CN114480370B - Nucleic acid extraction or purification reagents and methods - Google Patents
Nucleic acid extraction or purification reagents and methods Download PDFInfo
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Abstract
The present application relates to nucleic acid extraction or purification assaysAgents and methods. In particular, the application relates to a lysis binding solution which comprises Tris-HCl, guanidine hydrochloride, sodium perchlorate, ammonium chloride and EDTA-Na 2 Brij 30, polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer and water. The application also relates to a kit comprising reagents for preparing said lysis binding fluid and a method for extracting or purifying nucleic acids using said kit. The application has the advantages of simple and quick operation, and particularly can extract the total nucleic acid of various biological samples.
Description
Technical Field
The application 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 life, plays a role in storing and transmitting genetic information, and plays a decisive role in a series of important life phenomena such as growth, heredity, variation and the like. Since the first isolation of nucleic acids from leukocytes by swiss physicist Friedrich Miescher in 1869, researchers in various countries around the world have conducted diligent research and exploration on methods for extracting and purifying nucleic acids. Traditional methods of nucleic acid extraction include phenol extraction, alkaline lysis, CTAB extraction, boiling, and the like. Since 1979 Vogelstein and gillespei used glass fiber for recovering DNA fragments in agarose gel for the first time, novel nucleic acid extraction methods based on solid phase carriers have been developed, including adsorption membrane centrifugation, glass bead adsorption and magnetic bead extraction.
The magnetic bead extraction method is based on a principle similar to that of an adsorption membrane centrifugal column extraction method, and specific functional groups capable of adsorbing nucleic acid are modified on the surface of the magnetic beads, so that the purposes of cracking, combining, washing and eluting are achieved through different solution environments. Meanwhile, by utilizing the magnetic property of the magnetic beads, the directional movement and enrichment can be conveniently realized under the action of an external magnetic field, so that the purpose of separating nucleic acid from impurities is achieved, and further, 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 fluid, cultured cells, environment and the like, and the magnetic bead extraction method has a plurality of advantages in clinical molecular diagnosis: the automation and high-flux operation are easy to realize; the operation is simple, the time is short, and the repeated operations such as centrifugation and the like are not needed; the method does not use toxic reagents such as benzene, chloroform and the like in the traditional method, and is safe and nontoxic; the specific combination of the magnetic beads and the nucleic acid ensures that the magnetic beads have very high nucleic acid extraction efficiency, and can meet the project requirement of clinical molecular diagnosis on very high detection sensitivity requirements.
With the continuous development of magnetic bead extraction, nucleic acid extraction based on the magnetic bead technology is also becoming various, but different magnetic bead modification modes, different nucleic acid adsorption environments and the like have great influence on the efficiency and stability of extracting nucleic acid by the magnetic bead method, which is one of the reasons that the quantity of magnetic bead method nucleic acid extraction products on the market is very large, but the quality of the magnetic bead method nucleic acid extraction products is good and bad. The existing market lacks 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, and in addition, most of the nucleic acid extraction or purification reagents in the existing market still adopt four steps of cleavage-combination-washing-elution, and after the cleavage is finished, the combination of magnetic beads and nucleic acids can be carried out by adding a combination solution, so that the automation high flux is not easy to realize.
Chinese patent CN112226432 discloses a magnetic bead method rapid nucleic acid extraction kit, the operation is rapid and simple, the manual operation can be completed in one time only by 5 minutes, and after the sample is cracked, the binding liquid is not required to be added, and only the magnetic beads are required to be transferred, so that the requirement of instrument automation high flux is realized, but the kit can only be applied to virus nucleic acid in nasopharyngeal swab samples, and further application is limited.
Chinese patent CN106244583 discloses a method for extracting nucleic acid by magnetic bead method, which can be used for nucleic acid of biological samples such as whole blood, cell culture, bacterial culture, swab, tissue, saliva, etc., but it is found from the summary that the method is only suitable for extracting genomic DNA of the above sample types, and cannot be applied to extracting viral nucleic acid, and after sample cleavage, binding between nucleic acid and magnetic bead is performed by adding binding solution, which is not easy for automatic high-flux operation.
