CN113151397A - Nucleic acid extraction kit for extracting virus sample based on paramagnetic particle method - Google Patents
Nucleic acid extraction kit for extracting virus sample based on paramagnetic particle method Download PDFInfo
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Abstract
The invention discloses a nucleic acid extraction kit for extracting a virus sample based on a paramagnetic particle method, wherein the used magnetic beads are silicon hydroxyl magnetic beads and are formed by mixing a plurality of magnetic beads with different particle sizes, and the particle size is 10-1000 nm. The capability of capturing nucleic acid is strong, and the method is more suitable for extracting trace virus nucleic acid. The kit has easily obtained raw materials and low cost; the product is stored at normal temperature, and is convenient to transport and use; the extraction steps are simple, the cracking combination can be carried out only by adding the sample, the lysate and the magnetic beads, and imported raw materials such as protease K, CarrierRNA and the like do not need to be added. The virus DNA and RNA are extracted together, and the recovery rate is high; organic solvents such as phenol, chloroform and the like are not needed, so that the method is safe and has no toxic or side effect on operators; the operation is simple and convenient, centrifugation is not needed, and the requirements on instruments and equipment are not high; can match with a high-flux automatic extraction instrument; the method is suitable for extracting virus nucleic acid from environmental samples, such as micro virus samples of sewage, refrigerator, seafood meat, food inner and outer packages and the like.
Description
Technical Field
The invention relates to the technical field of nucleic acid extraction, in particular to a nucleic acid extraction kit for extracting a virus sample based on a paramagnetic particle method.
Background
Nucleic acids are carriers of genetic information and are the material basis for gene expression. Nucleic acid includes both DNA and RNA, and is widely present in all animal and plant cells and microorganisms, and nucleic acid in organisms is often present in a state of being bound to proteins. The structural change of nucleic acid molecule inevitably leads to the change of biological function, such as virus pathogenic action, caused by the invasion of virus nucleic acid into body cells, besides, malignant tumor, genetic lesion, metabolic disease and the like are closely related to the change of molecular structure and function of nucleic acid, so no matter the research of nucleic acid structure or function is carried out, the separation and purification of nucleic acid are needed firstly.
Common methods for extracting and purifying nucleic acid include phenol chloroform extraction, alkaline lysis, boiling, centrifugal column, and magnetic bead. The phenol-chloroform extraction method gradually exits the market due to the use of toxic organic solvents such as phenol and chloroform, low recovery rate of nucleic acid, complex operation and the like, and is also different in laboratory conditions and different in specific extraction and purification samples. Extraction methods such as boiling method and centrifugal column method are also used clinically to some extent, but the boiling method increases the risk of aerosol pollution because of the need of heating, and the centrifugal column method is difficult to realize automation because of the need of high-speed centrifuges and high cost. The magnetic bead method is a relatively common nucleic acid extraction method, and is generally considered to have the advantages of simplicity, convenience, high efficiency, high extraction concentration, high purity and the like for extracting nucleic acid of pathogens. In addition, different extraction methods can affect nucleic acid detection, especially in critical positive samples, to some extent due to differences in concentration and purity of the extracted product and different degrees of protection of DNA/RNA.
Compared with other methods, the magnetic bead method is simpler, more convenient, more efficient and higher in quality. The traditional nucleic acid extraction and purification kit (magnetic bead method) mainly comprises lysis solution, binding solution, washing solution I, washing solution II, eluent, nano magnetic beads and the like. The magnetic bead method nucleic acid extraction and purification technology utilizes a nanotechnology to improve and modify the surface of superparamagnetic nanoparticles to prepare superparamagnetic nano magnetic beads, takes the nano magnetic beads as a solid phase carrier, so that nucleic acid released by a sample can be specifically combined on the nano magnetic beads, realizes the separation and purification of the nucleic acid through the action of an external magnetic field, then washes and removes protein, polysaccharide and salt ions in a sample solution by using a washing solution, and separates the nucleic acid from the magnetic beads under the conditions of low salt and high pH, thereby obtaining a high-purity nucleic acid solution.
