CN111944157B - Nano magnetic bead and application thereof in 2019 novel coronavirus nucleic acid extraction, amplification and detection - Google Patents
Nano magnetic bead and application thereof in 2019 novel coronavirus nucleic acid extraction, amplification and detection Download PDFInfo
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- CN111944157B CN111944157B CN202010107141.9A CN202010107141A CN111944157B CN 111944157 B CN111944157 B CN 111944157B CN 202010107141 A CN202010107141 A CN 202010107141A CN 111944157 B CN111944157 B CN 111944157B
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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
Abstract
The invention provides a nano magnetic bead and application thereof in 2019 novel coronavirus nucleic acid extraction, amplification and detection. The preparation method of the magnetic bead comprises the following steps: adding a reducing agent into the iron precursor solution, stirring at 18-28 ℃, and collecting ferroferric oxide particles; dispersing ferroferric oxide particles in a solution of polylactic acid, polyhistidine and sodium alginate to form a mixed solution; and collecting the product after salting out the mixed solution. Ferroferric oxide particles synthesized under the mild stirring condition are grafted by polylactic acid, polyhistidine and sodium alginate by using a salting-out method to enable the ferroferric oxide particles to have charges and be combined with a hydrogen bond bridge chain to absorb nucleic acid. The magnetic bead can be directly amplified without elution in the extraction process of nucleic acid, subsequent amplification reaction is not influenced, the nucleic acid extraction time is shortened, the possibility of cross contamination is reduced, the loss of nucleic acid in the elution process is reduced, and the extraction process is simplified.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a nano magnetic bead and application thereof in 2019 novel coronavirus nucleic acid extraction, amplification and detection.
Background
When the real-time fluorescence RT-PCR is used for detecting virus nucleic acid, the control of different extraction reagents and manual extraction processes possibly has difference on the quantity and quality of finally extracted nucleic acid, and the quantity and quality of nucleic acid extracted from a specimen are crucial to nucleic acid amplification of the next step in the detection method, so that the final detection result is directly influenced. Therefore, the extraction and purification of the virus nucleic acid play a decisive role in the accuracy of the RT-PCR detection result.
However, the current mainstream viral nucleic acid extraction process is complex in operation and long in extraction time. In addition, the method is a phenol extraction/precipitation method, and toxic reagents such as chloroform, phenol and the like are used in the extraction process, so that the method is not beneficial to the health of experimenters. Therefore, the technology of extracting nucleic acid by magnetic bead method has been rapidly developed in recent years. The series of reagents do not contain toxic reagents such as chloroform/phenol and the like, the extracted and purified nucleic acid has good quality and high yield, and the automation is easy to realize. These advantages and features make magnetic bead nucleic acid extraction become the mainstream nucleic acid extraction method in the market.
The preparation method of the magnetic beads of the traditional magnetic bead method extraction reagent in China is complicated, and the synthesized magnetic beads need to be combined and incubated for at least more than two times when being used for nucleic acid extraction, so that the synthesized magnetic beads can be used for the next detection. The magnetic beads often cannot enter an amplification stage along with a nucleic acid sample due to the influence of the magnetic beads on the later-stage reaction, a specific solution is required to be added before the amplification stage for elution so that target nucleic acid and the magnetic beads are separated, impurities are further cleaned, the process is complicated, the time of a nucleic acid extraction process cannot be really reduced, cross contamination is easily caused, and the subsequent amplification detection is influenced. Therefore, it is necessary to provide a magnetic bead that can amplify a nucleic acid after extraction from a sample without elution.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, the invention provides a nano magnetic bead capable of amplifying nucleic acid without elution after the nucleic acid is extracted from a sample and application of the nano magnetic bead in extraction, amplification and detection of 2019 novel coronavirus nucleic acid.
In a first aspect, an embodiment of the present invention provides a method for preparing a magnetic nanobead, including the following steps:
adding a reducing agent into the iron precursor solution, stirring at 18-28 ℃, and collecting ferroferric oxide particles;
dispersing ferroferric oxide particles in a solution of polylactic acid, polyhistidine and sodium alginate to form a mixed solution;
and collecting the product after salting out the mixed solution.
