CN113735935A - Detection method of new coronavirus capable of enriching free nucleic acid by selectively adsorbing nucleoprotein - Google Patents
Detection method of new coronavirus capable of enriching free nucleic acid by selectively adsorbing nucleoprotein Download PDFInfo
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Abstract
A method for detecting new coronavirus rich in free nucleic acid by selective adsorption of nucleoprotein includes such steps as preparing selective adsorption material of new coronavirus protein by polymerization reaction with N protein of new coronavirus as template, carboxylated carbon nanotube as carrier and acrylamide as functional monomer, enriching N protein of new coronavirus and its associated nucleic acid, extracting the enriched nucleic acid, and detecting new coronavirus by reverse transcription-PCR method. The material has high adsorption capacity to the new coronavirus protein, the adsorption capacity reaches 88-140mg/g, and the material can be used for selective adsorption of the new coronavirus in the environments of air, water and the like. The first-order kinetic rate constant of the new coronavirus protein reaches 0.00015-0.00025/s, so that enough new coronavirus protein can be adsorbed in a short time for subsequent detection. And the sensitivity is far higher than that of the conventional nucleic acid detection method, and is 65-1000 times higher than that of the latter.
Description
Technical Field
The invention relates to a method for preparing a functional material for selectively adsorbing nucleoprotein (N protein) and a method for detecting low-concentration new coronavirus in environment by using the functional material; the functional material is prepared by utilizing the characteristic of a molecular imprinting selective recognition template, can selectively adsorb N protein of the new coronavirus in the environment, simultaneously enriches free nucleic acid of the new coronavirus connected with the N protein, and realizes efficient detection of the new coronavirus in the environment through subsequent detection and analysis of the N protein and the nucleic acid.
Background
The new coronavirus pneumonia (COVID-19) epidemic caused by the new coronavirus (SARS-CoV-2) spreads in most countries and regions, the number of infected people reaches one hundred million and eighty million, and a huge disaster is brought to public health and human development. In addition to infection in the population, the detection of new coronaviruses in environmental samples is increasing, and the cases of re-infection of the population with new coronaviruses in the environment are increasing, and the new coronaviruses become a main form of transmission. Therefore, the development of the detection of new coronaviruses in the environment is of great importance.
The current detection means of the new coronavirus mainly aims at human bodies and comprises a nucleic acid detection method, an immunological detection method, virus separation and identification and the like. The nucleic acid detection has high accuracy and can be positive in the early stage of infection, but the detection procedure is complex and time-consuming, and has higher requirements on sample storage, nucleic acid extraction, the proficiency of operators and the operation environment. The enzyme linked immunosorbent assay method is a qualitative and quantitative detection method which utilizes the specific combination of antigen and antibody to carry out immunoreaction, for example, the patent 'a novel coronavirus antibody detection kit and a preparation method thereof' (application number CN202011460161.0) discloses a novel coronavirus antibody detection kit and a preparation method thereof, wherein the kit comprises a reagent 1 and a reagent 2 which are independently packaged, and the reagent 2 at least comprises latex particles coated with novel coronaviruses and is used for carrying out antigen-antibody reaction with the novel coronavirus antibodies in a sample to be detected. However, the method has limited accuracy and slow detection, and often cannot realize early diagnosis. Virus isolation methods require several days of culture and are less specific and less sensitive than nucleic acid detection methods. Moreover, the methods are mainly used for detecting the new coronavirus in the human body, but the common concentration of the new coronavirus in the environment is low, the pollution medium is complex, and a plurality of influencing factors exist, so that the usability of the methods is further reduced. The development of a simple, convenient and rapid method with strong identification capability and high sensitivity, which is suitable for detecting the new coronavirus in the environment, has important significance.