In summary, although the prior art cannot provide a magnetic bead method nucleic acid extraction reagent capable of processing total nucleic acid of multiple biological sample types, the nucleic acid and the magnetic beads are combined without adding a binding solution after sample lysis is completed, and the magnetic bead method nucleic acid extraction reagent is suitable for an automatic high-throughput instrument.
Disclosure of Invention
The application aims to provide a nucleic acid extraction or purification reagent, which is simple and quick to operate and can extract total nucleic acid (genome DNA, virus DNA and virus RNA co-extraction) of multiple types of biological samples.
In order to achieve the above object of the present application, the present application provides in a first aspect a lysis-binding fluid comprising: tris-HCl, guanidine hydrochloride, sodium perchlorate, ammonium chloride, EDTA-Na 2 Brij 30, polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer and water.
In some preferred embodiments, the lysis-binding fluid comprises: 20-600mM Tris-HCl, 1-5M guanidine hydrochloride, 1-10M sodium perchlorate, 100-500mM ammonium chloride, 1-50mM EDTA-Na 2 0.1-20% Brij 30, 0.1-20% polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer and the balance water, and the pH of the lysis-binding solution is 5.0-8.5.
In other preferred embodiments, the lysis-binding fluid comprises: 50-500mM Tris-HCl, 2-4M guanidine hydrochloride, 1-5M sodium perchlorate, 100-300mM ammonium chloride, 5-50mM EDTA-Na 2 0.5-10% Brij 30,0.1-10% polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer and the balance water, and the pH of the lysis-binding solution is 6.0-8.0.
In other preferred embodiments, the lysis-binding fluid comprises: 100-200mM Tris-HCl, 2-3M guanidine hydrochloride, 2-5M sodium perchlorate, 100-200mM ammonium chloride, 10-40mMEDTA-Na 2 1-5% Brij 30,0.2-2% polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer and balance water, and the pH of the lysis-binding fluid is 6.5;
in other preferred embodiments, the lysis conjugate comprises 100mM Tris-HCl, 3M guanidine hydrochloride, 3M sodium perchlorate, 100mM ammonium chloride, 10mM EDTA-Na 2 2% Brij 30,0.25% of a polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer having a molecular weight of 4400 and the balance water, wherein the pH of the cleavage conjugate solution is 6.5.
In the lysis binding solution, tris-HCl can provide a buffer environment with stable pH, maintain the charge state of nucleic acid phosphate groups and protect DNA bases.
Guanidine hydrochloride is a chaotropic agent for destroying the three-dimensional structure of proteins, can quickly destroy cell membranes or viruses, release nucleic acids and denature the proteins, so that the nucleic acids can get rid of the entanglement of the proteins, and meanwhile, guanidine hydrochloride is a strong inhibitor of nuclease, so that the nuclease activity can be reduced and the nuclease activity can be inhibited.
Sodium perchlorate is also a protein denaturant, similar to guanidine hydrochloride, and provides a large number of monovalent cations (sodium ions) to establish the phosphate radical of nucleic acid and the salt bridge of the silicon hydroxyl surface of magnetic beads.
EDTA-Na 2 Is a divalent ion chelating agent capable of chelating Mg 2+ Or Ca 2+ And divalent cations, inhibiting the activity of metal-dependent enzymes such as DNase and RNase, can inhibit the degradation of nucleic acid by nucleases on free exposed nucleic acid after cleavage, thereby reducing the degradation of nucleic acid.
The polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer of the application exists as a nucleic acid protecting agent to prevent the degradation by nuclease, and can be combined with trace nucleic acid below pH 7.0 (such as 6.5) and separated from nucleic acid above pH7.5 (such as 8.0) without influencing the detection of downstream nucleic acid. Surprisingly, the addition of low concentrations (0.1-1%) of polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer 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.
Brij 30 (CAS: 9002-92-0) is a relatively mild nonionic surfactant (or detergent) that dissolves lipids and also has the effect of promoting hydrogen bonding and cationic bridge formation, often as an additive to stabilize proteins, particularly membrane proteins in their natural conformation. The present application in the examples attempted to replace with other conventional nonionic surfactants such as triton X-100 or tween 20, impressively Brij 30 had better cell lysis effect and nucleic acid quality and recovery in blood samples than the two surfactants described above, yielding a clear eluate.
