CN112246222B - Virus enrichment material, preparation method and application thereof, virus enrichment system and application thereof - Google Patents
Virus enrichment material, preparation method and application thereof, virus enrichment system and application thereof Download PDFInfo
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Abstract
The invention provides a virus enrichment material, a preparation method and application thereof, a virus enrichment system and application thereof, and belongs to the technical field of virus enrichment and adsorption. The virus enrichment material provided by the invention comprises graphene ferroferric oxide particles and CTAB attached to the surfaces of the graphene ferroferric oxide particles. The invention utilizes cationic surfactant CTAB to G-Fe3O4Modifying the compound to obtain CTAB-G-Fe3O4The surface of the composite material is filled with a large amount of positive charges, has good performances such as high potential, superparamagnetism, large specific surface area and the like, and can attract viruses with negative charges to realize the adsorption and enrichment of the viruses; the virus enrichment system established by the virus enrichment material can respectively enrich norovirus, rotavirus, adenovirus and new coronavirus, and has high enrichment efficiency.
Description
Technical Field
The invention relates to the technical field of virus enrichment and adsorption, in particular to a virus enrichment material, a preparation method and application thereof, a virus enrichment system and application thereof.
Background
Food-borne viruses infect people by contaminating food, causing a population gastroenteritis epidemic. The content of viruses in food is generally low. For example, naturally contaminated shellfish samples containing only 10 per gram of digested tissue2～104A virus of gene copy number. The virus content of the water environment and the food surface is low, which is not beneficial to the subsequent nucleic acid detection. Therefore, the virus is usually enriched before detection to increase the concentration for easy detection of the virus.
The current methods for enriching food-borne viruses include Polyethylene glycol (PEG) precipitation, ultracentrifugation, ultrafiltration, immunoconcentration, and cation separation. These enrichment methods are aimed at isolating viruses from large quantities of food and concentrating them into a smaller volume of liquid, while removing the substances that inhibit detection, such as polysaccharides, proteins and fatty acids, to facilitate subsequent nucleic acid detection.
Based on the principle that PEG can precipitate viruses in neutral buffer with high ionic strength, PEG precipitation is widely used to enrich viruses from eluents. The PEG precipitation method is simple to operate, and the virus can be precipitated only by adjusting the pH of the eluent to be neutral, improving the ionic strength of the eluent, adding PEG and standing overnight at 4 ℃. Reports show that 5-90% of virus can be recovered from the surface of food by combining an alkaline elution method and a PEG precipitation enrichment method, and the specific recovery efficiency is related to the components of the food. Currently, the CEN/TC275/WG6/TAG4 working group has combined alkaline elution and PEG pellet enrichment as the first Method of isolating Viruses from produce and soft fruits (David, LeescEN, WG6, TAG4(2010) International standardization of a Method for protection of Human Pathogenic Viruses in Mollussen Shell fish. food and environmental virology 2(3): 10.). The PEG precipitation method has low cost and easy operation, is widely used for precipitating virus from eluent, generally needs sedimentation, takes longer time, and also needs large-scale equipment such as a high-speed centrifuge and the like.
Ultracentrifugation is often used to concentrate viruses from eluents of various types of foods. During ultracentrifugation, centrifugal forces of up to 120000 Xg or 235000 Xg can precipitate virus particles in the eluate. However, the ultracentrifuge equipment is expensive and requires a professional to operate, which also makes it difficult to widely use this enrichment method.
The ultrafiltration is an enrichment method for concentrating viruses according to the difference of molecular weights of different components in eluent. The liquid and low molecular weight components are able to pass through the filter and the virus can be captured. Ultrafiltration can remove inhibitory components of molecular detection methods and improve detection sensitivity, but viral eluents must be purified before filtration to avoid clogging of the filter with food residues.
The immune concentration method is a method for efficiently enriching virus particles in an eluent by using a virus specific antibody or protein connected with magnetic beads based on the principle of antigen-antibody specific binding. It has been reported in a large number of documents that viruses in an eluate can be enriched by modifying the norovirus specific protein type porcine gastric mucin or HBGA on the surface of magnetic beads, and after an external magnetic field is applied, the viruses specifically bound to the proteins can be rapidly separated from the eluate. Park (Park Y, Cho YH, Jee Y, & Ko G (2008) immunological separation combined with real-time reverse transcription PCR assays for the detection of viruses in associated foods, applied and environmental microbiology 74(13): 4226. about.4230.) reports that 14.1% and 29.5% of type GI and type GII noroviruses can be recovered from strawberries, respectively, but that the immunological concentration uses a large amount of antibodies, increasing the virus detection cost.