The samples such as imported marine products, beef and mutton, sewage, environment of public health places and the like provide new challenges and requirements for virus extraction due to the characteristics of low virus carrying capacity, more sample impurities, complex components and the like. At present, most of nucleic acid extraction kits (magnetic bead method) for throat swabs, blood, tissues and other samples are available in the market, and are not suitable for virus detection of environmental samples in the outbreak situation.
In the market, virus nucleic acid extraction kits aiming at environmental samples are fewer, most of the existing nucleic acid extraction kits are complex in extraction steps and cannot realize virus DNA/RNA co-extraction, and for newly mutated viruses, DNA viruses or RNA viruses cannot be predicted, and an inappropriate kit is selected for nucleic acid extraction, so that the probability and difficulty of detection failure can be increased, and the detection cost is increased.
The kit components also have added imported raw materials such as: proteinase K, Carrier RNA etc. in the face of proruption public health incident, imported raw and other materials not only purchase the degree of difficulty big, the cycle length, still greatly increased the manufacturing cost of kit. When an emergent public health event happens, the supply of the goods source cannot be guaranteed by imported raw materials, the cost is high, and the production period of the kit is greatly prolonged; if domestic raw materials are adopted for replacement, the performance of the kit cannot be ensured.
In addition, the transportation and storage conditions of the proteinase K and Carrier RNA are relatively harsh, and the proteinase K and Carrier RNA need to be stored at the temperature of-20 ℃, so that the products are not easy to store and transport. In order to achieve more sufficient sample lysis, both the conventional nucleic acid extraction and purification kit and the improved and optimized kit are often used for separately performing lysis and combination steps, so that the extraction and purification process is long in time consumption and complex in operation, and the detection cost is increased.
Therefore, it is very important to develop a nucleic acid extraction kit which is simple and convenient to operate, does not need to use imported raw materials, can extract DNA/RNA together, and is suitable for multiple types of virus samples.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a nucleic acid extraction kit for extracting a virus sample based on a magnetic bead method, and the invention solves the technical problems that toxic organic solvents are used, DNA and RNA are less in co-extraction, protease K, Carrier RNA and other imported raw materials are additionally added for extraction, cracking and combination are separated, operation steps are complicated and the like in the prior art, so that the invention is more suitable for extracting and purifying the environmental micro-scale virus nucleic acid sample, and has the advantages of DNA and RNA co-extraction, safety, no toxicity, rapidness, high efficiency, simple and convenient operation, effective pollution avoidance and low cost.
The first purpose of the invention is to provide a silicon hydroxyl magnetic bead composition.
The second purpose of the invention is to provide the application of the silicon hydroxyl magnetic bead composition in preparing a nucleic acid extraction and/or purification kit.
The third purpose of the invention is to provide a nucleic acid extraction kit for extracting a virus sample based on a magnetic bead method.
In order to achieve the purpose, the invention is realized by the following scheme:
the invention claims a silicon hydroxyl magnetic bead composition, which comprises 20-60% by mass of silicon hydroxyl magnetic beads with the diameter of 50-200 nm, 20-40% by mass of silicon hydroxyl magnetic beads with the diameter of 300-500 nm, and 10-40% by mass of silicon hydroxyl magnetic beads with the diameter of 500-900 nm.
Preferably, the magnetic material comprises 50% by mass of silicon hydroxyl magnetic beads with the diameter of 50-200 nm, 40% by mass of silicon hydroxyl magnetic beads with the diameter of 300-500 nm and 10% by mass of silicon hydroxyl magnetic beads with the diameter of 500-900 nm.
The invention also protects the application of the silicon hydroxyl magnetic bead composition in preparing a nucleic acid extraction and/or purification kit.
