CN114438076A - Magnetic bead method virus nucleic acid extraction kit and use method thereof - Google Patents
Magnetic bead method virus nucleic acid extraction kit and use method thereof Download PDFInfo
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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
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Abstract
The invention discloses a kit for extracting viral nucleic acid by a paramagnetic particle method and a using method thereof, wherein the kit comprises lysis solution, paramagnetic particle solution, washing solution I, washing solution II, washing solution III and eluent, and the lysis solution consists of the following substances: Tris-HCl, guanidinium thiocyanate, EDTA, Triton-100, PEG-6000, SDS, isopropanol, water. The invention has the advantages that the lysate provided by the invention does not need to use proteinase K so as to save cost, the volatilization of an organic solvent can be accelerated and the harm to the health of operators is caused because the sample needs to be heated and cracked usually, and the lysate does not need to be heated so as to reduce the harm to the operators. The formula of the lysis solution for extracting the nucleic acid is obtained by researching the distribution ratio of the components of the lysis solution by the inventor.
Description
Technical Field
The invention relates to the field of medical treatment, in particular to a magnetic bead method virus nucleic acid extraction kit and a using method thereof.
Background
In recent years, molecular biology is rapidly developed, with the increasingly wide application of PCR and PR-PCR in virus detection, the workload of nucleic acid extraction is rapidly increased, the traditional method for extracting nucleic acid has complicated steps, needs to use methods such as phenol or chloroform for extraction, has long time and high toxicity, does not support automatic extraction, and thus cannot meet the requirement of large-scale disease detection.
Nucleic acid extraction technologies which are simpler and more convenient to operate than the traditional nucleic acid extraction method also appear in the market, and mainly comprise the following steps: chromatographic column method and magnetic bead method. Because the chromatographic column nucleic acid extraction technology needs a centrifugal column for matching use and needs automatic centrifugal equipment, the cost is higher, the prior chromatographic column nucleic acid extraction still mainly adopts manual extraction, and the efficiency is low.
The magnetic bead method for extracting nucleic acid only needs one magnetic bead and magnetic separation device (magnetic force frame) for separating nucleic acid, is simple to operate and low in cost, can be used for automatically extracting nucleic acid, and realizes large-batch sample extraction.
Disclosure of Invention
The invention aims to solve the problems, and designs a kit for extracting viral nucleic acid by a magnetic bead method and a using method thereof.
The technical scheme of the invention is that the invention provides a magnetic bead method virus nucleic acid extraction reagent, which is characterized by comprising the following components: lysis solution, magnetic bead solution, washing solution I, washing solution II, washing solution III and eluent.
In a first aspect, the lysis solution comprises Tris-HCl, guanidinium thiocyanate, EDTA, Triton-100, PEG-6000; SDS; isopropyl alcohol; wherein Tris-HCl is 0.1M-0.5M; guanidine thiocyanate 3-5M; EDTA 10-40 mm; PEG-6000 accounts for 2-5% of the total mass; SDS accounts for 1% -3% of the total mass; triton-100 accounts for 4.5-9% of the total mass; the isopropanol accounts for 5-15% of the total mass.
The magnetic bead liquid is purchased magnetic beads, and the concentration of the magnetic beads is 20-50mg/mL
The washing solution I comprises Tris-HCl and guanidine hydrochloride, wherein the guanidine hydrochloride is 1-3M, and the PH is 5.0-7.0
The washing liquid II is 60-75% ethanol
The washing liquid III is 75-85% ethanol
The eluent is TE buffer solution comprising EDTA and Tris-HCl
In a second aspect, the invention also provides a use method of the kit for extracting the viral nucleic acid by the magnetic bead method, which comprises the following steps:
adding lysis solution and magnetic beads into the sample, mixing at room temperature, and lysing
Placing the centrifuge tube containing the mixed solution in a magnetic frame, and discarding the supernatant after the magnetic beads are completely adsorbed
Adding the washing liquid I, uniformly mixing, placing the centrifuge tube filled with the mixed liquid in a magnetic frame, and discarding the supernatant after the magnetic beads are completely adsorbed
Adding the washing liquid II, uniformly mixing, placing the centrifuge tube filled with the mixed liquid in a magnetic frame, and discarding the supernatant after the magnetic beads are completely adsorbed
Adding the washing liquid III, uniformly mixing, placing the centrifuge tube filled with the mixed liquid in a magnetic frame, and discarding the supernatant after the magnetic beads are completely adsorbed
And (3) drying the magnetic beads, adding the eluent, incubating at 50-80 ℃ for elution, collecting nucleic acid liquid and storing.