The cation separation method is based on the principle that positively charged magnetic particles can bind to negatively charged viral capsids, and concentrates and purifies viral particles from food. However, the results obtained by cation separation are less consistent and require expensive equipment and professional operation, making widespread use difficult.
Therefore, the research on new virus enrichment reagents and methods is of great significance.
Disclosure of Invention
The invention aims to provide a virus enrichment material, a preparation method and application thereof, a virus enrichment system and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a virus enrichment material which comprises graphene ferroferric oxide particles and CTAB attached to the surfaces of the graphene ferroferric oxide particles.
Preferably, the mass ratio of CTAB to graphene ferroferric oxide particles is (5-6): 1.
the invention provides a preparation method of the virus enrichment material in the technical scheme, which comprises the following steps:
and dispersing the graphene ferroferric oxide particles in ultrapure water, mixing the obtained dispersion liquid with CTAB, and freeze-drying to obtain the virus enrichment material.
The invention provides the application of the virus-enriched material in the technical scheme or the virus-enriched material prepared by the preparation method in the technical scheme in enriching food-borne viruses or new coronaviruses.
The invention provides a virus enrichment system, which comprises a PBS buffer solution, a virus suspension and a virus enrichment material dispersion solution; the virus-enriched material in the virus-enriched material dispersion liquid is the virus-enriched material in the technical scheme or the virus-enriched material prepared by the preparation method in the technical scheme.
Preferably, the volume ratio of the PBS buffer solution to the virus suspension to the virus-enriched material dispersion is (1-100): (0-0.5): (0-1), and the volumes of the virus suspension and the virus-enriched material dispersion liquid are not 0.
Preferably, the concentration of the PBS buffer is 0.01mM, and the pH value is 7.4.
Preferably, the concentration of the virus-enriched material dispersion liquid is 0-0.25 mg/mL and is not 0.
The invention provides the application of the virus enrichment system in the technical scheme in virus enrichment.
Preferably, the virus is a food-borne virus or a neocoronavirus.
The invention provides a virus enrichment material which comprises graphene ferroferric oxide particles and CTAB attached to the surfaces of the graphene ferroferric oxide particles. The invention utilizes cationic surfactant CTAB to G-Fe3O4The complex is modified, and the CTAB is positively charged, so that the obtained CTAB-G-Fe3O4The surface of the composite material is filled with a large amount of positive charges, so that the composite material has high potential, and simultaneously has good performances such as superparamagnetism, large specific surface area and the like, and can attract viruses with negative charges to realize the adsorption and enrichment of the viruses;
the invention provides a virus enrichment system, which comprises a PBS buffer solution, a virus suspension and a virus enrichment material dispersion solution; also provides a large-volume virus enrichment detection system, which comprises PBS buffer solution/tap water/sea river water/sea water, virus suspension and virus enrichment material dispersion; the virus-enriched material in the virus-enriched material dispersion liquid is the virus-enriched material in the technical scheme or the virus-enriched material prepared by the preparation method in the technical scheme. The results of the examples show that the invention utilizes CTAB-G-Fe3O4A1.6 mL virus enrichment system established by the composite material can respectively enrich norovirus, rotavirus, adenovirus and new coronavirus of 6.12log, 6.19log, 5.91log and 6.37log, and has high enrichment efficiency; using CTAB-G-Fe3O4Compared with the conventional detection method, the 40mL virus enrichment detection system established by the composite material can obviously improve the detection rate of the virus.
Drawings
FIG. 1 is CTAB-G-Fe prepared in example 13O4Composite material SEM image and energy spectrum analysis image; FIG. 2 is CTAB-G-Fe prepared in example 13O4Composite material and G-Fe3O4XRD pattern of (a);
FIG. 3 is CTAB-G-Fe prepared in example 13O4Composite material and G-Fe3O4(ii) an infrared spectrum;
FIG. 4 is CTAB-G-Fe prepared in example 13O4XPS plot of composite material;
FIG. 5 is CTAB-G-Fe prepared in example 13O4Composite material and G-Fe3O4Zeta potential analysis chart of (1);
FIG. 6 is CTAB-G-Fe prepared in example 13O4N of composite material2An adsorption and desorption partial curve diagram;
FIG. 7 is CTAB-G-Fe prepared in example 13O4A magnetic analysis map of the composite;
FIG. 8 is a graph of enrichment efficiency of an enrichment material under different conditions;
FIG. 9 shows CTAB-G-Fe3O4The composite material has the enrichment efficiency on norovirus, rotavirus, adenovirus and new coronavirus pseudovirus.