The preparation method of the nano magnetic bead provided by the embodiment of the invention at least has the following beneficial effects:
the inventor unexpectedly finds that ferroferric oxide particles synthesized under the mild condition of stirring at the temperature of 18-28 ℃ are grafted by polylactic acid, polyhistidine and sodium alginate by a salting-out method to enable the ferroferric oxide particles to have charges, and are combined with a hydrogen bond bridge chain to be capable of adsorbing nucleic acid. The magnetic bead can be directly amplified without elution in the extraction process of nucleic acid, the subsequent amplification reaction is not influenced, the nucleic acid extraction time is shortened, the possibility of cross contamination is reduced, the loss of nucleic acid in the elution process is reduced, and the whole extraction process is greatly simplified. The magnetic beads prepared at 18-28 ℃ and under the salting-out condition have mild preparation conditions and good biocompatibility of surface coating molecules, so that the magnetic beads can be compatible with an amplification system; meanwhile, the magnetic beads are completely coated with iron cores after synthesis, and exposed metal ions can be avoided. Therefore, the subsequent amplification detection fluorescence detection is not influenced.
Meanwhile, the magnetic beads prepared by the method have the advantages of large specific surface area, quick magnetic response and good suspension property.
According to the preparation method of the nano magnetic bead, the iron precursor is at least one of ferric acetylacetonate, ferric chloride, ferric nitrate and ferric sulfate; the reducing agent is sodium borohydride.
According to some embodiments of the method for preparing magnetic beads of the present invention, the solvent of the iron precursor solution is at least one of water, methanol, ethanol, dimethyl sulfoxide, acetone, and isopropanol.
According to some embodiments of the method for preparing nano magnetic beads of the present invention, the solvent of the iron precursor solution is ethanol and water.
According to the preparation method of the nano magnetic bead, the volume ratio of ethanol to water is (0.5-2): 1.
according to the preparation method of the nanometer magnetic bead, the mass ratio of the ferric acetylacetonate to the sodium borohydride is (0.5-1): (2-3).
According to the preparation method of the nano magnetic bead, the mass ratio of the polylactic acid to the polyhistidine to the sodium alginate is (1.5-2.5): (1-2): (1-1.5).
According to some embodiments of the present invention, the salt solution used for salting out is selected from NaCl and Na2HPO3、NaH2PO3、KCl、K2HPO3、KH2PO3At least one of the salt solution, wherein the pH value of the salt solution is 4-10, and the concentration of the salt solution is 0.2-1.5 mol/L.
According to the preparation method of the nanometer magnetic beads, the salting-out condition is-20-30 ℃, and the salting-out time is 8-24 h.
In a second aspect, an embodiment of the present invention provides a nanobead obtained according to the above preparation method.
In a third aspect, an embodiment of the present invention provides an application of the aforementioned magnetic nanobead in nucleic acid extraction, amplification, and detection.
According to some embodiments of the invention, the application of the nano magnetic beads in 2019 novel coronavirus nucleic acid extraction, amplification and detection is provided.
In a fourth aspect, one embodiment of the present invention provides a nucleic acid extraction reagent, which includes the aforementioned nano magnetic bead.
In a fifth aspect, one embodiment of the present invention provides a nucleic acid extraction method including the steps of:
and mixing the nucleic acid extraction reagent with a sample to be extracted to obtain the nucleic acid adsorbate.
When the method is used for extracting nucleic acid, the nucleic acid adsorbate can be directly applied to the subsequent amplification step after being obtained, and meanwhile, the magnetic beads can also be eluted and then amplified.
When the nano magnetic beads in the nucleic acid extraction reagent provided by the invention are applied to nucleic acid extraction, an elution step is not needed, and the nano magnetic beads adsorbed with nucleic acid are directly amplified, so that the nucleic acid extraction time is shortened, the possibility of cross contamination is reduced, the loss of nucleic acid in an elution process is reduced, and the whole extraction process is greatly simplified.