Disclosure of Invention
The invention aims to prepare a functional material for selectively adsorbing the nuclear protein (N protein) of the new coronavirus, and detect the low-concentration new coronavirus in the environment by utilizing the functional material. The material has high adsorption capacity and specificity on the new coronavirus N protein, can effectively enrich the new coronavirus N protein and the connected nucleic acid thereof in the environment, and realizes the specific detection of the low-concentration new coronavirus in the environment. The technical scheme of the invention is as follows: the method comprises the steps of firstly, preparing a new coronavirus protein selective adsorption material by taking a new coronavirus N protein as a template, a carboxylated carbon nano tube as a carrier and acrylamide as a functional monomer through a polymerization reaction, enriching the new coronavirus N protein and the connected nucleic acid in the environment, extracting the enriched nucleic acid, and detecting the new coronavirus through a reverse transcription PCR method. The specific scheme is as follows:
1) dissolving a carboxylated carbon nanotube (CNTs-COOH) in deionized water, ultrasonically dispersing the solution into stable suspension, adding N-hydroxysuccinimide (NHS) and carbodiimide hydrochloride (EDAC), oscillating the suspension, centrifuging the oscillation, and cleaning the precipitate; the mass ratio of CNTs-COOH to NHS is (3-12) to 1, and the mass ratio of CNTs-COOH to EDAC is (5-20) to 1;
2) dissolving the precipitate obtained in the step 1) in a buffer solution containing the surface protein of the new coronavirus in the market, fully oscillating at the temperature of 2-8 ℃, adding a Tris solution, fully oscillating, centrifuging, and washing the precipitate with deionized water; the mass ratio of the CNTs-COOH to the new coronavirus surface protein is (25-100): 1; the mass ratio of CNTs-COOH to Tris is (0.1-0.5): 1;
3) after the precipitate in the step 2) is resuspended in PIPES solution, acrylamide (AAM) is added and sufficiently shaken, then N, N' -methylene bisacrylamide (NNMBA) and thiosulfate are added, and after shaking for 3-6 hours, centrifugation is carried out, and the precipitate is washed with deionized water. The mass ratio of the CNTs-COOH to the AAM is (0.5-2) to 1; the mass ratio of CNTs-COOH to NNMBA is (0.2-1) to 1; the mass ratio of the CNTs-COOH to the thiosulfate is (3-15) to 1.
4) Adding the precipitate obtained in the step 3) into a weak acid solution, oscillating for 3-12h, fully cleaning, and drying to obtain a new coronavirus Western blot material MIP;
5) immersing the MIP prepared in the step 4) into an environmental sample to be detected, stirring for 10-30min, and centrifuging to separate the MIP; then adding the MIP obtained by centrifugation into a commercial RNA extraction kit to extract enriched new coronavirus nucleic acid; amplifying the extracted new coronavirus nucleic acid by a commercial reverse transcription PCR kit, and detecting the content of the new coronavirus; the concentration of MIP added to the environmental sample was 100-1000 mg/L.
Preferred step 1): the concentration of the CNTs-COOH suspension in the step 1) is optimized to be 2-20 mg/mL;
preferred step 2): the concentration of the N protein of the new coronavirus in the step 2) is optimized to be 200-300 mg/L; the buffer solution comprises, but is not limited to PIPES buffer solution or PBS buffer solution, and the concentration is 0.01-1 mol/L; the thiosulfate is optimized into sodium thiosulfate, potassium thiosulfate and ammonium thiosulfate;
preferably step 3): in the step 4), the weak acid is optimized to be oxalic acid or acetic acid, and the concentration is optimized to be 0.5-2 mol/L;
preferably step 4): the environmental sample in step 5) above is optimized to be a water, soil or air sample, wherein the soil or air sample is first measured after the sample is immersed in water to transfer the new coronavirus to the water phase.
The invention has the beneficial effects that:
the MIP material for selectively adsorbing nucleoprotein has higher adsorption capacity on the new coronavirus protein, the adsorption quantity reaches 88-140mg/g, and the MIP material can be used for selectively adsorbing the new coronavirus in the environments such as air, water and the like.
The MIP material selectively adsorbing nucleoprotein has a fast adsorption rate to the new coronavirus protein, and the first-order kinetic rate constant reaches 0.00015-0.00025/s, so that enough new coronavirus protein can be adsorbed in a short time for subsequent detection.
The sensitivity of the novel coronavirus detection method provided by the invention is far higher than that of the conventional nucleic acid detection method, and is 65-1000 times higher than that of the latter nucleic acid detection method.
Drawings
FIG. 1: technical route map for material synthesis of the invention
FIG. 2: transmission electron micrographs of the material prepared in example 1;
FIG. 3: the infrared spectrum of the material prepared in example 1;
FIG. 4: comparison of the adsorption of the material prepared in example 1 and the control material to the new coronavirus surface protein;
FIG. 5: the adsorption isotherm fit of the material prepared in example 1 to the new coronavirus surface protein;
FIG. 6: the first order kinetic equation of adsorption of the material prepared in example 1 to the new coronavirus surface protein is fitted;
Detailed Description
The following detailed description of specific embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention easier to understand by those skilled in the art, and thus will clearly and clearly define the scope of the invention.