FIG. 10 shows CTAB-G-Fe3O4The composite material has the enrichment efficiency on norovirus, rotavirus, adenovirus and new coronavirus pseudoviruses in different water samples.
Detailed Description
The invention provides a virus enrichment material which comprises graphene ferroferric oxide particles and CTAB attached to the surfaces of the graphene ferroferric oxide particles.
In the present invention, the materials required are all commercially available products well known to those skilled in the art unless otherwise specified.
The virus-enriched material provided by the invention comprises graphene ferroferric oxide particles, the graphene ferroferric oxide particles are preferably prepared according to a method of the prior art (Zhan S, et al (2015) high effective removal of nutritional bacteria with magnetic graphene composition, ACS applied materials & interfaces 7(7):4290-4298), and the preparation method of the graphene ferroferric oxide particles preferably comprises the following steps:
mixing graphene oxide and ethylene glycol, and dispersing in an ultrasonic cleaning instrument for 30min to obtain graphene oxide suspension;
adding ferric acetylacetonate into the graphene oxide suspension, and dispersing in an ultrasonic cleaning instrument for 30min to obtain a suspension;
mixing ammonium acetate with the suspension, placing on a constant-temperature magnetic stirrer, uniformly stirring for 30min, transferring the obtained mixed liquid into a reaction kettle with a polytetrafluoroethylene lining, horizontally placing in an electrothermal blowing drying box after the reaction kettle is closed, carrying out solvent thermal reaction, after the reaction is finished, naturally cooling the reaction kettle to room temperature, transferring a product into a centrifuge tube, centrifuging, pouring out a supernatant, cleaning the obtained black precipitate with ultrapure water, precipitating black particles by using a magnet, pouring out the supernatant, and repeating for 7-8 times until the pH of the obtained cleaning liquid is neutral to obtain the graphene ferroferric oxide particles.
In the invention, the graphene oxide is preferably purchased from Shanghai Aladdin Biotechnology, Inc., and the volume ratio of the graphene oxide to the ethylene glycol is preferably 1: 1000; the mass ratio of the graphene oxide to the ferric acetylacetonate is preferably 1: 5; the mass ratio of the graphene oxide to the ammonium acetate is preferably 3: 100; the temperature of the solvothermal reaction is preferably 200 ℃, and the time is preferably 24 h; the rotating speed of the centrifugation is preferably 12000r/min, and the time is preferably 3 min. The other processes for preparing the graphene ferroferric oxide particles are not particularly limited, and can be carried out according to the processes well known in the art.
The virus enrichment material provided by the invention comprises CTAB (cetyl trimethyl ammonium bromide) attached to the surface of the graphene ferroferric oxide particles. In the invention, the mass ratio of CTAB to graphene ferroferric oxide particles is preferably (5-6): 1, more preferably 5.5: 1.
The invention provides a preparation method of the virus enrichment material in the technical scheme, which comprises the following steps:
and dispersing the graphene ferroferric oxide particles in ultrapure water, mixing the obtained dispersion liquid with CTAB, and freeze-drying to obtain the virus enrichment material.
The dispersion process is not particularly limited in the present invention, and may be carried out according to a process known in the art. The concentration of the dispersion liquid is not particularly limited, and the graphene ferroferric oxide particles can be fully dispersed.
In the invention, the mixing process of the dispersion and CTAB is preferably carried out on a constant-temperature magnetic stirrer, and the stirring time is preferably 3 h; the stirring speed is not particularly limited, and the uniformly dispersed mixed solution can be obtained.
In the invention, the mass ratio of the graphene ferroferric oxide particles to CTAB is preferably (5-6): 1, more preferably 5.5: 1.
after the mixing is finished, the black particles obtained by washing with excess ultrapure water are preferably used, redundant CTAB is washed away, the black particles obtained by washing are placed in a vacuum freeze dryer for freeze drying for 48 hours, and after the drying is finished, the black particles are stored in the dryer at normal temperature, so that the virus enrichment material is obtained.
In the virus enrichment material prepared by the invention, CTAB is used as a cationic surfactant and is crystallized on the surface of the graphene ferroferric oxide particles, so that the surface of the graphene ferroferric oxide particles modified by CTAB is filled with a large amount of positive charges and can attract viruses with negative charges to realize virus enrichment.