The nucleic acid extraction kit for extracting the virus sample based on the paramagnetic particle method comprises magnetic beads, lysis solution, washing solution I, washing solution II and eluent; wherein the magnetic beads are the silicon hydroxyl magnetic bead composition.
Preferably, the magnetic beads are the silicon hydroxyl magnetic bead composition with the concentration of 10-85 mg/ml.
More preferably, the magnetic beads are the silicon hydroxyl magnetic bead composition with the concentration of 25 mg/ml.
Preferably, the lysate contains: 2-8 mol/L of guanidinium isothiocyanate, 0.1-3 mol/L of sodium iodide, 0.01-0.2 mol/L of Tris-HCl, 1-20% (v/v) of nonionic surfactant, 0.1-10 mol/L of urea, 0.01-10 mg/ml of glycogen and 0.01-0.2 mol/L of metal ion complexing agent; the pH of the lysis solution is 4-7.
More preferably, the non-ionic surfactant is at least one of Tween20, Triton-X100, SDS, or NP 40.
More preferably, the lysate contains: 4mol/L of guanidinium isothiocyanate, 0.5mol/L of sodium iodide, 0.02mol/L of Tris-HCl, 1% (v/v) of nonionic surfactant, 0.5mol/L of urea, 2mg/ml of glycogen and 0.02mol/L of metal ion complexing agent; the lysate pH was 6.
Preferably, the washing solution I contains: 1-10% (v/v) of Tween20, 0.01-1 mol/L of sodium chloride, 0.01-0.3 mol/L of Tris-HCl, 0.2-5 mol/L of guanidine hydrochloride and 20-60% (v/v) of isopropanol.
More preferably, wash I contains: 3% (v/v) Tween20, 0.5mol/L sodium chloride, 2mol/L guanidine hydrochloride, 0.1mol/L Tris-HCl, 30% (v/v) isopropanol.
Preferably, the washing solution II is 60-80% (v/v) ethanol.
More preferably, Wash II is 80% (v/v) ethanol.
Preferably, the eluent is nuclease-free water or Tris-HCl-nuclease-free water with the concentration of 6-10 mol/L.
More preferably, the eluent is nuclease-free water.
Based on a large amount of experimental research and statistical analysis, the inventor finds that the effect of extracting the virus nucleic acid is better when the components of the kit are selected from the preferable values. The kit can effectively and quickly extract virus nucleic acid in environmental samples, blood plasma, blood serum, saliva, nasopharyngeal swabs and urine, and the technical scheme of scaling up/down on the basis of the kit belongs to the protection scope of the invention.
The invention further claims a method for extracting virus nucleic acid, a nucleic acid extraction kit using the nucleic acid extraction kit and/or the silicon hydroxyl magnetic bead composition.
Preferably, the method comprises the following steps:
s1, mixing a sample, a lysis solution and magnetic beads, fully and uniformly mixing, carrying out normal-temperature lysis for 10min, and uniformly mixing for 1 time every 30-60 s;
s2, centrifuging, carrying out magnetic separation, standing for 1-2 min, and discarding waste liquid;
s3, mixing the mixture with the washing liquid I, fully and uniformly mixing, centrifuging, carrying out magnetic separation, standing for 1-2 min, and discarding waste liquid;
s4, mixing with the washing liquid II, fully and uniformly mixing, centrifuging, carrying out magnetic separation, standing for 1-2 min, and discarding the waste liquid;
s5, repeating the step S4 twice;
s6, airing the magnetic beads under a magnetic separation condition;
s7, mixing with the eluent, fully and uniformly mixing, and incubating for 5min at 55 ℃.
