CN113926432B - Novel coronavirus nucleic acid extraction kit and nucleic acid extraction method - Google Patents
Novel coronavirus nucleic acid extraction kit and nucleic acid extraction method Download PDFInfo
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Abstract
The invention discloses a novel coronavirus nucleic acid extraction kit and a nucleic acid extraction method, belonging to the technical field of nucleic acid extraction, wherein the kit comprises a suspension prepared from novel biological nano magnetic beads, and the preparation method of the novel biological nano magnetic beads comprises the following steps: mixing Fe3O4@SiO2The nano magnetic beads are reacted with a modified silane coupling agent obtained by modifying chloromethyl trimethoxy silane with urea. The method for extracting the novel coronavirus nucleic acid by using the kit comprises the following steps: adding lysis solution into the sample, adding washing solution after the sample is lysed at room temperature, oscillating and centrifuging, taking supernate, adding novel biological nano magnetic bead suspension into the supernate, and adsorbing at room temperature; removing supernatant after adsorption, adding washing liquid and mixing uniformly; standing, removing supernatant and air drying; after air drying, adding an eluent to elute at room temperature to obtain an RNA solution. The kit can extract high-purity and high-concentration nucleic acid, and the extracted nucleic acid is not easy to degrade.
Description
Technical Field
The invention belongs to the technical field of nucleic acid extraction, and particularly relates to a novel coronavirus nucleic acid extraction kit and an extraction method.
Background
After the virus nucleic acid is extracted, purified and identified by PCR amplification, the detection result can be used as one of the bases of clinical reference. However, the extraction and purification of high quality viral nucleic acids is relatively difficult due to the presence of exogenous and endogenous nucleases and the relatively stable enzymatic activity thereof. The formula of the nucleic acid extraction reagent has great influence on the quality and yield of nucleic acid, so the research and development of the nucleic acid extraction reagent with high efficiency, rapidness and high yield is a subject continuously searched in the field. The magnetic bead method is used as an automatic nucleic acid extraction method, and the main principle is that nucleic acid is specifically adsorbed by magnetic beads in high-salt solution, and is eluted in low-salt solution after impurities such as protein, polysaccharide and the like are magnetically separated and rinsed, so that the purpose of extracting and purifying nucleic acid is achieved.
The conventional nucleic acid extraction technology has complex extraction process, long extraction time and low nucleic acid extraction efficiency, and cannot meet the clinical detection requirement; the commonly used virus nucleic acid extraction and purification techniques include Trizol method, centrifugal column method and the like. The Trizol method separates protein and nucleic acid by utilizing the difference of distribution positions of DNA, RNA and protein in an organic layer and an aqueous layer, has complex steps for extracting nucleic acid, poor repeatability of extracting different samples, needs at least 1.5 hours from sample cracking to nucleic acid obtaining, has complex operation and long time consumption, and is not suitable for clinical detection. The conventional centrifugal adsorption column method has relatively complex operation and long time consumption when extracting nucleic acid.
Disclosure of Invention
The invention aims to provide biological nanometer magnetic beads suitable for extracting nucleic acid, which have the effects of improving the extraction purity and stability of RNA and protecting RNA from degradation. Another objective of the invention is to provide a novel coronavirus nucleic acid extraction kit and a nucleic acid extraction method.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention discloses a novel coronavirus nucleic acid extraction kit, which is characterized by comprising biological nano magnetic beads; the biological nano magnetic beads are made of Fe3O4@SiO2Preparing nanometer magnetic beads and a modified silane coupling agent; the modified silane coupling agent is prepared by modifying chloromethyl trimethoxy silane with urea.
Preferably, the preparation method of the biological nanometer magnetic bead comprises the following steps:
FeC13·6H2O and FeC12·4H2Adding ammonia water into the hydrochloric acid solution of O under stirring, performing magnetic separation after stirring reaction, and washing the obtained solid substance to obtain Fe3O4；
Mixing Fe3O4Mixing with distilled water, mixing with isopropanol, adding PEG while stirring, adding distilled water and TEOS while stirring, magnetically separating, and washing the obtained solid to obtain Fe3O4@SiO2Nano magnetic beads;
reacting urea with chloromethyl trimethoxy silane under thermal reflux, carrying out reduced pressure distillation after the reaction, and collecting target fractions to obtain the modified silane coupling agent;
taking Fe3O4@SiO2Dispersing the nano magnetic beads in anhydrous toluene, slowly adding a modified silane coupling agent while stirring, stirring for reaction, carrying out magnetic separation, and washing the obtained solid substance to obtain the biological nano magnetic beads.
The preparation method adopts urea to modify the chloromethyl trimethoxy silane, and then the obtained modified silane coupling agent is used for modifying Fe3O4@SiO2The biological nano magnetic beads can better inhibit RNA enzyme and protect RNA adsorbed on the nano magnetic beads from being degraded, and even if the RNA is not eluted from the nano magnetic beads in time, the RNA can be stored for a long time and is not degraded, so that the stability of the RNA is improved; the preparation method can also improve the nucleic acid extraction effect of the nano magnetic beads and improve the RNA extraction purity.
Preferably, the concentration of the hydrochloric acid solution is 1-3M.
Preferably, the concentration of ammonia is 1-2M.
More preferably, FeC13·6H2O and FeC12·4H2The mass ratio of O is 1: 0.3-0.5.