In a second aspect, the application provides a nucleic acid extraction or purification kit comprising reagents for formulating the lysis binding fluid of the first aspect of the application.
In some preferred embodiments, the kit further comprises reagents for formulating a suspension of magnetic beads, reagents for formulating a washing solution, and reagents for formulating an eluent.
In other preferred embodiments, the magnetic bead suspension comprises a dispersion comprising 10 to 50% by volume of PEG300 and the balance water, and 5 to 20% by volume of superparamagnetic silica particles having a particle size of 0.1 to 10 μm, the magnetic bead suspension having a pH of 4.0 to 8.0; preferably, the magnetic bead suspension comprises a dispersion liquid and 10-15% by mass and volume of superparamagnetic silicon particles with the particle size of 0.5-5 μm, the pH of the magnetic bead suspension is 5.0-6.0, and the dispersion liquid comprises 20% by volume of PEG300 and the balance of water; more preferably, the magnetic bead suspension comprises a dispersion comprising 20% by volume of PEG300 and the balance water, and a mass volume percentage of superparamagnetic silicon particles having a particle size of 0.5-5 μm, the magnetic bead suspension having a pH of 5.0. The water is preferably nuclease-free water. The amount of superparamagnetic silica particles in the magnetic bead suspension is in mass volume percent, i.e. the mass of the superparamagnetic silica particles is in volume percent of the magnetic bead suspension.
In other preferred embodiments, the first universal washing solution comprises 10-100mM Tris-HCl,10-200mM lithium chloride, 1-20mM EDTA-Na 2 1-5M guanidine hydrochloride and 50-80% absolute ethanol; preferably, the first universal washing liquid is 10-50mM Tris-HCl,50-100mM lithium chloride, 2-10mM EDTA-Na 2 2-4M guanidine hydrochloride and 50% absolute ethanol; more preferably, the first universal washing solution comprises 50mM Tris-HCl,100mM lithium chloride, 2mM EDTA-Na 2 3M guanidine hydrochloride and 50% absolute ethanol.
In other preferred embodiments, the second general purpose cleaning solution comprises: 5-100mM Tris-HCl,5-100mM sodium chloride and 40-60% PEG300; preferably, the second universal washing solution comprises 10-20mM Tris-HCl,10-25mM sodium chloride and 50% PEG300; more preferably, the second universal washing solution comprises 10mM Tris-HCl,20mM sodium chloride and 50% PEG300.
In other preferred embodiments, the eluate comprises 10mM Tris-HCl and has a pH of 8.5.
In a fourth aspect, the present application provides a method of extracting or purifying nucleic acids from a biological sample, the method employing the lysis binding fluid of the first aspect of the application for extraction or purification.
Preferably, the method for extracting or purifying nucleic acid from a biological sample provided in the fourth aspect of the present application uses the kit according to the second aspect of the present application for extraction or purification.
In some preferred embodiments, the method comprises the steps of:
(1) Sample preparation: adding the biological sample into a centrifuge tube;
(2) Sample lysis: adding the lysis binding solution into the biological sample in the step (1), and heating at 50-80 ℃ for 3-20min to obtain sample lysis solution;
(3) Nucleic acid adsorption: adding the magnetic bead suspension into the sample lysate in the step (2), uniformly mixing and standing, and discarding the liquid after the magnetic beads are completely adsorbed to obtain the magnetic beads after adsorption;
(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 general-purpose 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: adding the eluent into the second cleaning 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 eluting.
In other preferred embodiments, the volume of the biological sample is 200 μl in the case of a liquid biological sample selected from the group consisting of blood, plasma, serum and saliva, and 200-400 μl in the case of various swab eluents; in step (2), adding 1-2 times the volume of the biological sample of the lysis buffer; in step (3), 5-30. Mu.L of the magnetic bead suspension is added; in step (4), 200-1000 μl of the first universal cleaning solution is added; in step (5), 200-1000 μl of the second universal cleaning solution is added; in step (6), 40-100. Mu.L of the eluent is added.
In other preferred embodiments, in step (1), in case of a liquid biological sample selected from the group consisting of blood, plasma, serum and saliva, the biological sample is used in a volume amount of 200 μl; in the case that the biological sample is various swab eluents, the volume usage of the biological sample is 300 mu L; in step (2), adding 2 times the volume of the lysis-binding solution, and heating at 50-80 ℃ for 5-10min; in step (3), 10-20. Mu.L of the magnetic bead suspension is added; in step (4), 500-600 μl of the first universal cleaning solution is added; in step (5), 500-600 μl of the second universal cleaning solution is added; in step (6), the elution is incubated at 60℃for 5min.
