CN113151397B - Nucleic acid extraction kit for extracting virus sample based on magnetic bead method - Google Patents
Nucleic acid extraction kit for extracting virus sample based on magnetic bead method Download PDFInfo
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Abstract
The invention discloses a nucleic acid extraction kit for extracting virus samples based on a magnetic bead method, wherein the magnetic beads are silicon hydroxyl magnetic beads, and are formed by mixing a plurality of magnetic beads with different particle diameters, and the particle diameter is 10-1000 nm. The capability of capturing nucleic acid is strong, and the method is more suitable for extracting the trace virus nucleic acid. The raw materials of the kit are easy to obtain, and the cost is low; the storage at normal temperature is convenient for transportation and use; the extraction step is simple, the cleavage and combination can be carried out by only adding a sample, a cleavage solution and magnetic beads, and no imported raw materials such as protease K, carrierRNA and the like are needed to be added. The virus DNA and RNA are extracted together, and the recovery rate is high; no organic solvents such as phenol, chloroform and the like are needed, so that the method is safe and has no toxic or side effect on operators; the operation is simple and convenient, the centrifugation is not needed, and the requirement on instruments and equipment is not high; can be matched with a high-flux automatic extractor; is suitable for extracting virus nucleic acid from environment samples such as sewage, ice chest, seafood meat, internal and external packaging of food and other trace virus samples.
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 magnetic bead method.
Background
Nucleic acids are vectors of genetic information, which are the material basis for gene expression. Nucleic acids, including both DNA and RNA, are widely found in all animal and plant cells and microorganisms, and nucleic acids in organisms are often present in a state of binding to proteins. The structural change of nucleic acid molecule can lead to biological function change, such as virus pathogenic effect, through invasion of virus nucleic acid into body cell, and in addition, malignant tumor, genetic lesion, metabolic disease, etc. are closely related to the change of nucleic acid molecular structure and function, so that it is necessary to separate and purify nucleic acid first for research on nucleic acid structure and function.
Common nucleic acid extraction and purification methods include phenol chloroform extraction, alkaline lysis, boiling, centrifugation, and magnetic bead. The phenol chloroform extraction method is gradually out of the market due to the fact that the phenol, chloroform and other toxic organic solvents are used, the recovery rate of nucleic acid is low, the operation is complex and other factors, and the laboratory conditions and the specific extraction and purification samples are different. The extraction methods such as the boiling method and the centrifugal column method have certain clinical application, but the boiling method increases the risk of aerosol pollution because of the need of heating, and the centrifugal column method has high cost because of the need of being provided with a high-speed centrifuge, and is difficult to realize automation. The magnetic bead method is a more common nucleic acid extraction method, and is widely considered to have the advantages of simplicity, high efficiency, high extraction concentration, high purity and the like for extracting the nucleic acid of pathogens. In addition, different extraction methods will affect the detection of nucleic acids, especially of critical positive samples, to some extent due to differences in the concentration and purity of the extracted products and the degree of protection of the DNA/RNA.
Compared with other methods, the magnetic bead method has the advantages of simpler operation, high efficiency and high quality. The traditional nucleic acid extraction and purification kit (magnetic bead method) mainly comprises a lysate, a binding solution, a washing solution I, a washing solution II, an eluent, nano magnetic beads and the like. The magnetic bead method nucleic acid extraction and purification technology utilizes a nano technology to modify and surface modify the surface of superparamagnetism nano particles to prepare superparamagnetism nano magnetic beads, uses the nano magnetic beads as a solid phase carrier, enables nucleic acid released by a sample to be specifically combined on the nano magnetic beads, realizes separation and purification of the nucleic acid through the action of an externally applied 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 of imported marine products, beef and mutton, sewage, the environment of public sanitary places and the like provide new challenges and requirements for virus extraction due to the characteristics of low virus load, more sample impurities, complex components and the like. At present, most of nucleic acid extraction kits (magnetic bead method) for samples such as throat swabs, blood, tissues and the like are not suitable for virus detection of environmental samples in the sudden epidemic situation.