Preferably, Fe3O4PEG and TEOS at a mass ratio of 0.01:1:0.8-1.2。
Preferably, the PEG is PEG 2000, PEG 4000 or PEG 6000.
Preferably, the mass ratio of urea to chloromethyltrimethoxysilane is 1: 0.8-1.2.
Preferably, Fe3O4@SiO2And the modified silane coupling agent in a mass ratio of 0.05-0.06: 1.
Preferably, the preparation method of the biological nanometer magnetic bead comprises the following steps:
based on the parts by weight, 1 part of FeC13·6H2O, 0.3-0.5 parts of FeC12·4H2O and 4-6 parts of 1-3M hydrochloric acid solution are added with 1.1-1.5 parts of 1-2M ammonia water under stirring, magnetic separation is carried out after stirring reaction for 30-40min, and the obtained solid matter is washed to obtain Fe3O4；
0.01 part by weight of Fe3O4Mixing with 0.4-0.6 part of distilled water, mixing with 30-50 parts of isopropanol, adding 1 part of PEG while stirring under oscillation, stirring for 10-15min, adding 2-5 parts of distilled water and 0.8-1.2 parts of TEOS, stirring, performing magnetic separation, and washing the obtained solid substance to obtain Fe3O4@SiO2Nano magnetic beads;
reacting 1 part of urea with 0.8-1.2 parts of chloromethyl trimethoxy silane under the heat reflux for 3-5h, carrying out reduced pressure distillation after the reaction, and collecting target fractions to obtain the modified silane coupling agent;
taking 0.05 to 0.06 weight portion of Fe3O4@SiO2Dispersing the nano magnetic beads in 10-15 parts of anhydrous toluene, slowly adding 1 part of modified silane coupling agent under stirring, stirring for reaction for 12-36h, carrying out magnetic separation, and washing the obtained solid substance to obtain the biological nano magnetic beads.
The invention also discloses a biological nanometer magnetic bead prepared by the method.
The invention also discloses application of the biological nano magnetic beads in nucleic acid extraction.
The invention also discloses the application of the biological nanometer magnetic beads in virus splitting.
The magnetic bead method for extracting nucleic acid can be matched with an automatic nucleic acid extractor except for manual extraction, so that the time and labor cost can be effectively saved, and the magnetic bead method is very suitable for extracting nucleic acid of a large batch of samples. Compared with the traditional non-magnetic method, the magnetic bead method has simple steps and does not need centrifugation, thereby really realizing high-flux automatic nucleic acid extraction. The method can remove most inhibiting factors such as egg, polysaccharide and phenols which interfere PCR reaction, and reduce false negative rate.
Another object of the present invention is to provide a novel coronavirus nucleic acid extraction kit which extracts nucleic acid at a high purity and a high concentration and in which the extracted nucleic acid is not easily degraded.
In order to achieve the purpose, the invention adopts the following technical scheme:
a novel coronavirus nucleic acid extraction kit comprises a suspension prepared from biological nano magnetic beads. The novel coronavirus nucleic acid extraction kit can efficiently extract nucleic acid with high purity and high concentration.
Preferably, the concentration of the suspension prepared from the biological nanometer magnetic beads is 50-55 mg/mL.
Preferably, glycine hydrochloride is added into the biological nanometer magnetic bead suspension. The glycine hydrochloride is added, so that the yield of the extracted nucleic acid can be effectively improved, the high-concentration nucleic acid can be obtained, the purity of the nucleic acid can be improved, and the subsequent detection and experiment can be conveniently carried out.
More preferably, glycine hydrochloride is added at a concentration of 1 to 3 mol/L.
Preferably, the novel coronavirus nucleic acid extraction kit further comprises lysis solution, washing solution 1, washing solution 2 and eluent.
Preferably, the components of the lysate include Tris-HCl and guanidinium isothiocyanate.
Guanidine isothiocyanate is a strong protein denaturant, can quickly destroy cell structures, lyses cells, releases nucleic acid from nucleoprotein, has strong denaturation on RNA enzyme, and is a good RNA enzyme inhibitor. Guanidine isothiocyanate was thus added to the lysate.
More preferably, the lysate has a concentration of Tris-HCl of 10-12mmol/L, pH of 7.0 and a concentration of guanidinium isothiocyanate of 3-5 mol/L.
Preferably, the components of the lysate are Tris-HCl and biological nano magnetic bead suspension. Because the modified urea grafted by the biological nano magnetic beads has the effects of cracking viruses, denaturing proteins and inhibiting RNA enzyme, the biological nano magnetic bead suspension can be used for replacing guanidinium isothiocyanate in the lysis solution.
Preferably, the washing solution 1 comprises a chloroform isoamyl alcohol mixed solution; the composition of washing solution 2 includes ethanol.
More preferably, the ratio of chloroform to isoamyl alcohol in the chloroform isoamyl alcohol solution in washing solution 1 is 24: 1.
More preferably, the volume fraction of ethanol in wash 2 is 70-75%.
Preferably, the eluent comprises Tris-HCl, EDTA and ethanol.
More preferably, the concentration of Tris-HCl in the eluent is 10-12mmol/L, pH is 6.5; the concentration of EDTA is 3-5 mol/L; the volume fraction of ethanol is 2-3%.