When the elution-free method is adopted for extracting nucleic acid, corresponding nucleic acid samples can be extracted within half an hour, the sensitivity can reach 10 virus particle loads by combining a general RT-PCR technology, the linear range spans five concentration gradients, and the performance is excellent.
In a sixth aspect, an embodiment of the present invention provides a nucleic acid extraction kit, which includes the above-mentioned nucleic acid extraction reagent.
According to some embodiments of the invention, the nucleic acid extraction kit further comprises a lysis solution, wherein the lysis solution comprises sodium dodecyl sulfate, urea, sodium chloride and sodium iodide, and the pH of the lysis solution is 4-8. Sodium dodecyl sulfate can destroy the structure of phosphatidic acid molecule layer, and is favorable to the release of nucleic acid, and sodium chloride may change the osmotic pressure of sample and make it easy to crack. The concentration of each component in the lysis solution can be reasonably adjusted according to the change of conditions such as samples and the like in the actual use process so as to improve the lysis efficiency.
According to the nucleic acid extraction kit of some embodiments of the present invention, the concentration of each component in the lysate is 0.1 to 10 wt% of sodium dodecyl sulfate, 0.1 to 10 wt% of urea, 0.01 to 1mol of sodium chloride, and 0.01 to 1mol of sodium iodide. When the lysate is matched with the nanometer magnetic beads to extract nucleic acid by an elution-free method, the extraction of the nucleic acid in the 2019 novel coronavirus sample can be completed within half an hour, the sensitivity can reach 10 virus particle loads by combining a general RT-PCR technology, the linear range spans four concentration gradients, and the performance is excellent.
According to some embodiments of the invention, the nucleic acid extraction kit further comprises an eluent, wherein the eluent is a Tris solution, and the pH of the eluent is 7-8. Tris elution can maintain the stability of nucleic acid and avoid degradation and other conditions, thereby reducing the accuracy of amplification and other operations. The concentration of Tris in the eluent is finely adjusted according to requirements on the basis of conventional setting.
The nucleic acid extraction kit according to some embodiments of the present invention, further comprises an ethanol wash. The use of an ethanol solution as a washing solution can remove impurities while maintaining the nucleic acid in a non-molten state in the solution.
Drawings
Fig. 1 is an electron microscope image of a nanobead according to an embodiment of the present invention.
Fig. 2 is a distribution diagram of the particle size of the nano magnetic beads according to an embodiment of the present invention.
Fig. 3 is a diagram illustrating a magnetic response result of a nanobead according to an embodiment of the present invention.
Fig. 4 is an amplification graph of the nanobead extracted 2019 of the novel coronavirus nucleic acid by using an elution method according to an embodiment of the present invention.
Fig. 5 is an amplification curve of the nanobead extracted 2019 of a novel coronavirus nucleic acid by an elution-free method according to an embodiment of the present invention.
Fig. 6 is an amplification graph of the nanobead extracted 2019 novel coronavirus nucleic acid by an automatic extractor according to an embodiment of the present invention.
FIG. 7 is an amplification curve diagram of a sensitivity experiment in which nanometer magnetic beads are used for extracting 2019 novel coronavirus nucleic acid by an elution-free method according to an embodiment of the present invention.
Fig. 8 is a line graph of a sensitivity experiment for extracting 2019 novel coronavirus nucleic acid from nanobeads by an elution-free method according to an embodiment of the present invention.
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and other embodiments obtained by those skilled in the art without inventive efforts based on the embodiments of the present invention belong to the protection scope of the present invention.