The existing majority of nucleic acid extraction kits have complex extraction steps, cannot realize virus DNA/RNA co-extraction, cannot predict whether the virus is DNA virus or RNA virus for new mutation, and can increase the probability and difficulty of detection failure and increase the detection cost by selecting an unsuitable kit for nucleic acid extraction.
The kit components also add inlet raw materials such as: proteinase K, carrier RNA, etc., in the face of sudden public health incident, the imported raw materials are not only purchased with great difficulty and long period, but also greatly increase the production cost of the kit. When burst public health event burst occurs, the imported raw materials cannot guarantee the supply of a cargo source, 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 preservation conditions of proteinase K and Carrier RNA are relatively harsh, and the proteinase K and Carrier RNA are required to be preserved at the temperature of-20 ℃, so that the proteinase K and Carrier RNA are not easy to preserve and transport. Whether a traditional nucleic acid extraction and purification kit or an improved and optimized kit, in order to enable a sample to be more fully cracked, the cracking and combining steps are often separated, so that the extraction and purification process is long in time consumption, complex in operation and high in detection cost.
Thus, it is important to develop a nucleic acid extraction kit that is easy to handle, does not require the use of imported raw materials, and allows the co-extraction of DNA/RNA, suitable for use in a plurality of types of virus samples.
Disclosure of Invention
Aiming at overcoming the defects of the prior art, the invention provides a nucleic acid extraction kit for extracting virus samples based on a magnetic bead method, which solves the technical problems that toxic organic solvents are used, DNA and RNA are less co-extracted, proteinase K, carrier RNA and other imported raw materials are required to be additionally added for extraction, the steps of splitting and combining are separated, the operation steps are complicated and the like in the prior art, so that the kit is more suitable for extracting and purifying environment-based trace virus nucleic acid samples, and has the advantages of DNA and RNA co-extraction, safety, no toxicity, rapidness, high efficiency, simplicity and convenience in operation, effective pollution avoidance and low cost.
It is a first object of the present invention to provide a silica-hydroxy magnetic bead composition.
The second object 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 object of the invention is to provide a nucleic acid extraction kit for extracting virus samples based on a magnetic bead method.
In order to achieve the above object, the present invention is realized by the following means:
the invention claims a silicon hydroxyl magnetic bead composition, which comprises 20-60% of silicon hydroxyl magnetic beads with the diameter of 50-200 nm, 20-40% of silicon hydroxyl magnetic beads with the diameter of 300-500 nm and 10-40% of silicon hydroxyl magnetic beads with the diameter of 500-900 nm by mass.
Preferably, the silica hydroxyl magnetic beads with the diameters of 50-200 nm, 40% with the diameters of 300-500 nm and 10% with the diameters of 500-900 nm are contained in the silica hydroxyl magnetic beads by mass.
The invention also provides 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 magnetic bead method comprises magnetic beads, a lysate, a washing solution I, a washing solution II and an 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 25mg/ml.
Preferably, the lysate contains: 2 to 8mol/L guanidine isothiocyanate, 0.1 to 3mol/L sodium iodide, 0.01 to 0.2mol/L Tris-HCl, 1 to 20% (v/v) nonionic surfactant, 0.1 to 10mol/L urea, 0.01 to 10mg/ml glycogen, 0.01 to 0.2mol/L metal ion complexing agent; the pH value of the lysate is 4-7.
More preferably, the nonionic surfactant is at least one of Tween20, triton-X100, SDS, or NP 40.
More preferably, the lysate contains: 4mol/L guanidine isothiocyanate, 0.5mol/L sodium iodide, 0.02mol/L Tris-HCl, 1% (v/v) nonionic surfactant, 0.5mol/L urea, 2mg/ml glycogen, 0.02mol/L metal ion complexing agent; the lysate pH was 6.