Preferably, the novel coronavirus nucleic acid extraction kit comprises five containers: the first container is biological nanometer magnetic bead suspension, the second container is lysis solution, the third container is washing solution 1, the fourth container is washing solution 2, and the fifth container is eluent.
Another object of the present invention is to provide a method for efficiently extracting high-quality nucleic acid using the novel coronavirus nucleic acid extraction kit of the present invention.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for extracting novel coronavirus nucleic acid by using the novel coronavirus nucleic acid extraction kit comprises the following steps:
adding lysis solution into a sample, carrying out lysis at room temperature, adding washing solution 1, oscillating and centrifuging, taking supernate, adding biological nano magnetic bead suspension into the supernate, and adsorbing at room temperature; removing supernatant after adsorption, adding washing solution 2 and mixing uniformly; standing, removing supernatant and air drying; after air drying, adding an eluent to elute at room temperature to obtain an RNA solution.
Preferably, a method for extracting novel coronavirus nucleic acid using the novel coronavirus nucleic acid extraction kit of the present invention comprises the steps of:
taking 2-4mL of sample, adding lysis solution, mixing uniformly, and performing room temperature lysis for 5-8 min; adding 200 plus 220 mu L of washing solution 1, oscillating for 30s, and centrifuging for 5-8min at 13000 r/min; centrifuging, taking 180-sample 200 mu L of supernatant to a new EP tube, adding 200-sample 220 mu L of biological nano magnetic bead suspension, oscillating and mixing uniformly, adsorbing at room temperature for 5-8min, standing for 20-30s, and removing the supernatant; adding 500-550 mu L of washing solution 2, uniformly mixing, standing for 20-30s, removing supernatant, and airing for 15-20min at room temperature; adding 70-75 μ L of eluent, mixing, eluting at room temperature for 10-12min, standing for 20-30s, transferring RNA solution to new EP tube, and storing at-80 deg.C.
Preferably, the novel coronavirus nucleic acid obtained by the above extraction method is stored at room temperature and 4 ℃.
The invention also discloses the modified silane coupling agent, which is obtained by reacting urea and chloromethyl trimethoxy silane.
The invention also discloses application of the modified silane coupling agent in preparation of biological nano magnetic beads.
The invention also discloses application of the modified silane coupling agent in improving the inhibition of RNA enzyme by nano magnetic beads.
The invention also discloses application of the modified silane coupling agent in improving the nucleic acid extraction effect of nano magnetic beads.
The invention has the following beneficial effects:
the surface of the biological nanometer magnetic bead is grafted with a modified silane coupling agent, which can crack cells and inhibit the activity of enzyme. The modified silane coupling agent is directly grafted to the surface of the nano magnetic bead, so that RNA enzyme can be better inhibited, and RNA adsorbed on the nano magnetic bead is protected from being degraded. Even if the RNA is not eluted from the nano magnetic beads in time, the RNA can be stored for a long time without being degraded. The novel coronavirus nucleic acid extraction kit can extract high-quality and high-concentration nucleic acid, and meanwhile, most of inhibiting factors such as egg, polysaccharide and phenolic substances which interfere PCR reaction can be removed by using the method for extracting nucleic acid by using the novel coronavirus nucleic acid extraction kit, so that the false negative rate is reduced. Because the synthesis of the magnetic sphere particles adopts low-price inorganic raw materials and organic raw materials, special instruments and equipment are not needed, and the synthesis and research and development costs are greatly reduced.
The invention aims to provide biological nanometer magnetic beads suitable for extracting nucleic acid, which have the effects of improving the extraction purity and stability of RNA and protecting RNA from degradation. Another objective of the invention is to provide a novel coronavirus nucleic acid extraction kit and a nucleic acid extraction method.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention discloses a preparation method of biological nanometer magnetic beads, which comprises the following steps:
FeC13·6H2O and FeC12·4H2Adding ammonia water into the hydrochloric acid solution of O under stirring, performing magnetic separation after stirring reaction, and washing the obtained solid substance to obtain Fe3O4；
Mixing Fe3O4Mixing with distilled water, mixing with isopropanol, adding PEG while stirring, adding distilled water and TEOS while stirring, magnetically separating, and washing the obtained solid to obtain Fe3O4@SiO2Nano magnetic beads;
reacting urea with chloromethyl trimethoxy silane under thermal reflux, carrying out reduced pressure distillation after the reaction, and collecting target fractions to obtain the modified silane coupling agent;
taking Fe3O4@SiO2Dispersing the nano magnetic beads in anhydrous toluene, slowly adding a modified silane coupling agent while stirring, stirring for reaction, carrying out magnetic separation, and washing the obtained solid substance to obtain the biological nano magnetic beads.
The preparation method adopts urea to modify the chloromethyl trimethoxy silane, and then the obtained modified silane coupling agent is used for modifying Fe3O4@SiO2The biological nano magnetic beads can better inhibit RNA enzyme and protect RNA adsorbed on the nano magnetic beads from being degraded, and even if the RNA is not eluted from the nano magnetic beads in time, the RNA can be stored for a long time and is not degraded, so that the stability of the RNA is improved; book (I)The preparation method can also improve the nucleic acid extraction effect of the nano magnetic beads and improve the RNA extraction purity.
Preferably, the concentration of the hydrochloric acid solution is 1-3M.
Preferably, the concentration of ammonia is 1-2M.