S7, carrying out magnetic separation, standing for 1-2 min, and taking supernatant.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a nucleic acid extraction kit for extracting a virus sample based on a paramagnetic particle method, wherein the used magnetic beads are silicon hydroxyl magnetic beads and are formed by mixing a plurality of magnetic beads with different particle sizes, and the particle size is 10-1000 nm. The capability of capturing nucleic acid is strong, and the method is more suitable for extracting trace virus nucleic acid. The kit has easily obtained raw materials and low cost; the product is stored at normal temperature, and is convenient to transport and use; the extraction steps are simple, the cracking combination can be carried out only by adding the sample, the lysate and the magnetic beads, and imported raw materials such as protease K, Carrier RNA and the like do not need to be added. The virus DNA and RNA are extracted together, and the recovery rate is high; organic solvents such as phenol, chloroform and the like are not needed, so that the method is safe and has no toxic or side effect on operators; the operation is simple and convenient, centrifugation is not needed, and the requirements on instruments and equipment are not high; can match with a high-flux automatic extraction instrument; the method is suitable for extracting virus nucleic acid from environmental samples, such as micro virus samples of sewage, refrigerator, seafood meat, food inner and outer packages and the like.
Drawings
FIG. 1 is a comparison of the extraction results of a sample of a freezer environment containing the novel coronavirus-pseudovirus.
FIG. 2 is a comparison of the extraction results of samples of sewage environment containing the novel coronavirus-pseudovirus.
FIG. 3 shows the comparison of the extraction effect of serum hepatitis B virus.
FIG. 4 is a comparison of the extraction efficiency of different concentrations of pseudoviruses.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 nucleic acid extraction kit for extracting viral sample based on the paramagnetic particle method
A, make up
Magnetic beads: the magnetic material comprises 50% by mass of silicon hydroxyl magnetic beads with the diameter of 50-200 nm, 40% by mass of silicon hydroxyl magnetic beads with the diameter of 300-500 nm and 10% by mass of mixed magnetic beads consisting of silicon hydroxyl magnetic beads with the diameter of 500-900 nm, wherein the concentration of the magnetic beads is 85 mg/ml.
Lysis solution: 1mol/L guanidinium isothiocyanate, 0.5mol/L sodium iodide, 0.02mol/L Tris-HCl, 1% (v/v) nonionic surfactant Tween20, 0.5mol/L urea, 1mg/ml glycogen and 0.02mol/L metal ion complexing agent; the pH of the lysate is 5;
washing solution I: 1% (v/v) Tween20, 0.5mol/L sodium chloride, 1mol/L guanidine hydrochloride, 0.05mol/L Tris-HCl, 20% (v/v) isopropanol;
washing solution II: 70% (v/v) ethanol solution;
eluent: nuclease-free water.
Second, use method
1. And (3) putting 200 mu l of a sample to be detected into a 1.5ml centrifuge tube, adding 400 mu l of lysis solution, adding 20 mu l of magnetic beads which are uniformly mixed by oscillation, and uniformly mixing for 10s by oscillation or reversing the mixture up and down for a plurality of times. For each formulation, the extraction was repeated 3 times.
2. Cracking at normal temperature for 10min, and uniformly mixing for 1 time every 30-60 s.
3. And (3) performing instantaneous centrifugation on the centrifugal tube to ensure that no magnetic bead is left on the tube cover, then placing the centrifugal tube on a magnetic frame for magnetic separation, standing for 1-2 min, uncovering, and discarding the waste liquid (completely sucking the tube cover and the residual liquid at the bottom of the tube).
4. Adding 600 mu l of washing liquid I, shaking and uniformly mixing for 30-60 s, performing instantaneous centrifugation to ensure that no magnetic bead remains on a tube cover, then placing an EP tube on a magnetic frame for magnetic separation, standing for 1-2 min, uncovering, and discarding waste liquid (completely sucking the tube cover and the tube bottom residual liquid).
5. Adding 600 mu l of washing liquid II, shaking and uniformly mixing for 30-60 s, performing instantaneous centrifugation to ensure that no magnetic bead remains on the tube cover, then placing the centrifugal tube on a magnetic frame for magnetic separation, standing for 1-2 min, uncovering, and discarding waste liquid (completely sucking the tube cover and the tube bottom residual liquid). This step was repeated twice.