Preferably, the washing liquid I contains: 1 to 10 percent (v/v) of Tween20, 0.01 to 1mol/L of sodium chloride, 0.01 to 0.3mol/L of Tris-HCl, 0.2 to 5mol/L of guanidine hydrochloride and 20 to 60 percent (v/v) of isopropanol.
More preferably, the washing liquid 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 liquid II is 60-80% (v/v) ethanol.
More preferably, the wash solution II is 80% (v/v) ethanol.
Preferably, the eluent is nuclease-free water or 6-10 mol/L Tris-HCl-nuclease-free water.
More preferably, the eluent is nuclease-free water.
The inventor finds that the effect of extracting the virus nucleic acid is better when the components of the kit are selected from the above preferred values on the basis of a large number of experimental researches and statistical analyses. The technical scheme of increasing/decreasing the ratio based on the kit of the invention belongs to the protection scope of the invention.
Further claimed is a method of viral nucleic acid extraction using said nucleic acid extraction kit, and/or said silica-hydroxymagnetic bead composition.
Preferably, the method comprises the following steps:
s1, mixing a sample, a pyrolysis liquid and magnetic beads, fully and uniformly mixing, performing pyrolysis at normal temperature for 10min, and uniformly mixing for 1 time every 30-60 s;
s2, centrifuging, magnetically separating, standing for 1-2 min, and discarding waste liquid;
s3, mixing the waste liquid with the washing liquid I, fully and uniformly mixing, centrifuging, magnetically separating, standing for 1-2 min, and discarding the waste liquid;
s4, mixing the waste liquid with the washing liquid II, fully and uniformly mixing, centrifuging, magnetically separating, standing for 1-2 min, and discarding the waste liquid;
s5, repeating the step S4 for two times;
s6, airing the magnetic beads under the magnetic separation condition;
s7, mixing the mixture with the eluent, fully and uniformly mixing, and incubating at 55 ℃ for 5min.
S7, magnetically separating, 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 virus samples based on a magnetic bead method, wherein the magnetic beads are silicon hydroxyl magnetic beads, and are formed by mixing a plurality of magnetic beads with different particle diameters, and the particle diameter is 10-1000 nm. The capability of capturing nucleic acid is strong, and the method is more suitable for extracting the trace virus nucleic acid. The raw materials of the kit are easy to obtain, and the cost is low; the storage at normal temperature is convenient for transportation and use; the extraction step is simple, the cleavage and combination can be carried out by only adding a sample, a cleavage solution and magnetic beads, and the addition of imported raw materials such as proteinase K, carrier RNA and the like is not required. The virus DNA and RNA are extracted together, and the recovery rate is high; no organic solvents such as phenol, chloroform and the like are needed, so that the method is safe and has no toxic or side effect on operators; the operation is simple and convenient, the centrifugation is not needed, and the requirement on instruments and equipment is not high; can be matched with a high-flux automatic extractor; is suitable for extracting virus nucleic acid from environment samples such as sewage, ice chest, seafood meat, internal and external packaging of food and other trace virus samples.
Drawings
FIG. 1 is a comparison of the extraction effect of a sample ice chest environment containing a novel coronavirus-pseudovirus.
FIG. 2 is a comparison of the extraction effect of a sample of sewage environment containing novel coronavirus-pseudovirus.
FIG. 3 shows comparison of the extraction effect of serum hepatitis B virus.
FIG. 4 is a comparison of the effect of pseudovirus extraction at different concentrations.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1A nucleic acid extraction kit for extracting a viral sample based on the magnetic bead method
1. Composition of the composition
Magnetic beads: the magnetic beads comprise 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 mixed magnetic beads with the diameter of 500-900 nm by mass, wherein the concentration of the magnetic beads is 85mg/ml.