More preferably, FeC13·6H2O and FeC12·4H2The mass ratio of O is 1: 0.3-0.5.
Preferably, Fe3O4The mass ratio of PEG to TEOS is 0.01:1: 0.8-1.2.
Preferably, the PEG is PEG 2000, PEG 4000 or PEG 6000.
Preferably, the mass ratio of urea to chloromethyltrimethoxysilane is 1: 0.8-1.2.
Preferably, Fe3O4@SiO2And the modified silane coupling agent in a mass ratio of 0.05-0.06: 1.
Preferably, the preparation method of the biological nanometer magnetic bead comprises the following steps:
based on the parts by weight, 1 part of FeC13·6H2O, 0.3-0.5 parts of FeC12·4H2O and 4-6 parts of 1-3M hydrochloric acid solution are added with 1.1-1.5 parts of 1-2M ammonia water under stirring, magnetic separation is carried out after stirring reaction for 30-40min, and the obtained solid matter is washed to obtain Fe3O4；
0.01 part by weight of Fe3O4Mixing with 0.4-0.6 part of distilled water, mixing with 30-50 parts of isopropanol, adding 1 part of PEG while stirring under oscillation, stirring for 10-15min, adding 2-5 parts of distilled water and 0.8-1.2 parts of TEOS, stirring, performing magnetic separation, and washing the obtained solid substance to obtain Fe3O4@SiO2Nano magnetic beads;
reacting 1 part of urea with 0.8-1.2 parts of chloromethyl trimethoxy silane under the heat reflux for 3-5h, carrying out reduced pressure distillation after the reaction, and collecting target fractions to obtain the modified silane coupling agent;
taking 0.05 to 0.06 weight portion of Fe3O4@SiO2Dispersing nano magnetic beads in 10-15 parts of anhydrous toluene, slowly adding 1 part of modified silane coupling agent under stirring, and stirring for reactionAnd carrying out magnetic separation after 12-36h, and washing the obtained solid substance to obtain the biological nano magnetic beads.
The invention also discloses a biological nanometer magnetic bead prepared by the method.
The invention also discloses application of the biological nano magnetic beads in nucleic acid extraction.
The magnetic bead method for extracting nucleic acid can be matched with an automatic nucleic acid extractor except for manual extraction, so that the time and labor cost can be effectively saved, and the magnetic bead method is very suitable for extracting nucleic acid of a large batch of samples. Compared with the traditional non-magnetic method, the magnetic bead method has simple steps and does not need centrifugation, thereby really realizing high-flux automatic nucleic acid extraction. The method can remove most inhibiting factors such as egg, polysaccharide and phenols which interfere PCR reaction, and reduce false negative rate.
Another object of the present invention is to provide a novel coronavirus nucleic acid extraction kit which extracts nucleic acid at a high purity and a high concentration and in which the extracted nucleic acid is not easily degraded.
In order to achieve the purpose, the invention adopts the following technical scheme:
a novel coronavirus nucleic acid extraction kit comprises a suspension prepared from biological nano magnetic beads. The novel coronavirus nucleic acid extraction kit can efficiently extract nucleic acid with high purity and high concentration.
Preferably, the concentration of the suspension prepared from the biological nanometer magnetic beads is 50-55 mg/mL.
Preferably, glycine hydrochloride is added into the biological nanometer magnetic bead suspension. The glycine hydrochloride is added, so that the yield of the extracted nucleic acid can be effectively improved, the high-concentration nucleic acid can be obtained, the purity of the nucleic acid can be improved, and the subsequent detection and experiment can be conveniently carried out.
More preferably, glycine hydrochloride is added at a concentration of 1 to 3 mol/L.
Preferably, the novel coronavirus nucleic acid extraction kit further comprises lysis solution, washing solution 1, washing solution 2 and eluent.
Preferably, the components of the lysate include Tris-HCl and guanidinium isothiocyanate.
Guanidine isothiocyanate is a strong protein denaturant, can quickly destroy cell structures, lyses cells, releases nucleic acid from nucleoprotein, has strong denaturation on RNA enzyme, and is a good RNA enzyme inhibitor. Guanidine isothiocyanate was thus added to the lysate.
More preferably, the lysate has a concentration of Tris-HCl of 10-12mmol/L, pH of 7.0 and a concentration of guanidinium isothiocyanate of 3-5 mol/L.
Preferably, the components of the lysate are Tris-HCl and biological nano magnetic bead suspension. Because the modified urea grafted by the biological nano magnetic beads has the effects of cracking viruses, denaturing proteins and inhibiting RNA enzyme, the biological nano magnetic bead suspension can be used for replacing guanidinium isothiocyanate in the lysis solution.
Preferably, the washing solution 1 comprises a chloroform isoamyl alcohol mixed solution; the composition of washing solution 2 includes ethanol.
More preferably, the ratio of chloroform to isoamyl alcohol in the chloroform isoamyl alcohol solution in washing solution 1 is 24: 1.
More preferably, the volume fraction of ethanol in wash 2 is 70-75%.
Preferably, the eluent comprises Tris-HCl, EDTA and ethanol.
More preferably, the concentration of Tris-HCl in the eluent is 10-12mmol/L, pH is 6.5; the concentration of EDTA is 3-5 mol/L; the volume fraction of ethanol is 2-3%.