6. And placing the centrifugal tube on a magnetic frame, airing for 5-10 min, and properly prolonging or shortening airing time according to indoor temperature and humidity.
7. Adding 50 μ l of eluent, shaking and mixing for 1min or gently blowing and mixing with a pipette gun, and incubating at 55 deg.C for 5 min.
8. After the incubation is finished, placing the centrifugal tube on a magnetic frame for magnetic separation, standing for 1-2 min, sucking the supernatant, storing in a new centrifugal tube, and performing downstream experiments.
A, make up
Magnetic beads: the magnetic material comprises 50% by mass of silicon hydroxyl magnetic beads with the diameter of 50-200 nm, 40% by mass of silicon hydroxyl magnetic beads with the diameter of 300-500 nm and 10% by mass of mixed magnetic beads consisting of silicon hydroxyl magnetic beads with the diameter of 500-900 nm, wherein the concentration of the magnetic beads is 50 mg/ml.
Lysis solution: 6mol/L guanidinium isothiocyanate, 0.5mol/L sodium iodide, 0.02mol/L Tris-HCl, 1% (v/v) nonionic surfactant Tween20, 0.5mol/L urea, 3mg/ml glycogen and 0.2mol/L metal ion complexing agent; the pH of the lysate is 7;
washing solution I: 5% (v/v) Tween20, 0.5mol/L sodium chloride, 3mol/L guanidine hydrochloride, 0.2mol/L Tris-HCl, 50% (v/v) isopropanol;
washing solution II: 60% (v/v) ethanol solution;
eluent: nuclease-free water.
Second, use method
The same as in example 1.
Embodiment 3 nucleic acid extraction kit for extracting virus sample based on paramagnetic particle method
A, make up
Magnetic beads: the magnetic material comprises 50% by mass of silicon hydroxyl magnetic beads with the diameter of 50-200 nm, 40% by mass of silicon hydroxyl magnetic beads with the diameter of 300-500 nm and 10% by mass of mixed magnetic beads consisting of silicon hydroxyl magnetic beads with the diameter of 500-900 nm, wherein the concentration of the magnetic beads is 25 mg/ml.
Lysis solution: 4mol/L guanidinium isothiocyanate, 0.5mol/L sodium iodide, 0.02mol/L Tris-HCl, 1% (v/v) nonionic surfactant Tween20, 0.5mol/L urea, 2mg/ml glycogen and 0.02mol/L metal ion complexing agent; the pH of the lysate is 6;
washing solution I: 3% (v/v) Tween20, 0.5mol/L sodium chloride, 2mol/L guanidine hydrochloride, 0.1mol/L Tris-HCl, 30% (v/v) isopropanol;
washing solution II: 80% (v/v) ethanol solution;
eluent: nuclease-free water.
Second, use method
The same as in example 1.
EXAMPLE 4 extraction of nucleic acid from sample of Virus stock solution containing New coronavirus
First, experiment method
A virus stock solution containing the novel coronaviruses was extracted using the kit of examples 1 to 3.
Fluorescent quantitative detection is carried out by using a novel coronavirus nucleic acid detection kit (PCR-fluorescent probe method) of the dalan gene. The amplification reaction system and the reaction procedure are shown in tables 1 and 2, respectively.
Table 1: amplification reaction system
| Name of reagent | Volume of |
| PCR reaction solution A | 17μl |
| PCR reaction solution B | 3μl |
| RNA template | 5μl |
Table 2:
second, experimental results
As shown in Table 3, the kits of examples 1 to 3 all had excellent nucleic acid extraction effects on the virus stocks containing the novel coronaviruses. Wherein, when the kit formula of the embodiment 3 is used for extracting the new corona pseudovirus nucleic acid, the extraction effect is the best, and the formula is used for extraction in subsequent experiments as the optimal kit formula. The extraction effect is about 1 time better than example 2 and 3 times better than example 1 (1 CT value ≈ 3.33 times, the smaller CT value, the higher concentration).