A kit for extracting viral nucleic acid by a magnetic bead method comprises: the reagent bottle, the reagent sealing cap and the sealing structure;
the reagent bottle is meshed with the actual sealing cap through threads, and the sealing structure is positioned between the sealing cap and the bottle mouth of the reagent bottle;
the reagent sealing cap comprises: sealing panel, panel plug, elastic top column;
the sealing panel is positioned on the panel plug, and the elastic top column is positioned between the sealing cap and the sealing panel;
further comprising: the sealing device comprises an annular sealing disc, an annular sealing strip and an annular connecting body;
the annular connector and the bottom of the actual sealing cap are integrally formed and extend outwards, the annular sealing disc is positioned on the reagent bottle, the bottom of the annular connector is provided with a circle of embedded groove, and the annular sealing strip is submerged in the embedded groove of the annular connector;
the same embedded groove of annular sealing strip width has also been seted up on the cyclic annular sealed dish, the embedded groove of cyclic annular sealed dish is entered into to the bottom of annular sealing strip.
According to the virus nucleic acid extraction kit prepared by the magnetic bead method and the using method, the lysis solution provided by the invention does not need proteinase K, so that the cost is saved, the volatilization of an organic solvent can be accelerated and the harm to the health of an operator is caused because the sample is generally cracked by heating, and the harm to the operator can be reduced because the lysis solution is not required to be heated. The formula of the lysis solution for extracting the nucleic acid is obtained by researching the distribution ratio of the components of the lysis solution by the inventor.
Drawings
FIG. 1 is a schematic structural diagram of a kit for extracting viral nucleic acid by a magnetic bead method according to the present invention;
FIG. 2 is a schematic diagram of a partial top view of a magnetic bead method viral nucleic acid extraction kit according to the present invention;
FIG. 3 is a schematic diagram of a partial top view of a magnetic bead method viral nucleic acid extraction kit according to the present invention;
FIG. 4 is a graph showing PCR results of nucleic acids extracted by 1000-fold dilution of the B stream;
FIG. 5 is a graph showing PCR results of nucleic acids extracted from influenza B virus diluted 10000 times
FIG. 6 is a graph showing PCR results for nucleic acid extracted from adenovirus at 1000-fold dilution;
FIG. 7 is a graph showing PCR results of nucleic acids extracted from adenovirus diluted 10000 times;
in the figure, 1, a reagent bottle; 2. a reagent sealing cap; 3. sealing the panel; 4. a panel plug; 5. an elastic top pillar; 6. an annular sealing disk; 7. an annular sealing strip; 8. an annular connector.
Detailed Description
The invention is described in detail below with reference to the accompanying drawings, and as shown in fig. 1-7, a kit for extracting viral nucleic acid by a magnetic bead method and a use method thereof.
The technical solution of the present invention will be further explained with reference to the drawings and the embodiments.
Example 1 influenza B virus nucleic acid extraction
The invention has the following steps:
diluting the influenza virus by 1000 times and 10000 times with a negative pharyngeal swab sample, respectively taking 200 mu L of the influenza virus, putting the 200 mu L of the influenza virus into a centrifugal tube, adding 700 mu L of lysis solution and 20 mu L of 20-50mg/ml magnetic bead solution, carrying out room-temperature lysis and uniform mixing for 5min, putting the centrifugal tube on a magnetic frame, carrying out magnetic attraction for 30s, and discarding the supernatant;
wherein, the components in the lysis solution are respectively as follows: 0.25M Tris-HCl, 3.17M guanidinium thiocyanate, 25mm EDTA, 3.6% PEG-6000, 2% SDS, 6% Triton-100, 9.5% isopropanol, and balance water
Adding 1ml of the washing solution I into the centrifugal tube, uniformly mixing for 1min, placing the centrifugal tube on a magnetic frame, absorbing magnetism for 30s, and removing a supernatant;
wherein, wash the interior component of liquid I respectively and be: 3M guanidine hydrochloride, 0.25M Tris-HCl, pH adjusted to 6.5
Adding 1ml of a washing solution II into the centrifugal tube, uniformly mixing for 1min, placing the centrifugal tube on a magnetic frame, absorbing magnetism for 30s, and removing a supernatant; the washing liquid II is 75% ethanol solution
Adding 1ml of the washing solution III into the centrifugal tube, uniformly mixing for 1min, placing the centrifugal tube on a magnetic frame, absorbing magnetism for 30s, and removing a supernatant; the washing liquid III is 85% ethanol solution
And finally, adding 60 mu L of eluent, heating and uniformly mixing at 56 ℃ for 10min, placing the centrifugal tube on a magnetic frame, absorbing magnetism for 30s, transferring the supernatant into a new clean centrifugal tube, and storing the extracted nucleic acid liquid at-20 ℃.