In some more specific embodiments, the method utilizes the kit of the second aspect of the application to extract or purify total nucleic acids (which may enable co-extraction or purification of genomic DNA, RNA, viral DNA, viral RNA) from blood, plasma, serum, saliva, and swab eluate samples, the method comprising the steps of:
(1) Sample preparation: samples were mixed and 200. Mu.L of various body fluids (blood, plasma, serum, saliva) or 200-400. Mu.L of various swab eluates were sampled in centrifuge tubes for detection.
(2) Sample lysis: adding 1-2 times of the volume of the lysis binding solution into the detection sample in the step (1), and heating for 3-20min at 50-80 ℃ to obtain a 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 the mixture on a magnetic rack, standing, and discarding the liquid after the magnetic beads are completely adsorbed to obtain the adsorbed magnetic beads;
(4) First cleaning: adding 200-1000 mu L of a first general cleaning solution into the magnetic beads obtained in the step (3), shaking and uniformly mixing for 1min, placing the mixture on a magnetic frame, standing until the magnetic beads are completely adsorbed, and discarding all the liquid;
(5) And (3) second cleaning: adding 200-1000 mu L of a second general-purpose cleaning solution into the magnetic beads obtained in the step (4), shaking and uniformly mixing for 1min, placing the mixture on a magnetic frame, standing until the magnetic beads are completely adsorbed, and discarding all the liquid;
(6) Sample elution: 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 rack, collecting the eluted nucleic acid solution after the magnetic beads are adsorbed, and storing at-80 ℃.
Compared with the prior art, the application has the following beneficial technical effects:
(1) A total nucleic acid extraction or purification reagent for multiple sample types is provided without the need to select different nucleic acid extraction or purification kits for different sample types.
(2) Can be matched with an own nucleic acid extractor to realize rapid high-flux nucleic acid extraction or purification.
(3) Genomic DNA, viral DNA and viral RNA in one sample can be simultaneously analyzed and detected.
(4) According to the scheme provided by the application, the second cleaning liquid does not contain volatile ethanol or isopropanol, so that the volatilization and drying of the alcohol are not needed, the time of a process is saved, and the pollution of aerosol is reduced.
(5) The extraction or purification scheme of the application does not contain proteinase K, and has more convenient operation and lower cost.
Drawings
FIG. 1 is a graph of amplification of OFR1ab fluorescence quantitative PCR of Covid-19 RNA extracted products in 20 replicate swab samples of example 4 of the present application.
Detailed Description
The application is further described below with reference to examples.
In order to more clearly describe embodiments of the present application or technical solutions in the prior art, in the following description, different "an embodiment" or "an embodiment" does not necessarily refer to the same embodiment. Various embodiments may be substituted or combined, and other implementations may be obtained from these embodiments by those of ordinary skill in the art without undue burden.
Definition of the definition
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 such 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, unless expressly stated otherwise, the ranges are intended to include the starting and ending values, as well as values or ranges of values therebetween. For example, "from 0.2 to 0.5" means 0.2, 0.3, 0.4, 0.5; the range between them is 0.2-0.3, 0.3-0.4, 0.2-0.4; increments therebetween such as 0.25, 0.35, 0.220, 0.325, 0.49; the increment range therebetween is, for example, 0.26-0.39, etc.
The term "sample" or "specimen" as used herein will be understood to mean any such specimen: the term "sample" or "sample" may include blood, plasma, serum, saliva, cerebrospinal fluid, a swab eluate, or a combination of one or more thereof.
It should be understood that there is a implied "about" prior to 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" means an insubstantial change in the amount of a component of a composition that does not have any significant effect on the effect or stability of the composition. Moreover, the use of "including," "comprising," 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 is inconsistent with the teachings of this disclosure, the teachings will control.