Table 3:
example 5 Effect of composition of magnetic beads having different particle diameters on viral nucleic acid extraction
First, experiment method
Nucleic acid was extracted from a storage solution containing a new coronavirus by using a different silicon hydroxy magnetic bead composition (a silicon hydroxy composition was prepared by mixing magnetic beads having different particle sizes) instead of the magnetic beads in the kit of example 3, and then quantitative fluorescence detection was performed by using a novel coronavirus nucleic acid detection kit (PCR-fluorescent probe method) containing the daan gene.
Silicon hydroxyl magnetic bead composition 1: the magnetic material comprises 50% of silicon hydroxyl magnetic beads with the diameter of 50-200 nm, 40% of silicon hydroxyl magnetic beads with the diameter of 300-500 nm and 10% of silicon hydroxyl magnetic beads with the diameter of 500-900 nm, wherein the concentration of the silicon hydroxyl magnetic bead composition is 25 mg/ml.
Silicon hydroxyl magnetic bead composition 2: the magnetic material comprises 40% of silicon hydroxyl magnetic beads with the diameter of 50-200 nm, 30% of silicon hydroxyl magnetic beads with the diameter of 300-500 nm and 30% of silicon hydroxyl magnetic beads with the diameter of 500-900 nm, and the concentration of the silicon hydroxyl magnetic bead composition is 25 mg/ml.
Silicon hydroxyl magnetic bead composition 3: the magnetic material comprises 30% of silicon hydroxyl magnetic beads with the diameter of 50-200 nm, 30% of silicon hydroxyl magnetic beads with the diameter of 300-500 nm and 40% of silicon hydroxyl magnetic beads with the diameter of 500-900 nm, wherein the concentration of the silicon hydroxyl magnetic bead composition is 25 mg/ml.
Second, experimental results
As a result, as shown in Table 4, different magnetic bead compositions showed excellent nucleic acid extraction effects for the virus preservation solutions containing the novel coronavirus. When the silicon hydroxyl magnetic bead composition 1 is used for extracting the new corona pseudovirus nucleic acid, the extraction effect is the best, the optimal magnetic bead combination is obtained, and the combination is used for extraction in subsequent experiments. The extraction effect is about 2 times better than that of the silicon hydroxyl magnetic bead composition 3 and 3 times better than that of the silicon hydroxyl magnetic bead composition 2 (1 CT value is approximately equal to 3.33 times, and the smaller the CT value is, the higher the concentration is).
TABLE 4
Example 6 Effect of magnetic beads of different particle sizes on viral nucleic acid extraction
First, experiment method
Three commercially available silicon hydroxyl magnetic beads (A, B, C) with a single particle size were selected instead of the silicon hydroxyl magnetic bead composition in the kit of example 3, and the concentration of the silicon hydroxyl magnetic bead composition was 25mg/ml of that in example 3, and the method of example 1 was used to extract nucleic acid from the storage solution containing the new coronaviruses. Then, a novel coronavirus nucleic acid detection kit (PCR-fluorescent probe method) of the Daan gene is used for carrying out fluorescent quantitative detection.
Second, experimental results
The results of the experiment are shown in Table 5.
Table 5:
the effect of the kit in the embodiment 3 is better than that of the commercially available magnetic bead A, magnetic bead B and magnetic bead C, and the extraction effect is about 3 times better than that of the magnetic bead B, 4 times better than that of the magnetic bead A and 5 times better than that of the magnetic bead A (1 CT value is about 3.33 times, the smaller the CT value is, the higher the concentration is).