Preferably, the novel coronavirus nucleic acid extraction kit comprises five containers: the first container is biological nanometer magnetic bead suspension, the second container is lysis solution, the third container is washing solution 1, the fourth container is washing solution 2, and the fifth container is eluent.
Another object of the present invention is to provide a method for efficiently extracting high-quality nucleic acid using the novel coronavirus nucleic acid extraction kit of the present invention.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for extracting novel coronavirus nucleic acid by using the novel coronavirus nucleic acid extraction kit comprises the following steps:
adding lysis solution into a sample, carrying out lysis at room temperature, adding washing solution 1, oscillating and centrifuging, taking supernate, adding biological nano magnetic bead suspension into the supernate, and adsorbing at room temperature; removing supernatant after adsorption, adding washing solution 2 and mixing uniformly; standing, removing supernatant and air drying; after air drying, adding an eluent to elute at room temperature to obtain an RNA solution.
Preferably, a method for extracting novel coronavirus nucleic acid using the novel coronavirus nucleic acid extraction kit of the present invention comprises the steps of:
taking 2-4mL of sample, adding lysis solution, mixing uniformly, and performing room temperature lysis for 5-8 min; adding 200 plus 220 mu L of washing solution 1, oscillating for 30s, and centrifuging for 5-8min at 13000 r/min; centrifuging, taking 180-sample 200 mu L of supernatant to a new EP tube, adding 200-sample 220 mu L of biological nano magnetic bead suspension, oscillating and mixing uniformly, adsorbing at room temperature for 5-8min, standing for 20-30s, and removing the supernatant; adding 500-550 mu L of washing solution 2, uniformly mixing, standing for 20-30s, removing supernatant, and airing for 15-20min at room temperature; adding 70-75 μ L of eluent, mixing, eluting at room temperature for 10-12min, standing for 20-30s, transferring RNA solution to new EP tube, and storing at-80 deg.C.
Preferably, the novel coronavirus nucleic acid obtained by the above extraction method is stored at room temperature and 4 ℃.
The invention also discloses the modified silane coupling agent, which is obtained by reacting urea and chloromethyl trimethoxy silane.
The invention also discloses application of the modified silane coupling agent in preparation of biological nano magnetic beads.
Preferably, the modified silane coupling agent is used for improving the RNA enzyme inhibition of the nano magnetic beads.
Preferably, the modified silane coupling agent is used for improving the nucleic acid extraction effect of the nano magnetic beads.
The invention has the following beneficial effects:
the surface of the biological nanometer magnetic bead is grafted with a modified silane coupling agent, which can crack cells and inhibit the activity of enzyme. The modified silane coupling agent is directly grafted to the surface of the nano magnetic bead, so that RNA enzyme can be better inhibited, and RNA adsorbed on the nano magnetic bead is protected from being degraded. Even if the RNA is not eluted from the nano magnetic beads in time, the RNA can be stored for a long time without being degraded. The novel coronavirus nucleic acid extraction kit can extract high-quality and high-concentration nucleic acid, and meanwhile, most of inhibiting factors such as egg, polysaccharide and phenolic substances which interfere PCR reaction can be removed by using the method for extracting nucleic acid by using the novel coronavirus nucleic acid extraction kit, so that the false negative rate is reduced. Because the synthesis of the magnetic sphere particles adopts low-price inorganic raw materials and organic raw materials, special instruments and equipment are not needed, and the synthesis and research and development costs are greatly reduced.
Drawings
FIG. 1 is an infrared spectrum of a modified silane coupling agent;
FIG. 2 is an infrared spectrum of biological nano magnetic beads;
FIG. 3 shows the real-time fluorescent quantitative reverse transcription PCR amplification result of a sample extracted by using the novel coronavirus nucleic acid extraction kit of the present invention; wherein 1 is the amplification curve cluster of the sample in example 1; 2 is the amplification curve cluster for the sample in example 2; 3 is the amplification curve cluster of the sample in example 3; 4 is the amplification curve cluster for the sample in comparative example 1; FIG. 5 is a cluster of amplification curves for the sample of comparative example 3.
Detailed Description
The exemplary embodiments will be described herein in detail, and the embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present disclosure. Rather, they are merely examples of methods consistent with certain aspects of the present disclosure, as detailed in the appended claims.
The experimental procedures in the following examples are, unless otherwise specified, either conventional or according to the manufacturer's recommendations. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Preparation of biological nano magnetic beads
Based on the parts by weight, 1 part of FeC13·6H2O, 0.38 parts of FeC12·4H2O and 5 parts of 1.5M hydrochloric acid solution are added with 1.12 parts of 1M ammonia water under stirring, magnetic separation is carried out after stirring reaction for 35min, and the obtained solid matter is washed to obtain Fe3O4；
0.01 part by weight of Fe3O4Mixing with 0.5 part of distilled water, mixing with 45 parts of isopropanol, adding 1 part of PEG while stirring under oscillation, stirring for 12min, adding 2.5 parts of distilled water and 1 part of TEOS, stirring, performing magnetic separation, and washing the obtained solid to obtain Fe3O4@SiO2Nano magnetic beads;
reacting 1 part of urea with 1 part of chloromethyl trimethoxy silane under the heat reflux for 3.5h, carrying out reduced pressure distillation after the reaction, and collecting target fractions to obtain the modified silane coupling agent;
taking 0.05 part of Fe by weight3O4@SiO2Dispersing the nano magnetic beads in 15 parts of anhydrous toluene, slowly adding 1 part of modified silane coupling agent while stirring, stirring for reaction for 20 hours, carrying out magnetic separation, and washing the obtained solid substance to obtain the biological nano magnetic beads.