Example 1
A preparation method of nano magnetic beads comprises the following steps:
1) dissolving 2g of ferric acetylacetonate in 50% ethanol water solution, charging N2Emptying, and ultrasonically oscillating for 10min to completely dissolve;
2) adding 4g of sodium borohydride, and mechanically stirring at the temperature of 18-28 ℃ until the solution is completely blackened;
3) continuously stirring for 3 hours, magnetically collecting the generated product, repeatedly cleaning with deionized water and ethanol to obtain ferric oxide particles, and storing the ferric oxide particles in 10mL of ethanol solution;
4) dispersing 5g of polylactic acid (PLA), 5g of Polyhistidine (PHA) and 5g of sodium Alginate (ALG) in 50mL of deionized water;
5) magnetically collecting the ferroferric oxide particles stored in the ethanol solution in the step 3), and dispersing the ferroferric oxide particles in 50mL of the solution in the step 4) to obtain a mixed solution;
6) slowly adding the mixed solution into 50mL of 1mol/L KCl solution, and preserving at-20 ℃ for 16h for salting out;
7) and magnetically collecting the generated nano magnetic beads, repeatedly washing with deionized water, and storing in 10mL of aqueous solution to obtain the nano magnetic bead aqueous solution serving as the nucleic acid extraction reagent.
Fig. 1 is an electron microscope image of a nanobead according to an embodiment of the present invention. As can be seen from the figure, the nano magnetic beads prepared in the embodiment have relatively uniform particle size and large specific surface area, and can efficiently adsorb nucleic acid. Referring to fig. 2, fig. 2 is a particle size distribution diagram of a magnetic nanoparticle according to an embodiment of the present invention, in which an abscissa represents a size of the magnetic nanoparticle, and an ordinate represents a proportion of the magnetic nanoparticle, as can be clearly seen from the diagram, the magnetic nanoparticle prepared in this embodiment has a single particle size, and through calculation, the average particle size of the magnetic nanoparticle is 235.4nm, a dispersion coefficient PDI is 0.345, and a small dispersion coefficient indicates that the magnetic nanoparticle has good suspensibility for extracting nucleic acid.
Fig. 3 is a diagram illustrating a magnetic response result of a nanobead according to an embodiment of the present invention. As can be seen from the figure, the uniformly dispersed nano magnetic bead solution recovers to be clear after 15 seconds under the action of the applied magnetic force, and the result shows that the magnetic response speed of the nano magnetic bead provided by the embodiment is relatively high.
Example 2
A nucleic acid extraction kit comprises 10mL of nano magnetic bead aqueous solution serving as a nucleic acid extraction reagent in embodiment 1 and 50mL of lysis solution. The composition of the lysis solution comprises 2.5 wt% of sodium dodecyl sulfate, 2.5 wt% of urea, 0.25mol/L of sodium chloride and 0.25mol/L of sodium iodide.
The preparation method of the lysate comprises the following steps:
12.5mL of a 10 wt% Sodium Dodecyl Sulfate (SDS) solution, 12.5mL of a 10 wt% urea solution, 12.5mL of a 1mol/L sodium chloride solution, and 12.5mL of a 1mol/L sodium iodide solution were prepared, and these solutions were stirred continuously until completely mixed to obtain a lysate.
Example 3
A nucleic acid extraction kit, which is different from the nucleic acid extraction kit in example 2, further comprises an eluent, wherein the eluent is 0.01nmol/L Tris solution.
In the following examples, samples containing 2019 novel coronavirus nucleic acids were extracted and amplified by several different nucleic acid extraction methods.
Example 4
Extraction of 2019 novel coronavirus nucleic acid by elution method
1) 200 μ L of serum 2019 novel coronavirus nucleic acid sample (containing 10)5Individual viruses) are added into a 1.5mL centrifuge tube, then 400 μ L of lysate and 16 μ L of nano magnetic bead water solution are added, and the mixture is shaken and mixed evenly. Placing the centrifuge tube in a rotary table to rotate at low speed or slightly shaking the oscillator to incubate for 10min, then magnetically attracting the centrifuge tube by a magnetic frame for 2min, and discarding the supernatant.
2) Adding 400 μ L ethanol lotion into centrifuge tube, sucking or point-shaking, mixing, reversing 3 times, rapidly vortexing, placing on magnetic frame, magnetically attracting for 1min, and discarding supernatant.