Fig. 1 shows a synthesis route of the MIP material for selectively adsorbing nucleoprotein in the present invention, i.e. carbon nanotubes are used as Carriers (CNTs), the surface layer of the carbon nanotubes needs to be carboxylated, and the carbon nanotubes can be purchased directly as finished products. After the carbon nano tube is treated by EDAC and NHS, an active site is formed, and can be easily connected with an amine group in the new coronavirus surface protein, and the new coronavirus N protein is fixed on the surface of the carbon nano tube in an amido bond mode. At the moment, acrylamide (AAM) is used as a functional monomer, N, N-methylene bisacrylamide (NNBMA) is used as a cross-linking agent, thiosulfate is used as an initiator, the polymerization reaction can be started at room temperature, and the obtained polyacrylamide polymer layer surrounds the surface protein of the new coronavirus. And (3) the new coronavirus surface protein is separated from the material after being denatured and eluted by oxalic acid (OAc) solution, and a blot site is left, so that a product loaded with the new coronavirus surface protein is obtained.
Example 1:
1) 10mg of commercially available carboxylated carbon nanotubes (CNTs-COOH, 20nm in diameter) were placed in a 5mL centrifuge tube, 1mL of deionized water was added and ultrasonically dispersed for 10min, 1mL of a mixed solution containing NHS (1.7mg) and EDAC (1.3mg) was added, and the mixture was shaken at room temperature for 45 min. After shaking, the solution was centrifuged, the supernatant was discarded, and the solution was washed 3 times with 2mL of deionized water, with a 10min duration for each centrifugation.
2) Adding 1mL of PIPES buffer solution (the concentration of the buffer solution is 0.1mol/L) containing 0.2mg of new coronavirus N protein into the precipitate in the last step, and oscillating for 4h at 4 ℃; then 1mL of Tris solution (containing 0.1g of Tris) was added and shaken at room temperature for 30 min. After shaking, the solution was centrifuged, the supernatant was discarded, and the solution was washed 3 times with 2mL of deionized water, with a 10min duration for each centrifugation.
3) The resulting product was resuspended in 1ml of LPIPES solution, and 10mg of AAM and 20mg of NNMBA were added thereto and sufficiently dissolved by shaking. Then 1.5mg of ammonium thiosulfate was added and shaken at room temperature for 3 hours. After shaking, the solution was centrifuged, the supernatant was discarded, and the solution was washed 3 times with 2mL of deionized water, with a 10min duration for each centrifugation.
4) 2mL of 1moL/L oxalic acid solution is added to the precipitate in the previous step, and the mixture is shaken at room temperature for 12 hours. After shaking, the solution was centrifuged, the supernatant was discarded, and the solution was washed 3 times with 2mL of deionized water, with a 10min duration for each centrifugation. The precipitate is then placed in an oven to dry, resulting in the MIP of the new coronavirus N-protein blot product.
5) Immersing 500mg of MIP prepared in the step 4) into 1L of sewage solution, stirring for 30min, and centrifuging to separate the MIP; then adding the MIP obtained by centrifugation into a commercial RNA extraction kit to extract RNA; and amplifying the extracted RNA by a commercial reverse transcription PCR kit, and detecting the content of the new coronavirus.
The transmission electron microscope of the MIP product in this example is shown in FIG. 2, which is a long fiber-like structure with a size of about 20 nm; the infrared spectrum image of the product is shown in figure 3, and the mass ratio of acrylamide is 25%; the comparison of the adsorption capacity of the product (MIP) on the N protein of the new coronavirus and the control product is shown in figure 4, and the adsorption capacity of the MIP is obviously higher than that of the control group (NIP and CNTs-COOH); the fitting graph of the adsorption isotherm of the product on the N protein of the new coronavirus is shown in FIG. 5, and the saturated adsorption capacity is 140g/g of material; the adsorption kinetic equation of the product on the N protein of the new coronavirus is fitted in a figure 6, and the adsorption rate is 0.00025/s. The content of new coronavirus detected by the method is 3.0 x 103copies/L, method for detection using traditional check kitThe detection sensitivity of the method is more than 1000 times of that of the traditional kit detection method when not detected.
Example 2:
1) 5mg of commercially available carboxylated carbon nanotubes (CNTs-COOH, diameter of 10nm) were placed in a 5mL centrifuge tube, 1mL of deionized water was added for ultrasonic dispersion, 1mL of a mixed solution containing NHS (1.7mg) and EDAC (1.3mg) was added, and the mixture was shaken at room temperature for 45 min. After shaking, the solution was centrifuged, the supernatant was discarded, and the solution was washed 3 times with 2mL of deionized water, with a 10min duration for each centrifugation.