Lysate: 1mol/L guanidine isothiocyanate, 0.5mol/L sodium iodide, 0.02mol/L Tris-HCl, 1% (v/v) nonionic surfactant Tween20, 0.5mol/L urea, 1mg/ml glycogen, 0.02mol/L metal ion complexing agent; the pH value of the lysate is 5;
washing liquid I:1% (v/v) Tween20, 0.5mol/L sodium chloride, 1mol/L guanidine hydrochloride, 0.05mol/L Tris-HCl, 20% (v/v) isopropanol;
washing liquid II:70% (v/v) ethanol solution;
eluent: nuclease-free water.
2. Application method
1. 200 μl of the sample to be tested is taken into a 1.5ml centrifuge tube, 400 μl of the lysate is added, 20 μl of magnetic beads mixed by shaking are added, and the mixture is mixed by shaking for 10s or inverted for several times. The extraction was repeated 3 times for each formulation.
2. Cracking for 10min at normal temperature, and uniformly mixing for 1 time every 30-60 s.
3. And (3) instantly centrifuging the centrifuge tube, ensuring that no magnetic beads remain on the tube cover, then placing the centrifuge tube on a magnetic rack for magnetic separation, standing for 1-2 min, uncapping, and discarding waste liquid (sucking the tube cover and the residual liquid at the bottom of the tube).
4. Adding 600 μl of washing solution I, shaking, mixing for 30-60 s, centrifuging instantly to ensure no magnetic bead residue on the tube cover, then placing the EP tube on a magnetic rack for magnetic separation, standing for 1-2 min, uncapping, and discarding the waste liquid (sucking the tube cover and the residual liquid at the bottom).
5. Adding 600 μl of washing solution II, shaking, mixing for 30-60 s, centrifuging instantly to ensure no magnetic bead residue on the tube cover, then placing the centrifuge tube on a magnetic rack for magnetic separation, standing for 1-2 min, uncapping, and discarding the waste liquid (sucking the tube cover and the residual liquid at the bottom of the tube). This procedure was repeated twice.
6. And placing the centrifuge tube on a magnetic rack, airing for 5-10 min, and properly prolonging or shortening the airing time according to the indoor temperature and humidity.
7. Add 50. Mu.l of eluent, shake mix for 1min or gently blow mix with a pipette and incubate for 5min at 55deg.C.
8. After the incubation is finished, the centrifuge tube is placed on a magnetic rack for magnetic separation, and is kept stand for 1-2 min, and the supernatant is sucked and stored in a new centrifuge tube for downstream experiments.
Example 2A nucleic acid extraction kit for extracting a viral sample based on the magnetic bead method
1. Composition of the composition
Magnetic beads: the magnetic beads comprise 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 mixed magnetic beads with the diameter of 500-900 nm by mass, wherein the concentration of the magnetic beads is 50mg/ml.
Lysate: 6mol/L guanidine isothiocyanate, 0.5mol/L sodium iodide, 0.02mol/L Tris-HCl, 1% (v/v) nonionic surfactant Tween20, 0.5mol/L urea, 3mg/ml glycogen, 0.2mol/L metal ion complexing agent; the pH of the lysate is 7;
washing liquid I:5% (v/v) Tween20, 0.5mol/L sodium chloride, 3mol/L guanidine hydrochloride, 0.2mol/L Tris-HCl, 50% (v/v) isopropanol;
washing liquid II:60% (v/v) ethanol solution;
eluent: nuclease-free water.
2. Application method
As in example 1.
Example 3 nucleic acid extraction kit for extracting virus sample based on magnetic bead method
1. Composition of the composition
Magnetic beads: the magnetic beads comprise 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 mixed magnetic beads with the diameter of 500-900 nm by mass, wherein the concentration of the magnetic beads is 25mg/ml.