Composition of novel coronavirus nucleic acid extraction kit
The components and concentrations of the novel coronavirus nucleic acid extraction kit are shown in tables 1 and 2.
TABLE 1 novel coronavirus nucleic acid extraction kit Components
TABLE 2 concentration of the components of the novel coronavirus nucleic acid extraction kit
Example 2
Preparation of biological nano magnetic beads
The same as in example 1.
Second, novel coronavirus nucleic acid extraction kit
The components and concentrations of the novel coronavirus nucleic acid extraction kit are shown in tables 3 and 4.
TABLE 3 novel coronavirus nucleic acid extraction kit Components
TABLE 4 concentration of the Components of the novel coronavirus nucleic acid extraction kit
Example 3
Preparation of biological nano magnetic beads
The same as in example 1.
Second, novel coronavirus nucleic acid extraction kit
The components and concentrations of the novel coronavirus nucleic acid extraction kit are shown in tables 5 and 6.
TABLE 5 novel coronavirus nucleic acid extraction kit Components
TABLE 6 concentration of the Components of the novel coronavirus nucleic acid extraction kit
Comparative example 1
Preparation of biological nano magnetic beads
Based on the parts by weight, 1 part of FeC13·6H2O, 0.38 parts of FeC12·4H2O and 5 parts of 1.5M hydrochloric acid solution are added with 1.12 parts of 1M ammonia water under stirring, magnetic separation is carried out after stirring reaction for 35min, and the obtained solid matter is washed to obtain Fe3O4；
0.01 part by weight of Fe3O4Mixing with 0.5 part of distilled water, mixing with 45 parts of isopropanol, adding 1 part of PEG while stirring, and stirringAdding 2.5 parts of distilled water and 1 part of TEOS after 12min, stirring, carrying out magnetic separation, and washing the obtained solid substance to obtain Fe3O4@SiO2Nano magnetic beads;
taking 0.05 part of Fe by weight3O4@SiO2Dispersing the nano magnetic beads in 15 parts of anhydrous toluene, slowly adding 1 part of chloromethyl trimethoxy silane under stirring, stirring for reacting for 20 hours, carrying out magnetic separation, and washing the obtained solid substance to obtain the biological nano magnetic beads.
Second, novel coronavirus nucleic acid extraction kit
The components and concentrations of the novel coronavirus nucleic acid extraction kit are shown in tables 7 and 8.
TABLE 7 novel coronavirus nucleic acid extraction kit Components
TABLE 8 concentration of the Components of the novel coronavirus nucleic acid extraction kit
Comparative example 2
Preparation of biological nano magnetic beads
As in comparative example 1.
Second, novel coronavirus nucleic acid extraction kit
The components and concentrations of the novel coronavirus nucleic acid extraction kit are shown in tables 9 and 10.
TABLE 9 novel coronavirus nucleic acid extraction kit Components
TABLE 10 concentration of the Components of the novel coronavirus nucleic acid extraction kit
Comparative example 3
Preparation of biological nano magnetic beads
As in comparative example 1.
Second, novel coronavirus nucleic acid extraction kit
The components and concentrations of the novel coronavirus nucleic acid extraction kit are shown in tables 11 and 12.
TABLE 11 novel coronavirus nucleic acid extraction kit Components
TABLE 12 concentration of the Components of the novel coronavirus nucleic acid extraction kit
Test example 1
Infrared characterization of modified silane coupling agent
Respectively characterizing the samples by adopting a Fourier infrared absorption spectrometer, tabletting and preparing the samples by using KBr, wherein the wave number range is 500-4000cm-1。
The infrared characterization of the modified silane coupling agent obtained in example 1 is shown in FIG. 1. 3300cm in FIG. 1-1An absorption characteristic peak of N-H appears nearby; 2930cm-1An absorption peak of a methyl methylene group appears nearby; 1720cm-1An absorption characteristic peak of C = O appears nearby; 1610cm-1An N-H absorption vibration peak appears nearby; 1130cm-1Nearby occurrence of Si-OCH3Absorption peak. Which indicates the successful modification of the silane coupling agent.
Second, infrared characterization of biological nanometer magnetic beads
Respectively characterizing the samples by adopting a Fourier infrared absorption spectrometer, tabletting and preparing the samples by using KBr, wherein the wave number range is 500-4000cm-1。
The infrared characterization of the biomagnetic beads obtained in example 1 is shown in FIG. 2. 600 cm can be seen from FIG. 2-1The characteristic absorption peak of Fe-O is nearby; 1000 cm-1The vicinity is a characteristic absorption peak of Si-O-Si; 1620 cm-1The nearby part is an absorption vibration peak of N-H; 1690 cm-1An absorption characteristic peak with C = O in the vicinity; 3390 cm-1The vicinity is a stretching vibration peak of-OH. Illustrating the successful grafting of the modified silane coupling agent to Fe3O4@SiO2And (4) successfully obtaining biological nano magnetic beads on the nano magnetic beads.