The eluent components are respectively: 1mm EDTA, 5mm Tris-HCl
The magnetic beads are silicon hydroxyl magnetic beads, and the particle size of the magnetic beads is 500nm
The invention can also be matched with a full-automatic nucleic acid extractor to extract the nucleic acid of the influenza virus throat swab quickly and efficiently, and the procedure is shown in the following table 1:
TABLE 1
The control kit is a German QIAGEN QIAamp Viral kit, the extraction sample volume is 200 μ L, and the elution volume is 60 μ L.
After extraction, 5 mu L of influenza B nucleic acid RNA extracting solution is taken as a template, 20 mu L of influenza B reaction solution and 25 mu L of total reaction volume are taken, and amplification detection is carried out by using a MA-688Y fluorescence quantitative PCR instrument. The results of the detection of the B stream by the nucleic acid extraction kit and the comparison kit are shown in Table 2, wherein FIG. 1 is a PCR result diagram of nucleic acid extracted by 1000-fold dilution of the B stream, and FIG. 2 is a PCR result diagram of nucleic acid extracted by 10000-fold dilution of the B stream.
TABLE 2 results of detection of influenza B virus by the nucleic acid extraction reagent of the present invention and the control reagent
As shown in Table 2, and FIGS. 1 and 2, QIAGEN, Germany, has a high market share and is widely used, and is once recognized as the "gold standard" for nucleic acid extraction reagents at home and abroad. The analysis can obtain that the extraction efficiency of the invention has the level of the similar products and meets the requirements of the subsequent tests.
EXAMPLE 2 adenovirus nucleic acid extraction
The invention has the following steps:
1) diluting adenovirus 1000 times and 10000 times by using a negative excrement sample, respectively placing 200 mu L into a centrifuge tube, adding 700 mu L of lysis solution and 20 mu L of 20-50mg/ml magnetic bead solution, uniformly mixing for 5min at room temperature, placing the centrifuge tube on a magnetic frame, absorbing magnetism for 30s, and discarding the supernatant;
wherein, the components in the lysis solution are respectively as follows: 0.25M Tris-HCl, 3.17M guanidinium thiocyanate, 25mm EDTA, 3.6% PEG-6000, 2% SDS, 6% Triton-100, 9.5% isopropanol, and balance water
2) Adding 1ml of the washing solution I into the centrifugal tube, uniformly mixing for 1min, placing the centrifugal tube on a magnetic frame, absorbing magnetism for 30s, and removing a supernatant;
wherein, wash the interior component of liquid I respectively and be: 3M guanidine hydrochloride, 0.25M Tris-HCl, pH adjusted to 6.5
3) Adding 1ml of a washing solution II into the centrifugal tube, uniformly mixing for 1min, placing the centrifugal tube on a magnetic frame, absorbing magnetism for 30s, and removing a supernatant; the washing liquid II is 65% ethanol solution
4) Adding 1ml of the washing solution III into the centrifugal tube, uniformly mixing for 1min, placing the centrifugal tube on a magnetic frame, absorbing magnetism for 30s, and removing a supernatant; the washing liquid III is 85% ethanol solution
5) And finally, adding 60 mu L of eluent, heating and uniformly mixing at 56 ℃ for 10min, placing the centrifugal tube on a magnetic frame, absorbing magnetism for 30s, transferring the supernatant into a new clean centrifugal tube, and storing the extracted nucleic acid liquid at-20 ℃.
The eluent components are respectively: 1mm EDTA, 5mm Tris-HCl
The magnetic beads are silicon hydroxyl magnetic beads, and the particle size of the magnetic beads is 500nm
The invention can also be matched with a full-automatic nucleic acid extractor to extract the nucleic acid of the adenovirus feces quickly and efficiently, and the procedure is shown in the following table 1:
TABLE 1
The control kit is nucleic acid extraction kit by Shandong micromagnet bead method, the sample amount is 200 μ L, and the elution volume is 70 μ L.
After extraction, 5 mu L of the adenovirus nucleic acid DNA extracting solution is taken as a template, 20 mu L of adenovirus reaction solution and 25 mu L of total reaction volume are taken, and amplification detection is carried out by using a MA-688Y fluorescence quantitative PCR instrument. The results of adenovirus detection by the nucleic acid extraction kit and the comparison kit of the present invention are shown in table 3, fig. 3 is a PCR result diagram of nucleic acid extracted by 1000-fold dilution of adenovirus, and fig. 4 is a PCR result diagram of nucleic acid extracted by 10000-fold dilution of adenovirus.