Lysate: 4mol/L guanidine isothiocyanate, 0.5mol/L sodium iodide, 0.02mol/L Tris-HCl, 1% (v/v) nonionic surfactant Tween20, 0.5mol/L urea, 2mg/ml glycogen, 0.02mol/L metal ion complexing agent; the pH value of the lysate is 6;
washing liquid I:3% (v/v) Tween20, 0.5mol/L sodium chloride, 2mol/L guanidine hydrochloride, 0.1mol/L Tris-HCl, 30% (v/v) isopropanol;
washing liquid II:80% (v/v) ethanol solution;
eluent: nuclease-free water.
2. Application method
As in example 1.
EXAMPLE 4 extraction of sample nucleic acid from Virus stock containing novel coronavirus
1. Experimental method
The kit of examples 1 to 3 was used to extract a virus stock solution containing a novel coronavirus.
Fluorescent quantitative detection is carried out by using a novel coronavirus nucleic acid detection kit (PCR-fluorescent probe method) of the da' an gene. The amplification reaction system and the reaction procedure are shown in tables 1 and 2, respectively.
Table 1: amplification reaction system
| Reagent name | Volume of |
| PCR reaction solution A | 17μl |
| PCR reaction solution B | 3μl |
| RNA template | 5μl |
Table 2:
2. experimental results
As shown in Table 3, the kits of examples 1 to 3 all have excellent nucleic acid extraction effects on the virus preservation solution containing the novel coronavirus. Wherein, when the kit formula of the embodiment 3 is used for extracting the novel coronavirus nucleic acid, the extraction effect is the best, and the kit formula is the best, and the subsequent experiments are carried out by using the formula for extraction. The extraction effect was about 1-fold better than example 2 and 3-fold better than example 1 (1 CT value. Apprxeq. 3.33-fold, smaller CT value, higher concentration).
Table 3:
EXAMPLE 5 Effect of the composition of magnetic beads of different particle size on the viral nucleic acid extraction Effect
1. Experimental method
The magnetic beads in the kit of example 3 were replaced with different silica-hydroxyl magnetic bead compositions (silica-hydroxyl compositions were mixed with magnetic beads of different particle sizes), nucleic acid extraction was performed on the preservation solution containing the novel coronavirus, and then fluorescence quantitative detection was performed using the novel coronavirus nucleic acid detection kit of the da' an gene (PCR-fluorescent probe method).
Silica hydroxyl magnetic bead composition 1: contains 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, 10% of silicon hydroxyl magnetic beads with the diameter of 500-900 nm, and the concentration of the silicon hydroxyl magnetic bead composition is 25mg/ml.
Silica hydroxyl magnetic bead composition 2: contains 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, 30% of silicon hydroxyl magnetic beads with the diameter of 500-900 nm, and the concentration of the silicon hydroxyl magnetic bead composition is 25mg/ml.
Silica hydroxyl magnetic bead composition 3: contains 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, 40% of silicon hydroxyl magnetic beads with the diameter of 500-900 nm, and the concentration of the silicon hydroxyl magnetic bead composition is 25mg/ml.
2. Experimental results
As shown in Table 4, the different magnetic bead compositions have excellent nucleic acid extraction effects on the virus preservation solution containing the novel coronavirus. Wherein, when the silicon hydroxyl magnetic bead composition 1 is used for extracting the novel coronavirus nucleic acid, the extraction effect is the best, and the combination is used for extraction in the subsequent experiments. The extraction effect was about 2 times better than that of the dihydroxy magnetic bead composition 3 and 3 times better than that of the dihydroxy magnetic bead composition 2 (1 CT value: 3.33 times, the smaller the CT value, the higher the concentration).
TABLE 4 Table 4
EXAMPLE 6 Effect of magnetic beads of different particle size on viral nucleic acid extraction Effect
1. Experimental method
Three commercially available silica hydroxyl magnetic beads (A, B, C) with conventional single particle size were used to replace the silica hydroxyl magnetic bead composition in the kit of example 3, the concentration of each silica hydroxyl magnetic bead composition was 25mg/ml of that of example 3, and the preservation solution containing the novel coronavirus was subjected to nucleic acid extraction in the same manner as in example 1. Then, the novel coronavirus nucleic acid detection kit (PCR-fluorescent probe method) of the dalan gene is used for fluorescence quantitative detection.