2) Adding 1mL of PBS buffer solution (the concentration of the buffer solution is 1mol/L) containing 0.2mg of new coronavirus N protein into the precipitate in the last step, and oscillating for 4h at 6 ℃; then 1ml of Tris solution (containing 0.01g of Tris) was added thereto, and the mixture was shaken at room temperature for 30 min. After shaking, the solution was centrifuged, the supernatant was discarded, and the solution was washed 3 times with 2mL of deionized water, with a 10min duration for each centrifugation.
3) The resulting product was resuspended in 1mL of PIPES solution, and 10mg of AAM and 25mg of NNMBA were added thereto and sufficiently dissolved by shaking. Then 1.5mg of ammonium thiosulfate was added and shaken at room temperature for 3 hours. After shaking, the solution was centrifuged, the supernatant was discarded, and the solution was washed 3 times with 2mL of deionized water, with a 10min duration for each centrifugation.
4) 2mL of 0.5moL/L acetic acid solution was added to the precipitate from the previous step, followed by shaking at room temperature for 3 hours. After shaking, the solution was centrifuged, the supernatant was discarded, and the solution was washed 3 times with 2mL of deionized water, with a 10min duration for each centrifugation. The precipitate is then placed in an oven to dry, resulting in the MIP of the new coronavirus N-protein blot product.
5) Immersing 50mg of MIP prepared in the step 4) into 500mL of soil leachate fertilized with a certain excrement, stirring for 10min, and centrifuging to separate the MIP; then adding the MIP obtained by centrifugation into a commercial RNA extraction kit to extract RNA; and amplifying the extracted RNA by a commercial reverse transcription PCR kit, and detecting the content of the new coronavirus.
The diameter of the product is 10nm, the mass ratio of acrylamide is 25%, and the adsorption capacity and the adsorption rate of the product to the new coronavirus S protein are 125g/g material and 0.00022/S respectively; the content of new coronavirus detected by the method is 7.5 x 105copies/L, the content of new coronavirus detected by using the traditional nucleic acid kit detection method is 1.2 x 103The detection sensitivity of the method is 650 times that of the traditional kit detection method.
Example 3:
1) 20mg of commercially available carboxylated carbon nanotubes (CNTs-COOH, diameter 30nm) were placed in a 5mL centrifuge tube, 2mL deionized water was added and ultrasonically dispersed for 10min, 1mL of a mixed solution containing NHS (6.6mg) and EDAC (4mg) was added, and the mixture was shaken at room temperature for 45 min. After shaking, the solution was centrifuged, the supernatant was discarded, and 3 times of centrifugation with 3mL of deionized water, each time for 10 min.
2) Adding 1mL of PIPES buffer solution (the concentration of the buffer solution is 0.01mol/L) containing 0.2mg of new coronavirus N protein into the precipitate in the last step, and oscillating for 4h at the temperature of 3 ℃; then 1mL of Tris solution (containing 0.1g of Tris) was added and shaken at room temperature for 30 min. After shaking, the solution was centrifuged, the supernatant was discarded, and 3 times of centrifugation with 3mL of deionized water, each time for 10 min.
3) The resulting product was resuspended in 1mL of PIPES solution, and 10mg of AAM and 20mg of NNMBA were added and sufficiently dissolved by shaking. Then 2mg of sodium thiosulfate was added and shaken at room temperature for 3 h. After shaking, the solution was centrifuged, the supernatant was discarded, and 3 times of centrifugation with 3mL of deionized water, each time for 10 min.
4) 2mL of 2moL/L oxalic acid solution is added to the precipitate in the previous step, and the mixture is shaken at room temperature for 8 hours. After shaking, the solution was centrifuged, the supernatant was discarded, and 3 times of centrifugation with 3mL of deionized water, each time for 10 min. The precipitate is then placed in an oven to dry, resulting in the MIP of the new coronavirus N-protein blot product.
5) Immersing 25mg of MIP prepared in the step 4) into 500mL of aerosol-immersed aqueous solution, stirring for 20min, and centrifuging to separate the MIP; then adding the MIP obtained by centrifugation into a commercial RNA extraction kit to extract RNA; and amplifying the extracted RNA by a commercial reverse transcription PCR kit, and detecting the content of the new coronavirus.