Embodiments in the specification that are described as "comprising" various components are also considered to be "consisting of" or "consisting essentially of" the components unless specifically indicated; embodiments described in the specification 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 nitrogen-containing heterocyclic bases or base analogs, wherein the nucleosides are linked together by phosphodiester bonds or other bonds 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 comprised of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid linkages. The nucleic acid may include modified bases to alter the function or behavior of the nucleic acid, such as adding 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 the present application, "extracting", "separating" or "purifying" refers to the removal of one or more components of a sample or the separation of other sample components. The sample component comprises the target nucleic acid, which is often in a generally aqueous solution phase, which may also comprise cellular fragments, proteins, carbohydrates, lipids, salt ions, metal ions, and other nucleic acids. "extract", "isolate" or "purify" does not mean any degree of purification. Typically, at least 70% or at least 80% or at least 90% of the target nucleic acids are isolated or purified from the other sample components.
Examples
For a further understanding of the present application, preferred embodiments of the application are described below in conjunction with the examples, but it should be understood that these descriptions are merely intended to illustrate further features and advantages of the application, and are not limiting of the claims of the application. The experimental methods described in the following examples are all conventional methods unless otherwise specified; such materials, unless otherwise specified, are commercially available.
Preparative 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 composition of the lysis binding solution is as follows: 100mM Tris-HCl, 3M guanidine hydrochloride, 3M sodium perchlorate, 100mM ammonium chloride, 10mM EDTA-Na 2 2% Brij 30,0.25% polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer (molecular weight=4400), balance water, pH of the cleavage conjugate solution was 6.5.
2) The magnetic bead suspension consists of superparamagnetic silicon particles with the particle size of 0.5-5 mu m, the mass fraction of the superparamagnetic silicon particles is 15%, the pH value of the magnetic bead suspension is 5.0, and the dispersion is water without the nuclease, wherein the mass fraction of the water is 20% of PEG300.
3) The first general cleaning liquid consists of the following components: 50mM Tris-HCl, containing a total of two amino acids,100mM lithium chloride, 2mM EDTA-Na 2 3M guanidine hydrochloride, 50% absolute ethanol.
4) The second general cleaning liquid consists of the following components: 10mM Tris-HCl,20mM sodium chloride, 50% PEG300.
5) The eluent comprises the following components: an aqueous solution (pH 8.5) containing 10mM Tris-HCl.
Comparative preparation 1:
the cleavage and binding solution did not contain a polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer, and the remaining components were the same as those in preparation example 1.
Comparative preparation 2:
brij 30 in the lysate was replaced with an equal volume of tween 20 and the remaining components were identical to those of preparation 1.
Comparative preparation 3:
brij 30 in the lysis conjugate was replaced with an equal volume of triton X-100, and the remaining components were identical to those of preparation 1.
Example 1: blood genomic DNA extraction
In this example, genomic DNA from different blood samples was extracted using the nucleic acid extraction or purification kits of preparation 1 and comparative preparation 2 and comparative preparation 3, as follows:
(1) Sample preparation: mixing the samples uniformly, taking 200 mu L of blood sample, and adding the 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 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 the mixture on a magnetic rack, standing, and discarding the liquid after the magnetic beads are completely adsorbed to obtain the magnetic beads after adsorption;
(4) First cleaning: adding 500 mu L of a first general cleaning solution into the magnetic beads after adsorption obtained in the step (3), shaking and uniformly mixing for 1min, then placing the magnetic beads on a magnetic frame, standing until the magnetic beads are completely adsorbed, and discarding all the liquid to obtain first cleaning magnetic beads;
(5) And (3) second cleaning: adding 500 mu L of a second general-purpose cleaning solution into the first cleaning magnetic beads obtained in the step (4), vibrating and uniformly mixing for 1min, placing the mixture on a magnetic frame, standing until the magnetic beads are completely adsorbed, and discarding all the liquid to obtain second cleaning magnetic beads;
(6) Sample elution: adding 100 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 rack, collecting the nucleic acid solution obtained after elution after the magnetic beads are adsorbed, and storing at-80 ℃.
The nucleic acid concentration and purity were measured by comparing the above-extracted 6 human blood samples, and the results of nucleic acid extraction in preparation example 1 and comparative preparation example were compared, and the measurement results 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 application is significantly superior to that of the comparative preparation. Furthermore, the inventors observed that the DNA eluents extracted in comparative preparation 2 and comparative preparation 3 were not clear in color, and exhibited pale yellow or pale red, whereas the DNA eluate of preparation 1 was clear and colorless.