2. Experimental results
The experimental results are shown in table 5.
Table 5:
the effect of using the kit of example 3 was better than that of commercially available magnetic beads A, B and C, and the extraction effect was about 3 times better than that of magnetic bead B, 4 times better than that of magnetic bead A and 5 times better than that of magnetic bead A (1 CT value approximately equal to 3.33 times, the smaller CT value, the higher the concentration).
Example 7 extraction detection of novel coronavirus-pseudovirus in a sample of an ice chest Environment
1. Experimental method
Soaking cotton swab in sample tube in virus preserving liquid, taking 5cm×5cm area as sampling point on inner wall of refrigerator, randomly taking 5 points, drawing well-shaped on sampling point, placing swab back into virus preserving liquid after sampling, adding a certain amount of new coronavirus, shaking, and mixing to obtain 1×10 4 The copies/ml ice chest environmental simulation sample containing pseudoviruses.
The above ice chest environmental simulation samples were extracted using the kit of example 3 and the virus DNA/RNA extraction kit of castoreum, respectively: the extraction method using the kit of example 3 was as in example 1; the virus DNA/RNA extraction kit from the beaver organism company was used to extract 200. Mu.l of sample size (consistent with the samples used in the experimental group) and the extraction was performed according to the kit instructions, with an elution volume of 50. Mu.l.
After the extraction, fluorescence quantitative detection is carried out by using a novel coronavirus nucleic acid detection kit (PCR-fluorescence probe method) of the da' an gene.
2. Experimental results
The experimental results are shown in table 6 and fig. 1.
Table 6:
as shown by experimental results, the effect of the reagent for extracting the novel coronavirus nucleic acid from the ice chest environmental simulation sample is better than that of a control reagent, the extraction effect is about 3 times that of the control reagent (1 CT value is about 3.33 times, and the smaller the CT value is, the higher the concentration is).
Example 8 detection of novel coronavirus-pseudovirus extraction in wastewater Environment samples
1. Experimental method
Adding a certain amount of new coronavirus into the sewage sample sampled from the sewer of the kitchen market, vibrating and mixing uniformly to obtain 1×10 4 copies/ml sewage environment simulation sample containing pseudoviruses.
The above sewage environmental simulation samples were extracted using the kit of example 3 and the virus DNA/RNA extraction kit of castup bio company, respectively: the extraction method using the kit of example 3 was as in example 1; the virus DNA/RNA extraction kit from the beaver organism company was used to extract 200. Mu.l of sample size (consistent with the samples used in the experimental group) and the extraction was performed according to the kit instructions, with an elution volume of 50. Mu.l.
After the extraction, fluorescence quantitative detection is carried out by using a novel coronavirus nucleic acid detection kit (PCR-fluorescence probe method) of the da' an gene.
2. Experimental results
The experimental results are shown in table 7 and fig. 2.
Table 7:
the experimental result shows that the effect of the reagent for extracting the novel coronavirus nucleic acid from the sewage environment simulation sample is better than that of a control reagent, the extraction effect is about 5 times that of the control reagent (1 CT value is about 3.33 times, and the smaller the CT value is, the higher the concentration is).
Example 9 detection of hepatitis B Virus in serum
1. Experimental method
Serum containing hepatitis B virus (200. Mu.l) was extracted using the kit of example 3 and the virus DNA/RNA extraction kit of beaver organism company, respectively: the extraction method using the kit of example 3 was as in example 1; the virus DNA/RNA extraction kit from the beaver organism company was used to extract 200. Mu.l of sample size (consistent with the samples used in the experimental group) and the extraction was performed according to the kit instructions, with an elution volume of 50. Mu.l.
After the extraction, fluorescence quantitative detection is carried out by using a novel coronavirus nucleic acid detection kit (PCR-fluorescence probe method) of the da' an gene.