Example 7 detection of extraction of a novel coronavirus-pseudovirus in a freezer Environment sample
First, experiment method
Soaking cotton swab in sampling tube in virus preservation solution, randomly taking 5 points on the inner wall of the freezer with an area of 5cm × 5cm as a sampling point, drawing well on the sampling point, returning the swab to the virus preservation solution after sampling, adding a certain amount of new coronavirus, shaking and mixing uniformly to obtain 1 × 104copies/ml mock virus containing freezer environment mock sample.
The above freezer environment simulation samples were extracted using the kit of example 3 and the beaver bio virus DNA/RNA extraction kit, respectively: the extraction method using the kit of example 3 was as in example 1; the virus DNA/RNA extraction kit of beaver biological company is used for extraction, the sample volume (same as the sample used in the experimental group) is 200 mul, the extraction is carried out according to the kit instruction, and the elution volume is 50 mul.
After the extraction is completed, fluorescent quantitative detection is performed by using a novel coronavirus nucleic acid detection kit (PCR-fluorescent probe method) of the dalan gene.
Second, experimental results
The results of the experiment are shown in table 6 and fig. 1.
Table 6:
according to experimental results, the effect of extracting the new corona pseudovirus nucleic acid from the freezer environment simulation sample of the reagent is better than that of the contrast reagent, and the extraction effect is about 3 times of that of the contrast reagent (1 CT value is approximately equal to 3.33 times, and the smaller the CT value is, the higher the concentration is).
Example 8 extraction and detection of novel coronavirus-pseudovirus in a sample of a Sewage Environment
First, experiment method
Adding a certain amount of new coronaviruses into a sewage sample sampled from a sewer of a vegetable market, shaking and uniformly mixing to prepare 1 multiplied by 104copies/ml sewage environment simulation sample containing pseudovirus.
The above sewage environment simulation samples were extracted using the kit of example 3 and the beaver bio-inc virus DNA/RNA extraction kit, respectively: the extraction method using the kit of example 3 was as in example 1; the virus DNA/RNA extraction kit of beaver biological company is used for extraction, the sample volume (same as the sample used in the experimental group) is 200 mul, the extraction is carried out according to the kit instruction, and the elution volume is 50 mul.
After the extraction is completed, fluorescent quantitative detection is performed by using a novel coronavirus nucleic acid detection kit (PCR-fluorescent probe method) of the dalan gene.
Second, experimental results
The results of the experiment are shown in table 7 and fig. 2.
Table 7:
according to experimental results, the effect of the reagent for extracting the new coronaviruses from the sewage environment simulation sample is superior to that of the contrast reagent, and the extraction effect is about 5 times of that of the contrast reagent (1 CT value is approximately equal to 3.33 times, and the smaller the CT value is, the higher the concentration is).
EXAMPLE 9 detection of hepatitis B Virus extraction from serum
First, experiment method
Serum containing hepatitis B virus (200. mu.l) was extracted using the kit of example 3 and the beaver bio-corporation virus DNA/RNA extraction kit, respectively: the extraction method using the kit of example 3 was as in example 1; the virus DNA/RNA extraction kit of beaver biological company is used for extraction, the sample volume (same as the sample used in the experimental group) is 200 mul, the extraction is carried out according to the kit instruction, and the elution volume is 50 mul.
After the extraction is completed, fluorescent quantitative detection is performed by using a novel coronavirus nucleic acid detection kit (PCR-fluorescent probe method) of the dalan gene.
Second, experimental results
The results of the experiment are shown in table 8 and fig. 3.
Table 8:
the experimental result shows that the effect of extracting serum hepatitis B virus nucleic acid of the reagent is better than that of the contrast reagent, and the extraction effect is about 5 times of that of the contrast reagent (1 CT value is approximately equal to 3.33 times, and the smaller the CT value is, the higher the concentration is).