TABLE 1 genomic DNA extraction test results of example 1 blood samples
Example 2: genomic DNA extraction of saliva samples and swab eluate samples
Experiments genomic DNA was extracted from different saliva samples and sample of test eluate using the nucleic acid extraction or purification kit of preparation example 1 and the comparative kit (QIAamp DNA mini Kits, qiagen, cat # 51304), the specific procedure being essentially the same as in example 1, but in step (1) sample preparation, the subject saliva sample was taken as 200 μl and in step 6 the elution step the eluent volume was 50 μl. In addition, the swab eluate was freshly collected, and after scraping oral cells with a flocked swab, it was resuspended in 600 μl of saline, and 200 μl of each of the kit of preparation example 1 and the control kit of the present application was used for nucleic acid extraction. In the elution step in step 6, the eluent volume was 50. Mu.L.
The nucleic acid concentration and purity were measured by comparing the above-extracted 6 human saliva samples with 6 swab eluates, and compared with the nucleic acid extraction results of the comparative kit, the measurement results 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 application is significantly better than that of the comparative inlet kit.
TABLE 2 genomic DNA extraction test results of saliva samples and swab samples
Example 3: co-extraction of viral DNA and RNA in multiple sample types
This example uses the nucleic acid extraction or purification kit of preparation example 1 and the kit of comparative preparation example 1 to co-extract multiple sample types of viral DNA and RNA, DNA virus selected porcine epidemic diarrhea virus (PEDV quality control), and RNA virus selected Covid-19 pseudovirus quality control. The DNA virus or RNA virus with the same titer is respectively added into blood, plasma, saliva and swab eluents, and about 5000 copies of the virus is added into every 200 mu L, and the specific steps are as follows:
(1) Sample preparation: mixing different types of samples, respectively taking 200 mu L of blood, plasma, saliva and swab eluent, and adding into a 2mL centrifuge tube (added with virus quality control agent);
(2) Sample lysis: adding 2 times of the volume of the lysis binding solution into the sample 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 the mixture on a magnetic rack, standing, and discarding the liquid after the magnetic beads are completely adsorbed to obtain the magnetic beads after adsorption;
(4) First cleaning: adding 500 mu L of a first general cleaning solution into the magnetic beads obtained in the step (3), vibrating and uniformly mixing for 1min, placing the magnetic beads on a magnetic rack, standing until the magnetic beads are completely adsorbed, and discarding all the liquid to obtain first cleaning magnetic beads;
(5) And (3) second cleaning: adding 500 mu L of a second general-purpose cleaning solution into the first cleaning magnetic beads obtained in the step (4), vibrating and uniformly mixing for 1min, placing the mixture on a magnetic frame, standing until the magnetic beads are completely adsorbed, and discarding all the liquid to obtain second cleaning magnetic beads;
(6) Sample elution: 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 rack, collecting the nucleic acid solution obtained after elution after the magnetic beads are adsorbed, and storing at-80 ℃.
The above-extracted 4 parts of blood, 4 parts of plasma, 4 parts of saliva sample and 4 parts of viral DNA and RNA in the swab eluate were subjected to fluorescent quantitative PCR for detecting the concentration of nucleic acid, and the PEDV fluorescent quantitative PCR kit Covid-19 fluorescent quantitative PCR kit (OFR 1ab gene) was obtained from Shenzhen Biotechnology Co., ltd and Guangzhou Dan Gene Co., ltd, respectively, and the results of nucleic acid extraction were compared by Ct value and with the comparative reagent, and the detection results are shown in Table 3 below. As can be seen from table 3, the extraction effect of the nucleic acid extraction or purification kit of the present application is significantly better than that of comparative preparation 1, which can be attributed to the addition of polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer, which improves the enrichment ability for trace viral nucleic acids.