TABLE 3 results of adenovirus detection using nucleic acid extraction reagents of the invention and control reagents
As shown in the results in Table 3 and FIGS. 3 and 4, the detection result of the kit for extracting adenovirus nucleic acid is equivalent to the detection effect of the contrast kit for extracting adenovirus which is on the market at home.
Example 3 influenza B virus nucleic acid extraction
The invention has the following steps:
1) diluting the virus B1000 times with negative saliva sample, placing 200 μ L into a centrifuge tube, adding 700 μ L lysis solution and 20 μ L magnetic bead solution of 20-50mg/ml, splitting at room temperature, mixing for 5min, placing the centrifuge tube on a magnetic frame, magnetizing for 30s, and discarding the supernatant;
wherein, the components in the lysis solution are respectively as follows: 0.25M Tris-HCl, 3.17M guanidinium thiocyanate, 25mm EDTA, 3.6% PEG-6000, 2% SDS, 6% Triton-100, 9.5% isopropanol, and balance water
2) Adding 1ml of the washing solution I into the centrifugal tube, uniformly mixing for 1min, placing the centrifugal tube on a magnetic frame, absorbing magnetism for 30s, and removing a supernatant;
wherein, wash the interior component of liquid I respectively and be: 3M guanidine hydrochloride, 0.25M Tris-HCl, pH adjusted to 6.5
3) Adding 1ml of a washing solution II into the centrifugal tube, uniformly mixing for 1min, placing the centrifugal tube on a magnetic frame, absorbing magnetism for 30s, and removing a supernatant; the washing liquid II is 75% ethanol solution
4) Adding 1ml of the washing solution III into the centrifugal tube, uniformly mixing for 1min, placing the centrifugal tube on a magnetic frame, absorbing magnetism for 30s, and removing a supernatant; the washing liquid III is 85% ethanol solution
5) And finally, adding 60 mu L of eluent, heating and uniformly mixing at 56 ℃ for 10min, placing the centrifugal tube on a magnetic frame, absorbing magnetism for 30s, transferring the supernatant into a new clean centrifugal tube, and storing the extracted nucleic acid liquid at-20 ℃.
The eluent components are respectively: 1mm of EDTA and 5mm of Tris-HCl.
The magnetic beads are silicon hydroxyl magnetic beads, and the particle size of the magnetic beads is 500 nm.
And (5) after extraction, carrying out quantitative and purity detection on the RNA. Taking 5 μ L of each sample, detecting with the above eluent as blank, measuring A260/A280 value of the sample with ultramicro ultraviolet spectrophotometer (NANODOEP ONE), detecting nucleic acid purity with the ratio, and comparing with nucleic acid extraction kit by Shandong micromagnetic bead method to obtain the following results shown in Table 4
TABLE 4
As can be seen from Table 4, the concentration and purity of the extracted nucleic acids are good in the kit and the control kit, the ratio of the high-quality nucleic acid A260/A280 is 1.8-2.0, and the purity ratios of the kit and the control kit are all 1.8-2.0, which indicates that the sample has no protein and other pollutants. The concentration and the purity are equivalent to those of a control sample, so that the method can effectively extract the virus nucleic acid in the biological sample, and the extracted nucleic acid can be used for the next experiment;
example 3
The kit comprises: the reagent bottle comprises a reagent bottle 1, a reagent sealing cap 2 and a sealing structure;
the reagent bottle 1 is engaged with an actual sealing cap through threads, and the sealing structure is positioned between the sealing cap and the bottle mouth of the reagent bottle 1;
the reagent sealing cap 2 comprises: a sealing panel 3, a panel plug 4 and an elastic top column 5;
the sealing panel 3 is positioned on the panel plug 4, and the elastic top column 5 is positioned between the sealing cap and the sealing panel 3;
further comprising: an annular sealing disc 6, an annular sealing strip 7 and an annular connecting body 8;
the annular connector 8 and the bottom of the actual sealing cap are integrally formed and extend outwards, the annular sealing disc 6 is positioned on the reagent bottle 1, the bottom of the annular connector 8 is provided with a circle of embedded groove, and the annular sealing strip 7 is submerged in the embedded groove of the annular connector 8;
the same embedded groove of 7 width with annular sealing strip has also been seted up on the cyclic annular sealed dish 6, the embedded groove of cyclic annular sealed dish 6 is entered into to the bottom of annular sealing strip 7.