3) Adding 400 μ L of lotion, slightly sucking or shaking, mixing, reversing the upside down for 3 times, recovering liquid attached to tube wall by instantaneous centrifugation, placing on magnetic frame, magnetically attracting for 1min, and discarding supernatant. The instantaneous centrifugation and the magnetic attraction can be repeated once to remove a little residual liquid on the tube wall.
4) Adding 30 mu L of eluent head into the centrifuge tube, sucking or gently scraping the nano magnetic beads adsorbed on the wall of the centrifuge tube, mixing uniformly, incubating at 56 ℃ for 5min, and reversing up and down three times every one minute or mixing uniformly by vortex.
5) And (3) after the liquid attached to the tube wall is recovered by instantaneous centrifugation, placing the centrifuge tube on a magnetic frame, magnetically attracting for 1min, and recovering the supernatant to obtain the nucleic acid solution.
Taking an elution method to extract 2019 novel coronavirus nucleic acid 15 mu L to an octaplex tube, adding a PCR reaction solution, and carrying out RT-PCR amplification detection, wherein the result is shown in figure 4. Fig. 4 is an amplification graph of the nanobead extracted 2019 of the novel coronavirus nucleic acid by using an elution method according to an embodiment of the present invention. As can be seen from the figure, the basic amplification requirement can be achieved by separating the nano magnetic beads from the nucleic acid and then amplifying the nucleic acid after the extraction of the viral nucleic acid is completed according to the conventional elution method.
Example 5
Extraction of 2019 novel coronavirus nucleic acid by elution-free method
1) 200 μ L of serum 2019 novel coronavirus nucleic acid sample (containing 10)5Individual viruses) are added into a 1.5mL centrifuge tube, then 400 μ L of lysate and 16 μ L of nano magnetic bead water solution are added, and the mixture is shaken and mixed evenly. Placing the centrifuge tube in a rotary table to rotate at low speed or slightly shaking the oscillator to incubate for 10min, then magnetically attracting the centrifuge tube by a magnetic frame for 2min, and discarding the supernatant.
2) Adding 400 μ L ethanol lotion into centrifuge tube, sucking or point-shaking, mixing, reversing 3 times, rapidly vortexing, placing on magnetic frame, magnetically attracting for 1min, and discarding supernatant.
3) Adding 400 μ L of lotion, slightly sucking or shaking, mixing, reversing the upside down for 3 times, recovering liquid attached to tube wall by instantaneous centrifugation, placing on magnetic frame, magnetically attracting for 1min, and discarding supernatant. And repeating instantaneous centrifugation and magnetic attraction once to remove a little residual liquid on the tube wall to obtain the nanometer magnetic beads absorbed with nucleic acid.
Adding prepared 15 μ L of PCR mixed reaction solution, sucking or lightly scraping the nano magnetic beads adsorbed on the wall of the centrifuge tube with a gun head, mixing uniformly, transferring to an eight-connected tube, and performing RT-PCR amplification detection, wherein the result is shown in FIG. 5. Fig. 5 is an amplification graph of nano magnetic beads extracted 2019 novel coronavirus nucleic acid by an elution-free method according to an embodiment of the present invention. As can be seen from the combination of FIG. 4 and FIG. 5, when the method is used for directly carrying out the amplification with the nano magnetic beads without an elution step, the nano magnetic beads in the method do not influence the later-stage amplification reaction, and the amplification results of the nano magnetic beads and the amplification results are similar.
Example 6
Extraction of 2019 novel coronavirus nucleic acid by automatic extractor
1) 400 mu L of lysis solution and 16 mu L of nano magnetic bead aqueous solution are sequentially added into an 1/7 th row A-H hole of a 96-well plate, and then 200 mu L of serum 2019 novel coronavirus nucleic acid sample (containing 10 mu L of serum 2019) is respectively added5Individual viruses).
2) 400 μ L of each wash was added to 2/8 th column A-H wells of a 96-well plate, and 400 μ L of each wash was added to 3/9 th column A-H wells.
3) 50 μ L of eluent was added to the 96-well plate in column 6/12, A-H wells.
4) And (3) putting the 96-well plate added with the solution into a Kaplan magnetic bead method nucleic acid extractor, and selecting a default program for carrying out an experiment.