The diameter of the product is 30nm, the mass of acrylamide accounts for 20%, and the adsorption capacity and the adsorption rate of the product to the new coronavirus N protein are respectively 88g/g material and 0.00016/s; the content of new coronavirus detected by the method is 2.4 x 103copies/L, is not detected by using the traditional nucleic acid kit detection method, and the detection sensitivity of the method is the traditional testThe kit detection method is more than 1000 times.
Example 4:
1) placing 15mg of commercial carboxylated carbon nanotubes (CNTs-COOH, size 20nm) in a 5mL centrifuge tube, adding 0.75mL deionized water, ultrasonically dispersing for 10min, adding 1mL mixed solution containing NHS (1.3mg) and EDAC (0.75mg), and shaking at room temperature for 45 min. After shaking, the solution was centrifuged, the supernatant was discarded, and the solution was washed 3 times with 2mL of deionized water, with a 10min duration for each centrifugation.
2) Adding 1mL of PBS buffer solution (the concentration of the buffer solution is 0.5mol/L) containing 0.3mg of new coronavirus N protein into the precipitate in the last step, and oscillating for 4h at 4 ℃; then 1mL of Tris solution (containing 0.08g of Tris) was added and the mixture was shaken at room temperature for 30 min. After shaking, the solution was centrifuged, the supernatant was discarded, and the solution was washed 3 times with 2mL of deionized water, with a 10min duration for each centrifugation.
3) The resulting product was resuspended in 1mL of PIPES solution, and 15mg of AAM and 30mg of NNMBA were added thereto and sufficiently dissolved by shaking. Then 2mg of sodium thiosulfate was added and shaken at room temperature for 3 h. After shaking, the solution was centrifuged, the supernatant was discarded, and the solution was washed 3 times with 2mL of deionized water, with a 10min duration for each centrifugation.
4) 2mL of 1moL/L acetic acid solution was added to the precipitate from the previous step, followed by shaking at room temperature for 6 hours. After shaking, the solution was centrifuged, the supernatant was discarded, and the solution was washed 3 times with 2mL of deionized water, with a 10min duration for each centrifugation. The precipitate is then placed in an oven to dry, resulting in a new coronavirus western blot product MIP.
5) Immersing 80mg of MIP prepared in the step 4) into 1L of certain sewage solution, stirring for 30min, and centrifuging to separate the MIP; then adding the MIP obtained by centrifugation into a commercial RNA extraction kit to extract RNA; and amplifying the extracted RNA by a commercial reverse transcription PCR kit, and detecting the content of the new coronavirus.
The diameter of the product is 20nm, the mass ratio of acrylamide is 25%, and the adsorption capacity and the adsorption rate of the product to the new coronavirus N protein are respectively 132g/g material and 0.00023/s; the content of new coronavirus detected by the method is 3.6 x 105copies/L, the content of new coronavirus detected by using the traditional nucleic acid kit detection method is 0.43 x 103The detection sensitivity of the method is that the detection of the traditional kit is realized830 times of the method.
Claims (8)
1. A detection method of new coronavirus with free nucleic acid enriched by selective adsorption of nucleoprotein is characterized in that: firstly, preparing a new coronavirus N protein selective adsorption material by utilizing the characteristic of a molecular imprinting selective recognition template, further enriching the new coronavirus N protein and the connected nucleic acid in the environment by utilizing the material, extracting the enriched nucleic acid, and detecting the new coronavirus existing in the environment by a reverse transcription PCR method; the selective adsorbing material for the new coronavirus N protein is prepared by taking the new coronavirus N protein as a template, a carboxylated carbon nanotube as a carrier and acrylamide as a functional monomer through polymerization reaction.
2. The method for detecting a novel coronavirus capable of enriching a free nucleic acid by selectively adsorbing a nucleoprotein, according to claim 1, characterized in that: the structure of the novel coronavirus N protein selective adsorption material is divided into an inner layer and an outer layer, wherein the inner layer is a carbon nano tube, the outer layer is a polyacrylamide polymerization layer, and virus surface western blotting is inlaid in the polyacrylamide polymerization layer; the diameter of the new coronavirus N protein selective adsorption material is 10-30nm, and the mass ratio of acrylamide is 20-25%; the adsorbing capacity of the new coronavirus N protein selective adsorbing material to the new coronavirus surface protein reaches 88-140g/g, and the adsorption kinetic rate constant reaches 0.00015-0.00025/s.