Test example 2
Novel coronavirus nucleic acid extraction
The novel coronavirus nucleic acid extraction kit provided by the invention is used for extracting nucleic acid from nasal swabs, throat swabs, saliva and urine samples containing the novel coronavirus. Taking 2mL of sample, adding 50 mu L of lysate, mixing uniformly, and performing room-temperature lysis for 5 min; adding 200 mu L of washing solution 1, oscillating for 30s, and centrifuging for 5min at 13000 r/min; adding 200 μ L of supernatant into a new EP tube, adding 200 μ L of biological nanometer magnetic bead suspension, shaking, mixing, adsorbing at room temperature for 5min, standing for 20s, and removing supernatant; adding 500 μ L of washing solution 2, mixing, standing for 20s, removing supernatant, and air drying at room temperature for 15 min; adding 70 μ L of eluent, mixing, eluting at room temperature for 10min, standing for 20s, transferring RNA solution to new EP tube, and storing at-80 deg.C.
Test example 3
Detection of nucleic acid extraction Effect of novel coronavirus
Using the novel coronavirus nucleic acid extraction kits of example 1, example 2, comparative example 1, comparative example 2 and comparative example 3, the novel coronavirus nucleic acid in the sample was extracted according to the method described in test example 2, the concentration of RNA was measured after extraction, and the absorbance of the eluted RNA was measured at 260nm and 280nm, respectively, to determine whether the purity of the extracted RNA could be subjected to PCR amplification reaction. Then, a sample capable of undergoing PCR amplification reaction was subjected to PCR amplification using a novel fluorescent quantitative detection kit for coronavirus nucleic acid amplification (PCR) manufactured by Jienon Biotech, Shanghai (batch No.: GZTR2020005, national institutes of record 20203400058). The samples were throat swabs, nasal swabs, saliva and urine carrying the novel coronavirus.
Real-time fluorescent quantitative reverse transcription PCR amplification system (25 mL): 12.5mL of reaction solution, 7.0mL of sterile deionized water, 1.0mL of enzyme mixed solution, 2.5mL of fluorescent probe and 2.0mL of virus RNA extracted from a sample. The amplification procedure was: 5min at 42 ℃; 10s at 95 ℃; the fluorescence signal was collected at 95 ℃ for 5s and 60 ℃ for 35s (end of this period), for 40 cycles. And (4) judging a result: the Ct value is less than or equal to 37, and the result is judged to be positive; if the Ct value is more than 37 and less than 40, the sample is suspicious, nucleic acid needs to be extracted again, and if the sample is still suspicious, the sample is positive; if the Ct value is more than or equal to 40 or no Ct value, the result is negative.
The results of measuring the absorbance of the eluted RNA at the RNA concentrations and 260nm and 280nm are shown in Table 13.
TABLE 13 RNA extraction concentration and purity
As can be seen from Table 13, RNA of sufficient purity could not be extracted using the novel coronavirus nucleic acid extraction kit of comparative example 2, and thus PCR amplification detection could not be performed. The novel coronavirus nucleic acid extraction kits in example 1, example 2, example 3, comparative example 1 and comparative example 3 can successfully extract RNA with qualified purity, wherein the purity of the RNA extracted in examples 1-3 is obviously higher than that of the RNA extracted in comparative examples 1-3, respectively, which shows that the biological nano magnetic beads are favorable for obtaining high-purity RNA.
Comparing the concentrations of the RNAs extracted in example 1 and example 3 and comparative example 1 and comparative example 3, respectively, the concentrations of the RNAs extracted in example 1 and comparative example 1 were higher than those extracted in example 3 and comparative example 3, respectively, and it is demonstrated that the addition of glycine hydrochloride was effective in increasing the amount of RNA extracted regardless of whether bio-nanobead was used.
The real-time fluorescent quantitative reverse transcription PCR amplification results are shown in FIG. 3.
As can be seen from FIG. 3, the novel coronavirus nucleic acid extraction kit of examples 1-3 has a good extraction effect on the sample, and can successfully detect that the sample is positive. The novel coronavirus nucleic acid detection kits in comparative examples 1 and 3 extract the nucleic acid of the virus in the sample, but cannot successfully detect the positive sample. Therefore, the novel coronavirus nucleic acid extraction kit can successfully extract the virus nucleic acid and can be normally detected. The comparison between example 1 and example 2 shows that the amplification curves are similar, and only the CT value is different, which indicates that only the nucleic acid extraction amount is slightly different between example 1 and example 2, and indicates that the biological nano-magnetic beads of the present invention can be used as RNase inhibitor in the lysis solution instead of guanidinium isothiocyanate.
Test example 4
Effect of storage time after extraction of novel coronavirus nucleic acids on nucleic acid sample integrity
Extraction of coronavirus nucleic acid
The novel coronavirus nucleic acid extraction kits of examples 1 to 3 and comparative example 3 were used to extract the novel coronavirus nucleic acid from the sample by the following method
Taking 2mL of sample, adding 50 mu L of lysate, mixing uniformly, and performing room-temperature lysis for 5 min; adding 200 mu L of washing solution 1, oscillating for 30s, and centrifuging for 5min at 13000 r/min; adding 200 μ L of supernatant into a new EP tube, adding 200 μ L of biological nanometer magnetic bead suspension, shaking, mixing, adsorbing at room temperature for 5min, standing for 20s, and removing supernatant; adding 500 μ L of washing solution 2, mixing, standing for 20s, removing supernatant, and air drying at room temperature for 15 min; elution was not performed. Respectively storing at normal temperature, 4 deg.C and-80 deg.C, taking out a batch of samples every 1, 3, 5, 7, 14 and 28 days to determine whether RNA is degraded. The measurement results are shown in Table 14.