2. Experimental results
The experimental results are shown in table 8 and fig. 3.
Table 8:
as shown by experimental results, the serum hepatitis B virus nucleic acid extraction effect of the reagent is better than that of a control reagent, and the extraction effect is about 5 times that of the control reagent (1 CT value is about 3.33 times, and the smaller the CT value is, the higher the concentration is).
Example 10 detection limit of nucleic acid extraction
1. Experimental method
The novel coronavirus-pseudovirus standard (from Jin Weizhi) was diluted to 5.0X10 with virus stock 4 copies/ml、5.0×10 3 copies/ml、5.0×10 2 copies/ml、5.0×10 1 copies/ml。
The above samples (200 μl) were extracted using the kit of example 3 and the virus DNA/RNA extraction kit of castanopsis grossedentata, respectively: the extraction method using the kit of example 3 was as in example 1; the virus DNA/RNA extraction kit from the beaver organism company was used to extract 200. Mu.l of sample size (consistent with the samples used in the experimental group) and the extraction was performed according to the kit instructions, with an elution volume of 50. Mu.l.
After the extraction, fluorescence quantitative detection is carried out by using a novel coronavirus nucleic acid detection kit (PCR-fluorescence probe method) of the da' an gene.
2. Experimental results
The experimental results are shown in table 9 and fig. 4.
Table 9:
as shown by experimental results, the inventive reagent has better effect of extracting different concentrations of novel coronavirus than that of the control reagentThe control reagent was 5-6 times better (1 CT value. Apprxeq. 3.33 times, smaller CT value, higher concentration). Novel coronavirus nucleic acid detection kit (PCR-fluorescent probe method) matched with da' an gene is used for detection, and the minimum detection limit can reach 10copies (5 multiplied by 10) 1 The addition of copies/ml of the novel coronavirus is 200. Mu.l), so that the method is more suitable for nucleic acid extraction of a trace virus sample.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (3)
1. A nucleic acid extraction kit for extracting a virus sample based on a magnetic bead method is characterized by comprising magnetic beads, a lysate, a washing solution I, a washing solution II and an eluent; the magnetic beads are a silicon hydroxyl magnetic bead composition with the concentration of 25mg/mL, and the silicon hydroxyl magnetic bead composition contains 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 by mass;
the lysate contains: 4mol/L guanidine isothiocyanate, 0.5mol/L sodium iodide, 0.02mol/L Tris-HCl, 1% (v/v) Tween20, 0.5mol/L urea, 2mg/mL glycogen, 0.02mol/L metal ion complexing agent; the pH value of the lysate is 6;
the washing liquid I contains: 3% (v/v) Tween20, 0.5mol/L sodium chloride, 2mol/L guanidine hydrochloride, 0.1mol/L Tris-HCl, 30% (v/v) isopropanol;
the washing liquid II is 80% (v/v) ethanol;
the eluent is water without nuclease or Tris-HCl-water without nuclease with the concentration of 6-10 mol/L.
2. The kit of claim 1, wherein the eluent is nuclease-free water.
3. A method for extracting viral nucleic acid, characterized in that the nucleic acid extraction kit according to any one of claims 1 to 2 is used.
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| CN114292841B (en) * | 2021-12-30 | 2023-06-09 | 苏州白垩纪生物科技有限公司 | Nucleic acid extraction reagent and nucleic acid extraction method |
| CN114164207B (en) * | 2022-02-11 | 2022-05-17 | 济凡生物科技（北京）有限公司 | Alcohol-free lysate, kit and extraction method for extracting virus nucleic acid |
| GB202210963D0 (en) * | 2022-07-27 | 2022-09-07 | Earlham Entpr Ltd | Methods for extraction and sequencing of nucleic acid |
| CN115786326B (en) * | 2022-11-04 | 2023-10-03 | 重庆医科大学 | Virus nucleic acid extraction kit and method for extracting complete virus particle nucleic acid by using same |
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