15 μ L of the eluted product was transferred to an octal tube, and added to the PCR reaction solution to perform RT-PCR amplification detection, the results are shown in FIG. 6. Fig. 6 is an amplification curve of the nanobead extracted 2019 of a novel coronavirus nucleic acid by an elution-free method according to an embodiment of the present invention. As can be seen from FIGS. 4 to 6, the objective of nucleic acid extraction and successful amplification can be achieved by either elution or elution-free or direct extraction of viral nucleic acid by an automatic extractor.
Example 7
2019 novel coronavirus nucleic acid sensitivity test
1) 200 μ L of serum 2019 novel coronavirus nucleic acid samples (each containing 10. mu.L of serum)5、104、103、10210 and 0 virus) are added into a 1.5mL centrifuge tube, then 400 μ L of lysis solution and 16 μ L of nano magnetic bead water solution are added, and the mixture is stirred and mixed evenly. Placing the centrifuge tube in a rotating disc to rotate at a low speed or slightly shaking an oscillator for incubationAfter 10min, the magnetic frame is magnetically attracted for 2min, and the supernatant is discarded.
2) Adding 400 μ L lotion into centrifuge tube, sucking or point-shaking, mixing, reversing 3 times, rapidly vortexing, placing on magnetic frame, magnetically attracting for 1min, and discarding supernatant.
3) Adding 400 μ L of lotion, slightly sucking or shaking, mixing, reversing the upside down for 3 times, recovering liquid attached to tube wall by instantaneous centrifugation, placing on magnetic frame, magnetically attracting for 1min, and discarding supernatant. The instantaneous centrifugation and the magnetic attraction can be repeated once to remove a little residual liquid on the tube wall.
4) Adding prepared 15 mu L of PCR mixed reaction solution, sucking or lightly scraping the nano magnetic beads adsorbed on the wall of the centrifugal tube by using a gun head, uniformly mixing, transferring to an eight-connection tube, and carrying out RT-PCR amplification detection.
The amplification results are shown in fig. 7 and fig. 8, wherein fig. 7 and fig. 8 are an amplification curve and a linear graph, respectively, of a sensitivity experiment in which the nano-magnetic beads provided by an embodiment of the present invention extract 2019 novel coronavirus nucleic acid by an elution-free method. As can be seen from FIG. 7 and FIG. 8, the logarithmic values of the different template concentrations of the viral nucleic acid and the Ct value of the amplification in this example show a good linear relationship (R)20.9987), i.e. between 10 and 105Under different concentration gradients, the virus particle load extraction results are in a linear relation, and the sensitivity can be as low as 10 virus particle loads.
From the results, the preparation method of the reagent is simple and rapid, and the prepared nano magnetic beads have large specific surface area, fast magnetic response and good suspension property. Different from the conventional magnetic beads, when the nano magnetic beads provided by the embodiment of the invention are used for extracting nucleic acid, an elution step is not needed in the extraction process, and the nano magnetic beads are directly amplified, so that the possibility of cross contamination is reduced, the loss of nucleic acid in the elution process is reduced, and the whole extraction process is greatly simplified. The novel 2019 coronavirus nucleic acid can be extracted within half an hour, the sensitivity can reach 10 virus particle loads by combining a general RT-PCR technology, a linear range spans four concentration gradients, and the performance is excellent.
Example 8
A preparation method of nano magnetic beads comprises the following steps:
1) dissolving 2g ferric chloride in 30% ethanol water solution, charging N2Emptying, and ultrasonically oscillating for 10min to completely dissolve;
2) adding 4g of sodium borohydride, and mechanically stirring at the temperature of 18-28 ℃ until the solution is completely blackened;
3) continuously stirring for 3 hours, magnetically collecting the generated product, repeatedly cleaning with deionized water and ethanol to obtain ferric oxide particles, and storing the ferric oxide particles in 10mL of ethanol solution;
4) dispersing 5g of polylactic acid (PLA), 5g of Polyhistidine (PHA) and 5g of sodium Alginate (ALG) in 50mL of deionized water;
5) magnetically collecting the ferroferric oxide particles stored in the ethanol solution in the step 3), and dispersing the ferroferric oxide particles in 50mL of the solution in the step 4) to obtain a mixed solution;
6) slowly adding 50mL of NaH with the concentration of 1mol/L into the mixed solution2PO3Preserving the solution at 20 ℃ for 8h for salting out;
7) and magnetically collecting the generated nano magnetic beads, repeatedly washing with deionized water, and storing in 10mL of aqueous solution to obtain the nano magnetic bead aqueous solution serving as the nucleic acid extraction reagent.