TABLE 3 detection results of viral DNA and RNA extraction of multiple sample types
Example 4: repeatability of viral RNA extraction from swab samples
In this example, covid-19 pseudovirus quality control was added to a freshly prepared 20 swab sample suspension (normal saline) using the nucleic acid extraction or purification kit of preparation example 1. About 5000 copies of virus were added per 200 μl, and the specific extraction steps were as follows:
(1) Sample preparation: mixing the sample swab heavy suspension sample, and adding 300 mu L of swab eluent into a 2mL centrifuge tube (virus quality control agent is added);
(2) Sample lysis: adding 2 times of the volume of the lysis binding solution into the sample in the step (1), and heating at 60 ℃ for 5min 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 the mixture on a magnetic rack, standing, and discarding the liquid after the magnetic beads are completely adsorbed to obtain the magnetic beads after adsorption;
(4) First cleaning: adding 500 mu L of a first general cleaning solution into the magnetic beads after adsorption obtained in the step (3), shaking and uniformly mixing for 1min, then placing the magnetic beads on a magnetic frame, standing until the magnetic beads are completely adsorbed, and discarding all the liquid to obtain first cleaning magnetic beads;
(5) And (3) second cleaning: adding 500 mu L of a second general-purpose cleaning solution into the first cleaning magnetic beads obtained in the step (4), vibrating and uniformly mixing for 1min, placing the mixture on a magnetic frame, standing until the magnetic beads are completely adsorbed, and discarding all the liquid to obtain second cleaning magnetic beads;
(6) Sample elution: 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 rack, collecting the nucleic acid solution obtained after elution after the magnetic beads are adsorbed, and storing at-80 ℃.
The viral RNA in the 20 swab eluates thus extracted was subjected to fluorescent quantitative PCR to detect the concentration of nucleic acid, and the results were shown in FIG. 1 by comparing Ct values with the curves. As can be seen from FIG. 1, the 20 repeated verifications show that the CV value of Ct value is 1.8%, which shows that the application has very good stability and sensitivity for extracting the novel coronavirus RNA.
The above description of the embodiments is only for aiding in the understanding of the method of the present application and its core ideas. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the application can be made without departing from the principles of the application and these modifications and adaptations are intended to be within the scope of the application as defined in the following claims. The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present application. 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 application. Thus, the present application 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 needed. The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (18)
1. A lysis conjugate, the lysis conjugate comprising: 20-600mM Tris-HCl, 1-5M guanidine hydrochloride, 1-10M sodium perchlorate, 100-500mM ammonium chloride, 1-50mM EDTA-Na 2 0.1-20% Brij 30, 0.1-20% polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer and the balance water, and the pH of the lysis-binding solution is 5.0-8.5.
2. The lysis conjugate of claim 1, wherein:
the lysis conjugate comprises: 50-500mM Tris-HCl, 2-4M guanidine hydrochloride, 1-5M sodium perchlorate, 100-300mM ammonium chloride, 5-50mM EDTA-Na 2 0.5-10% brij 30,0.1-10% polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer and the balance water, and the pH of the lysis-binding fluid is 6.0-8.0.
3. The lysis conjugate of claim 1, wherein:
the lysis conjugate comprises: 100-200mM Tris-HCl, 2-3M guanidine hydrochloride, 2-5M sodium perchlorate, 100-200mM ammonium chloride, 10-40mM EDTA-Na 2 1-5% brij 30,0.2-2% polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer and balance water, and the pH of the lysis-binding fluid is 6.5.
4. The lysis conjugate of claim 1, wherein:
the lysis binding solution comprises 100mM Tris-HCl, 3M guanidine hydrochloride, 3M sodium perchlorate, 100mM ammonium chloride and 10mM EDTA-Na 2 2% Brij 30,0.25% of a polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer having a molecular weight of 4400 and the balance water, wherein the pH of the lysate is 6.5.
5. The lysis conjugate of any of claims 1 to 4, wherein:
the molecular weight of the polyethylene glycol-polypropylene glycol-polyethylene glycol segmented copolymer is 2000-20000.
6. The lysis conjugate of claim 5, wherein:
the molecular weight of the polyethylene glycol-polypropylene glycol-polyethylene glycol segmented copolymer is 4000-10000.
7. A nucleic acid extraction or purification kit comprising reagents for formulating the lysis conjugate of any of claims 1 to 6.
8. A method for extracting or purifying nucleic acid from a biological sample, comprising:
the method employs the lysis conjugate of any of claims 1 to 6 for extraction or purification.
9. The method according to claim 8, wherein:
the method employs the kit of claim 7 for extraction or purification.