In the above, the opening of the reagent bottle 1 and the reagent sealing cap 2 are sealed by the panel plug 4, the panel plug 4 is disc-shaped, is buckled at the opening of the reagent bottle 1, and is sealed by the reagent sealing cap 2 and the elastic top pillar 5;
meanwhile, the bottom of the reagent sealing cap 2 is provided with an annular sealing strip 7 through an annular connecting body 8, and the bottom of the annular sealing strip 7 can enter an embedded groove on the annular sealing disc, so that the internal liquid of the reagent bottle 1 is sealed;
the technical solutions described above only represent the preferred technical solutions of the present invention, and some possible modifications to some parts of the technical solutions by those skilled in the art all represent the principles of the present invention, and fall within the protection scope of the present invention.
Claims (9)
1. The kit for extracting the viral nucleic acid by the paramagnetic particle method is characterized by comprising a lysis solution, a paramagnetic particle solution, a washing solution I, a washing solution II, a washing solution III and an eluent, wherein the lysis solution consists of the following substances: Tris-HCl, guanidine thiocyanate, EDTA, Triton-100, PEG-6000, SDS, isopropanol and water;
the washing solution I comprises the following raw materials: Tris-HCl, guanidine hydrochloride;
the washing liquid II and the washing liquid III comprise the following raw materials: ethanol;
the eluent comprises the following raw materials: EDTA, Tris-HCl.
2. The kit for extracting viral nucleic acid by using the magnetic bead method according to claim 1, wherein the lysis solution comprises the following substances: 0.1M-0.5M Tris-HCl, 3-5M guanidinium thiocyanate, 10-40mm EDTA, 2-5% PEG-6000, 1% -3% SDS, 4.5-9% Triton-100, 5-15% isopropanol, and the balance water.
3. The kit for extracting viral nucleic acid according to claim 1, wherein the magnetic bead solution is purchased magnetic beads, and the concentration of the magnetic beads is 20-50 mg/mL.
4. The kit for extracting viral nucleic acid by using the magnetic bead method according to claim 1, wherein the washing solution I comprises the following substances: 1-3M guanidine hydrochloride, 0.1-0.5M Tris-HCl.
5. The kit for extracting viral nucleic acid by using the magnetic bead method according to claim 1, wherein the washing solution II and the washing solution III are composed of the following substances: 60-85% ethanol.
6. The kit for extracting viral nucleic acid by magnetic bead method according to claim 1, wherein the eluent comprises the following components: 1-5mmEDTA, 4-10 mmTris-HCl.
7. The use of the kit for extracting viral nucleic acid according to claim 1 to 6, comprising the following steps:
s1, adding lysis solution and magnetic beads into the biological sample, mixing and lysing;
s2, placing the centrifuge tube filled with the mixed solution in a magnetic frame, and discarding the supernatant after the magnetic beads are completely adsorbed;
s3, adding the washing solution I, mixing uniformly, placing the centrifugal tube filled with the mixed solution in a magnetic frame, and discarding the supernatant after the magnetic beads are completely adsorbed
S4, adding the washing liquid II, uniformly mixing, placing the centrifuge tube filled with the mixed liquid in a magnetic frame, and discarding supernatant after the magnetic beads are completely adsorbed;
s5, adding the washing solution III, uniformly mixing, placing the centrifuge tube filled with the mixed solution in a magnetic frame, and discarding supernatant after magnetic beads are completely adsorbed;
s6, drying the magnetic beads, adding the eluent, incubating at 50-80 ℃ for elution, collecting nucleic acid liquid and storing.
8. The kit of claim 7, wherein the biological sample comprises saliva, throat swab, serum, plasma, or feces.
9. The kit for extracting viral nucleic acid by magnetic bead method according to claim 1, comprising: the reagent bottle, the reagent sealing cap and the sealing structure;
the reagent bottle is meshed with the actual sealing cap through threads, and the sealing structure is positioned between the sealing cap and the bottle mouth of the reagent bottle;
the reagent sealing cap comprises: sealing panel, panel plug, elastic top column;
the sealing panel is positioned on the panel plug, and the elastic top column is positioned between the sealing cap and the sealing panel;
further comprising: the sealing device comprises an annular sealing disc, an annular sealing strip and an annular connecting body;
the annular connector and the bottom of the actual sealing cap are integrally formed and extend outwards, the annular sealing disc is positioned on the reagent bottle, the bottom of the annular connector is provided with a circle of embedded groove, and the annular sealing strip is submerged in the embedded groove of the annular connector;
the same embedded groove of annular sealing strip width has also been seted up on the cyclic annular sealed dish, the embedded groove of cyclic annular sealed dish is entered into to the bottom of annular sealing strip.
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| CN116004608A (en) * | 2022-08-01 | 2023-04-25 | 南京诺唯赞生物科技股份有限公司 | Method and composition for rapidly extracting nucleic acid |
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