3. The method for detecting a novel coronavirus capable of enriching a free nucleic acid by selective adsorption of a nucleoprotein, according to claim 1 or 2, characterized in that the specific steps are as follows:
1) dissolving carboxylated carbon nanotube CNTs-COOH in water to be dispersed into stable suspension, adding N-hydroxysuccinimide NHS and carbodiimide hydrochloride EDAC, oscillating, centrifuging, and cleaning to obtain a precipitate;
the mass ratio of CNTs-COOH to NHS is (3-12) to 1, and the mass ratio of CNTs-COOH to EDAC is (5-20) to 1;
2) adding the precipitate in the step 1) into a buffer solution containing new coronavirus N protein, fully oscillating, adding a Tris solution of Tris, oscillating, centrifuging, and cleaning to obtain a precipitate, wherein the mass ratio of CNTs-COOH to new coronavirus N protein is (25-100): 1; the mass ratio of CNTs-COOH to Tris is (0.1-0.5): 1;
3) after the sediment in the step 2) is resuspended, adding acrylamide AAM to fully oscillate, then adding N, N' -methylene bisacrylamide NNMBA and thiosulfate to oscillate for 2-8 hours, centrifuging and cleaning to obtain sediment, wherein the mass ratio of CNTs-COOH to AAM is (0.5-2): 1; the mass ratio of CNTs-COOH to NNMBA is (0.2-1) to 1; the mass ratio of the CNTs-COOH to the thiosulfate is (3-15) to 1;
4) adding the precipitate obtained in the step 3) into a weak acid solution, oscillating for 3-12h, fully cleaning, and drying to obtain a new coronavirus N protein selective adsorption material MIP;
5) immersing the MIP prepared in the step 4) into an environmental sample to be detected, stirring for 10-30min, and centrifuging to separate the MIP; then adding the MIP obtained by centrifugation into a commercial RNA extraction kit to extract enriched new coronavirus nucleic acid; amplifying the extracted new coronavirus nucleic acid by a commercial reverse transcription PCR kit, and detecting the content of the new coronavirus; the concentration of MIP added to the environmental sample was 100-1000 mg/L.
4. The method for detecting a novel coronavirus capable of enriching a free nucleic acid by selective adsorption of a nucleoprotein, as claimed in claim 3, characterized in that the concentration of the suspension of CNTs-COOH in step 1) is 2-20 mg/mL.
5. The method for detecting the novel coronavirus capable of enriching the free nucleic acid by selectively adsorbing the nucleoprotein as claimed in claim 3, wherein the buffer solution in the step 2) is any solution capable of stabilizing the protein, including but not limited to PIPES buffer solution or PBS buffer solution, and the concentration is 0.01-1 mol/L; the concentration of the N protein of the new coronavirus is 200-300 mg/L.
6. The method for detecting a novel coronavirus capable of enriching a free nucleic acid by selectively adsorbing a nucleoprotein, as claimed in claim 3, wherein the thiosulfate in the step 3) is sodium thiosulfate, potassium thiosulfate or ammonium thiosulfate.
7. The method for detecting the novel coronavirus capable of enriching the free nucleic acid by selectively adsorbing the nucleoprotein as claimed in claim 3, wherein the weak acid in the step 4) is oxalic acid or acetic acid, and the concentration is 0.5-2 mol/L.
8. The method according to claim 3, wherein the environmental sample in step 5) is water, soil or air, wherein the soil or air is first immersed in water to transfer the isolated nucleic acids to the aqueous phase for determination.
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| CN101377463A (en) * | 2008-10-22 | 2009-03-04 | 南开大学 | Method and application for preparing carbon nano tube of histidine label protein function |
| CN103520737A (en) * | 2013-10-15 | 2014-01-22 | 西北农林科技大学 | Carbon nano tube carrier vaccination-free vaccine as well as preparation method and application thereof in preparation of aquatic immune offspring seed |
| CN111085178A (en) * | 2018-10-24 | 2020-05-01 | 中国石油化工股份有限公司 | Preparation method and application of acrylamide polymer containing carbon nano-tube |
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| CN101377463A (en) * | 2008-10-22 | 2009-03-04 | 南开大学 | Method and application for preparing carbon nano tube of histidine label protein function |
| CN103520737A (en) * | 2013-10-15 | 2014-01-22 | 西北农林科技大学 | Carbon nano tube carrier vaccination-free vaccine as well as preparation method and application thereof in preparation of aquatic immune offspring seed |
| CN111085178A (en) * | 2018-10-24 | 2020-05-01 | 中国石油化工股份有限公司 | Preparation method and application of acrylamide polymer containing carbon nano-tube |
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