10. The method according to claim 9, characterized in that it comprises the steps of:
(1) Sample preparation: adding the biological sample into a centrifuge tube;
(2) Sample lysis: adding the lysis binding solution into the biological sample in the step (1), and heating at 50-80 ℃ for 3-20min to obtain sample lysis solution;
(3) Nucleic acid adsorption: adding a magnetic bead suspension into the sample lysate in the step (2), uniformly mixing and standing, and discarding the liquid after the magnetic beads are completely adsorbed to obtain the magnetic beads after adsorption;
(4) First cleaning: adding a 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 a second general-purpose 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: adding an eluent into the second cleaning magnetic beads obtained in the step (5), uniformly mixing, incubating and eluting at 50-80 ℃ for 5-10min, and collecting a nucleic acid solution obtained after eluting;
wherein:
the magnetic bead suspension comprises a dispersion liquid and 5-20% by volume of superparamagnetic silicon particles with the particle diameter of 0.1-10 mu m, wherein the pH value of the magnetic bead suspension is 4.0-8.0, and the dispersion liquid comprises 10-50% by volume of PEG300 and the balance of water;
the first general washing liquid comprises 10-100mM Tris-HCl,10-200mM lithium chloride, 1-20mM EDTA-Na 2 1-5M guanidine hydrochloride and 50-80% absolute ethanol;
the second general washing liquid comprises 5-100mM Tris-HCl,5-100mM sodium chloride and 40-60% PEG300;
the eluate contained 10mM Tris-HCl and had a pH of 8.5.
11. The method according to claim 10, wherein:
the magnetic bead suspension comprises a dispersion liquid and superparamagnetic silicon particles with the mass volume percentage of 10-15% and the particle diameter of 0.5-5 mu m, the pH value of the magnetic bead suspension is 5.0-6.0, and the dispersion liquid comprises 20 volume% of PEG300 and the balance of water.
12. The method according to claim 10, wherein:
the magnetic bead suspension comprises a dispersion liquid and a mass volume percentage of superparamagnetic silicon particles with the particle diameter of 0.5-5 mu m, wherein the pH value of the magnetic bead suspension is 5.0, and the dispersion liquid comprises 20 volume percent of PEG300 and the balance of water.
13. The method according to claim 10, wherein:
the first general cleaning solution comprises 10-50mM Tris-HCl,50-100mM lithium chloride and 2-10mM EDTA-Na 2 2-4M guanidine hydrochloride and 50% absolute ethanol.
14. The method according to claim 10, wherein:
the first universal washing liquid comprises 50mM Tris-HCl,100mM lithium chloride and 2mM EDTA-Na 2 3M guanidine hydrochloride and 50% absolute ethanol.
15. The method according to claim 10, wherein:
the second universal washing solution comprises 10-20mM Tris-HCl,10-25mM sodium chloride and 50% PEG300.
16. The method according to claim 10, wherein:
the second universal washing solution contained 10mM Tris-HCl,20mM sodium chloride and 50% PEG300.
17. The method according to claim 10, wherein:
in the case of a liquid biological sample selected from the group consisting of blood, plasma, serum and saliva, the volume of the biological sample is 200 μl, and in the case of various swab eluents, the volume of the biological sample is 200-400 μl;
in step (2), adding 1-2 times the volume of the biological sample of the lysis buffer;
in step (3), 5-30. Mu.L of the magnetic bead suspension is added;
in step (4), 200-1000 μl of the first universal cleaning solution is added;
in step (5), 200-1000 μl of the second universal cleaning solution is added;
in step (6), 40-100. Mu.L of the eluent is added.
18. The method according to claim 10, wherein:
in step (1), in the case of a liquid biological sample selected from the group consisting of blood, plasma, serum and saliva, the biological sample is used in a volume amount of 200 μl; in the case that the biological sample is various swab eluents, the volume usage of the biological sample is 300 mu L;
in step (2), adding 2 times the volume of the lysis-binding solution, and heating at 50-80 ℃ for 5-10min;
in step (3), 10-20. Mu.L of the magnetic bead suspension is added;
in step (4), 500-600 μl of the first universal cleaning solution is added;
in step (5), 500-600 μl of the second universal cleaning solution is added;
in step (6), the elution is incubated at 60℃for 5min.
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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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