TABLE 14 degradation of RNA at different storage temperatures
+: the RNA is not degraded;
-: RNA is degraded
As can be seen from Table 14, examples 1 and 3, in the case of using the biological nanobead suspension of the present invention and guanidinium isothiocyanate, RNA could be stored at 4 ℃ for 14 days, at room temperature for 3 days, and at-80 ℃ for 28 days without elution after RNA extraction. It can be seen from comparative example 2 that the extracted RNA can be preserved at 4 ℃ and normal temperature for one day even if a suspension of biomagnetic beads is used in the lysate instead of guanidinium isothiocyanate. In contrast, in comparative examples 1 and 3, in the case where the biological nanobead suspension was not used, no matter whether guanidinium isothiocyanate was used in the lysate or not, the RNA could be stored only at-80 ℃ without degradation after extraction. In comparative example 2, RNA could not be extracted successfully without using biomicromagnetic beads and guanidium isothiocyanate. The biological nano magnetic beads can effectively protect RNA from being degraded at normal temperature and in a storage environment of 4 ℃; simultaneously, guanidine isothiocyanate in the lysate can be replaced.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A novel coronavirus nucleic acid extraction kit is characterized by comprising a suspension prepared from biological nano magnetic beads;
the preparation method of the biological nanometer magnetic bead comprises the following steps:
FeC13·6H2O and FeC12·4H2Adding ammonia water into the hydrochloric acid solution of O under stirring, performing magnetic separation after stirring reaction, and washing the obtained solid substance to obtain Fe3O4；
Subjecting said Fe to3O4Mixing with distilled water, mixing with isopropanol, adding PEG while stirring, adding distilled water and TEOS while stirring, magnetically separating, and washing the obtained solid to obtain Fe3O4@SiO2Nano magnetic beads;
reacting urea with chloromethyl trimethoxy silane under thermal reflux, carrying out reduced pressure distillation after the reaction, and collecting target fractions to obtain the modified silane coupling agent;
subjecting said Fe to3O4@SiO2Dispersing the nano magnetic beads in anhydrous toluene, slowly adding the modified silane coupling agent under stirring, stirring for reaction, carrying out magnetic separation, and washing the obtained solid substance to obtain biological nano magnetic beads;
the mass ratio of the urea to the chloromethyl trimethoxy silane is 1: 0.8-1.2;
said Fe3O4@SiO2The mass ratio of the modified silane coupling agent to the modified silane coupling agent is 0.05-0.06: 1;
the concentration of the suspension prepared from the biological nano magnetic beads is 50-55 mg/mL;
the concentration of glycine hydrochloride added into the suspension prepared from the biological nano magnetic beads is 1-3 mol/L;
the novel coronavirus nucleic acid extraction kit further comprises lysis solution, washing solution 1, washing solution 2 and eluent.
2. The kit for extracting coronavirus nucleic acid according to claim 1, wherein the Fe is Fe3O4The mass ratio of PEG to TEOS is 0.01:1: 0.8-1.2.
3. The novel coronavirus nucleic acid extraction kit of claim 1, wherein the components of the lysate comprise Tris-HCl and guanidinium isothiocyanate; the washing solution 1 comprises chloroform isoamyl alcohol mixed solution; the washing solution 2 comprises ethanol; the eluent comprises Tris-HCl, EDTA and ethanol.
4. The kit for extracting nucleic acid from a novel coronavirus according to claim 3, wherein the concentration of Tris-HCl in the lysate is 10 to 12mmol/L, pH of 7.0, and the concentration of guanidine isothiocyanate is 3 to 5 mol/L; the ratio of chloroform to isoamyl alcohol in the chloroform isoamyl alcohol solution in the washing solution 1 is 24: 1; the volume fraction of ethanol in the washing solution 2 is 70-75%; the concentration of Tris-HCl in the eluent is 10-12mmol/L, pH to be 6.5; the concentration of EDTA is 3-5 mol/L; the volume fraction of ethanol is 2-3%.
5. The novel coronavirus nucleic acid extraction kit of claim 3, wherein the kit comprises five containers: the first container is biological nanometer magnetic bead suspension, the second container is lysis solution, the third container is washing solution 1, the fourth container is washing solution 2, and the fifth container is eluent.
6. A method for extracting novel coronavirus nucleic acid using the kit of any one of claims 1-5, comprising the steps of:
adding lysis solution into a sample, carrying out lysis at room temperature, adding washing solution 1, oscillating and centrifuging, taking supernate, adding biological nano magnetic bead suspension into the supernate, and adsorbing at room temperature; removing supernatant after adsorption, adding washing solution 2 and mixing uniformly; standing, removing supernatant and air drying; after air drying, adding an eluent to elute at room temperature to obtain an RNA solution.
7. Use of biological nano magnetic beads in the novel coronavirus nucleic acid extraction kit as claimed in claim 1 for extracting nucleic acid.
8. Use of biological nanobagnetic beads in the novel coronavirus nucleic acid extraction kit as claimed in claim 1 for virus lysis.
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