Example 10 detection Limit for nucleic acid extraction
First, experiment method
Diluting the new coronavirus-pseudovirus standard (purchased from Jinwei Zhi) to 5.0 × 104copies/ml、 5.0×103copies/ml、5.0×102copies/ml、5.0×101copies/ml。
The above samples (200. mu.l) were extracted using the kit of example 3 and the beaver bio-Inc's viral DNA/RNA extraction kit, respectively: the extraction method using the kit of example 3 was as in example 1; the virus DNA/RNA extraction kit of beaver biological company is used for extraction, the sample volume (same as the sample used in the experimental group) is 200 mul, the extraction is carried out according to the kit instruction, and the elution volume is 50 mul.
After the extraction is completed, fluorescent quantitative detection is performed by using a novel coronavirus nucleic acid detection kit (PCR-fluorescent probe method) of the dalan gene.
Second, experimental results
The results of the experiment are shown in table 9 and fig. 4.
Table 9:
according to experimental results, the reagent has better effect of extracting the new corona pseudoviruses with different concentrations than the control reagent, and the extraction effect is 5-6 times better than that of the control reagent (1 CT value is approximately equal to 3.33 times, and the smaller the CT value is, the higher the concentration is). The detection is carried out by a novel coronavirus nucleic acid detection kit (PCR-fluorescent probe method) matched with the gene of the dapan, and the minimum detection limit can reach 10copies (5 multiplied by 10)1copies/ml of new coronaviruses, and the addition amount is 200 mu l), so the method is more suitable for nucleic acid extraction of micro-virus samples.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. A silicon hydroxyl magnetic bead composition is characterized by comprising 20-60% by mass of silicon hydroxyl magnetic beads with the diameter of 50-200 nm, 20-40% by mass of silicon hydroxyl magnetic beads with the diameter of 300-500 nm, and 10-40% by mass of silicon hydroxyl magnetic beads with the diameter of 500-900 nm.
2. The composition of claim 1, wherein the composition comprises 50% by mass of the silicon hydroxyl magnetic beads having a diameter of 50 to 200nm, 40% by mass of the silicon hydroxyl magnetic beads having a diameter of 300 to 500nm, and 10% by mass of the silicon hydroxyl magnetic beads having a diameter of 500 to 900 nm.
3. Use of the silicon hydroxy magnetic bead composition of claim 1 or 2 in the preparation of a nucleic acid extraction and/or purification kit.
4. A nucleic acid extraction kit for extracting a virus sample based on a paramagnetic particle method is characterized by comprising magnetic beads, lysis solution, washing solution I, washing solution II and eluent; wherein the magnetic beads are the silicon hydroxyl magnetic bead composition of claim 1.
5. The nucleic acid extraction kit according to claim 4, characterized in that: the concentration of the magnetic beads is 10-85 mg/ml.
6. The nucleic acid extraction kit according to claim 4, wherein the lysate contains: 2-8 mol/L of guanidinium isothiocyanate, 0.1-3 mol/L of sodium iodide, 0.01-0.2 mol/L of Tris-HCl, 1-20% (v/v) of nonionic surfactant, 0.1-10 mol/L of urea, 0.01-10 mg/ml of glycogen and 0.01-0.2 mol/L of metal ion complexing agent; the pH of the lysis solution is 4-7.
7. The nucleic acid extraction kit according to claim 4, wherein the washing solution I contains: 1-10% (v/v) Tween20, 0.01-1 mol/L sodium chloride, 0.01-0.3 mol/L Tris-HCl, 0.2-5 mol/L guanidine hydrochloride, and 20-60% (v/v) isopropanol.
8. The nucleic acid extraction kit according to claim 4, wherein the washing solution II is 60-80% (v/v) ethanol.
9. The nucleic acid extraction kit of claim 4, wherein the eluent is nuclease-free water or 6-10 mol/L Tris-HCl-nuclease-free water.
10. A method for extracting viral nucleic acid, comprising using the nucleic acid extraction kit of claim 3 and/or the silicon hydroxy magnetic bead composition of claim 1 or 2.
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