably, the second general wash comprises 10mM Tris-HCl, 20mM sodium chloride and 50% PEG 300; and/or
The eluent contained 10mM Tris-HCl and had a pH of 8.5.
7. A method for extracting or purifying nucleic acid from a biological sample, comprising:
the method employs the lysis binding solution of any one of claims 1 to 3 for extraction or purification;
preferably, the method is performed by extraction or purification using the kit of any one of claims 4 to 6.
8. The method according to claim 7, characterized in that it comprises the steps of:
(1) sample preparation: adding a biological sample into a centrifuge tube;
(2) sample lysis: adding the lysis binding solution into the biological sample obtained in the step (1), and heating at the temperature of 50-80 ℃ for 3-20min to obtain sample lysate;
(3) nucleic acid adsorption: adding the magnetic bead suspension into the sample lysis solution obtained in the step (2), uniformly mixing and standing, and removing liquid after the magnetic beads are completely adsorbed to obtain adsorbed magnetic beads;
(4) first cleaning: adding the first general cleaning solution into the adsorbed magnetic beads obtained in the step (3), uniformly mixing, standing until the magnetic beads are completely adsorbed, and discarding the liquid to obtain first cleaning magnetic beads;
(5) and (3) second cleaning: adding the second universal cleaning solution into the first cleaning magnetic beads obtained in the step (4), uniformly mixing, standing until the magnetic beads are completely adsorbed, and discarding the liquid to obtain second cleaning magnetic beads;
(6) sample elution: and (5) adding the eluent into the second washed magnetic beads obtained in the step (5), uniformly mixing, incubating and eluting at 50-80 ℃ for 5-10min, and collecting the nucleic acid solution obtained after elution.
9. The method of claim 8, wherein:
in the case of a liquid biological sample selected from the group consisting of blood, plasma, serum and saliva, the volume usage of the biological sample is 200 μ L, in the case of various swab elutions, 200-400 μ L;
in the step (2), adding 1-2 times of the lysis binding solution by volume of the biological sample;
in the step (3), 5-30 μ L of the magnetic bead suspension is added;
in the step (4), adding 200-;
in the step (5), adding 200-;
in step (6), 40-100. mu.L of the eluent is added.
10. The method of claim 7, wherein:
in the step (1), in the case where the biological sample is a liquid biological sample selected from the group consisting of blood, plasma, serum and saliva, the volume usage of the biological sample is 200 μ L; in the case where the biological sample is a variety of swab elutions, the volume usage of the biological sample is 300 μ L;
in the step (2), 2 times of the volume of the cracking binding solution is added, and the mixture is heated for 5-10min at 50-80 ℃;
in the step (3), 10-20 μ L of the magnetic bead suspension is added;
in the step (4), 500-;
in the step (5), 500-;
in step (6), the elution was incubated at 60 ℃ for 5 min.
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| CN101748116A (en) * | 2009-12-22 | 2010-06-23 | 陕西北美基因股份有限公司 | Method for extracting large numbers of DNA with magnetic particulate |
| CN107002147A (en) * | 2014-12-11 | 2017-08-01 | 通用电气公司 | Method for capturing nucleic acid |
| CN111057705A (en) * | 2019-12-31 | 2020-04-24 | 首都医科大学附属北京胸科医院 | Kit for extracting free nucleic acid and use method |
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| CN101748116A (en) * | 2009-12-22 | 2010-06-23 | 陕西北美基因股份有限公司 | Method for extracting large numbers of DNA with magnetic particulate |
| CN107002147A (en) * | 2014-12-11 | 2017-08-01 | 通用电气公司 | Method for capturing nucleic acid |
| CN111057705A (en) * | 2019-12-31 | 2020-04-24 | 首都医科大学附属北京胸科医院 | Kit for extracting free nucleic acid and use method |
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| CN115873664A (en) * | 2022-12-06 | 2023-03-31 | 上海领骏生物科技有限公司 | Cleaning solution for nucleic acid extraction magnetic beads and use method thereof |
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