The nano magnetic bead aqueous solution is used for extracting 2019 novel coronavirus nucleic acid by the method in the embodiment 6 and amplifying, and the amplification result is good.
Example 9
A preparation method of nano magnetic beads comprises the following steps:
1) dissolving 2g of ferric acetylacetonate in 50% ethanol water solution, charging N2Emptying, and ultrasonically oscillating for 10min to completely dissolve;
2) adding 4g of sodium borohydride, and mechanically stirring at the temperature of 18-28 ℃ until the solution is completely blackened;
3) continuously stirring for 3 hours, magnetically collecting the generated product, repeatedly cleaning with deionized water and ethanol to obtain ferric oxide particles, and storing the ferric oxide particles in 10mL of ethanol solution;
4) dispersing 3g of polylactic acid (PLA), 3g of Polyhistidine (PHA) and 3g of sodium Alginate (ALG) in 50mL of deionized water;
5) magnetically collecting the ferroferric oxide particles stored in the ethanol solution in the step 3), and dispersing the ferroferric oxide particles in 50mL of the solution in the step 4) to obtain a mixed solution;
6) slowly adding the mixed solution into 50mL of NaCl solution with the concentration of 1mol/L, and preserving at the temperature of minus 10 ℃ for 16h for salting out;
7) and magnetically collecting the generated nano magnetic beads, repeatedly washing with deionized water, and storing in 10mL of aqueous solution to obtain the nano magnetic bead aqueous solution serving as the nucleic acid extraction reagent.
The nano magnetic bead aqueous solution is used for extracting 2019 novel coronavirus nucleic acid by the washing-free method in the embodiment 5 and amplifying, and the amplification result is good.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
Claims (10)
1. A preparation method of nano magnetic beads is characterized by comprising the following steps:
adding a reducing agent into the iron precursor solution, stirring at 18-28 ℃, and collecting ferroferric oxide particles;
dispersing the ferroferric oxide particles in a solution of polylactic acid, polyhistidine and sodium alginate to form a mixed solution;
salting out the mixed solution and collecting the product.
2. The preparation method according to claim 1, wherein the iron precursor is at least one of ferric acetylacetonate, ferric chloride, ferric nitrate, and ferric sulfate; the reducing agent is sodium borohydride.
3. The preparation method according to claim 2, wherein the iron precursor is ferric acetylacetonate, and the mass ratio of the ferric acetylacetonate to the sodium borohydride is (0.5-1): (2-3).
4. The preparation method according to claim 1, wherein the mass ratio of the polylactic acid to the polyhistidine to the sodium alginate is (1.5-2.5): (1-2): (1-1.5).
5. Nanobead obtained by the preparation process according to any one of claims 1 to 4.
6. Use of the nanobead of claim 5 in the preparation of 2019 products for extraction, amplification and detection of novel coronavirus nucleic acids.
7. A nucleic acid extraction reagent comprising the nanobead of claim 5.
8. A method for extracting nucleic acid, comprising the steps of:
mixing the nucleic acid extraction reagent of claim 7 with a sample to be extracted to obtain a nucleic acid adsorbate.
9. A nucleic acid extraction kit comprising the nucleic acid extraction reagent according to claim 7.
10. The nucleic acid extraction kit according to claim 9, further comprising a lysis solution, wherein the lysis solution comprises sodium dodecyl sulfate, urea, sodium chloride and sodium iodide, and the pH of the lysis solution is 4-8.
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