The invention provides the application of the virus-enriched material in the technical scheme or the virus-enriched material prepared by the preparation method in the technical scheme in enriching food-borne viruses or new coronaviruses. The method of the present invention is not particularly limited, and the method may be applied according to a method known in the art.
The invention provides a virus enrichment system, which comprises a PBS buffer solution, a virus suspension and a virus enrichment material dispersion solution; the virus-enriched material in the virus-enriched material dispersion liquid is the virus-enriched material in the technical scheme or the virus-enriched material prepared by the preparation method in the technical scheme.
In the present invention, the concentration of the PBS buffer is preferably.0.01 mM, and the pH value is preferably 7.4. In the present invention, the dispersing agent of the virus-enriched material dispersion is preferably a PBS buffer (concentration of 0.01mM, pH 7.4); the composition of the PBS buffer is not particularly limited in the present invention, and may be any PBS buffer known in the art. In the invention, the concentration of the virus-enriched material dispersion liquid is preferably 0-0.25 mg/mL but not 0, and more preferably 0.05-0.20 mg/mL; the virus suspension is a virus sample liquid to be detected, the concentration of the virus suspension is calculated by gene copy number, preferably the gene copy number is 100～107Per mL; the virus in the virus suspension is preferably a food-borne virus, preferably a norovirus, rotavirus or adenovirus, or a neocoronavirus pseudovirus.
In the invention, the volume ratio of the PBS buffer solution to the virus suspension to the virus-enriched material dispersion is preferably (1-100): (0-0.5): (0-1), and the volumes of the virus suspension and the virus-enriched material dispersion liquid are not 0; more preferably 1.4:0.1:0.1 and 39.4:0.1:0.5, and in example 2 of the present invention, the volumes of the PBS buffer, the virus suspension and the virus-enriched material dispersion are specifically 1.4mL, 100. mu.L and 100. mu.L, respectively; in example 3 of the present invention, the volumes of the PBS buffer, the virus suspension and the virus-enriched material dispersion were specifically 39.4mL, 100. mu.L and 500. mu.L, respectively.
The preparation method of the virus enrichment system is not specially limited, and the virus enrichment system can be obtained by directly mixing the PBS buffer solution, the virus suspension and the virus enrichment material dispersion.
The invention provides the application of the virus enrichment system in the technical scheme in virus enrichment.
In the present invention, the virus is preferably a food-borne virus or a new coronavirus pseudovirus; the food-borne virus is preferably a norovirus, rotavirus or adenovirus.
In the present invention, the method for the use of the virus-enriched system in the enrichment of viruses preferably comprises the steps of:
adding PBS buffer solution and virus suspension into a centrifugal tube, uniformly mixing, adding virus enrichment material dispersion liquid, then flatly placing the centrifugal tube in a constant-temperature shaking table, incubating for 0-60 min (namely adsorption time) at 150r/min, carrying out virus enrichment, and separating a virus enrichment material in an incubated system from a liquid phase through a magnet to obtain supernatant;
taking the obtained supernatant, extracting virus RNA aiming at RNA virus, and carrying out reverse transcription reaction of the virus RNA; extracting virus DNA aiming at DNA virus; and then determining the residual content of the virus in the supernatant through the established quantitative PCR reaction system, and simultaneously establishing a control group, namely only adding a PBS buffer solution and a virus suspension into the virus enrichment system without adding a virus enrichment material dispersion solution, and determining the residual content of the virus in the supernatant through the quantitative PCR reaction system after the incubation is finished.
The enrichment efficiency calculation formula is as follows:
wherein the total virus copy number is the virus copy number in the supernatant when the virus-enriched material dispersion is added.
Preferred use of the invention
The Viral RNAminii Kit is preferably used for extracting RNA from RNA viruses by PrimeScript from TaKaRaTM1st Strand cDNA Synthesis Kit for reverse transcription of viral RNA; preferably, DNA of the DNA virus is extracted using a genomic DNA extraction kit for the virus of the UNlQ-10 column type, manufactured by Shanghai Bioengineering Co., Ltd. The process for extracting viral RNA and the process for extracting viral DNA by reverse transcription reaction are not particularly limited in the present invention, and may be performed according to a process known in the art.
When the virus is norovirus, rotavirus or adenovirus, the quantitative PCR reaction system is not particularly limited, and the quantitative PCR reaction system corresponding to different virus types known in the art can be adopted. In the present embodiment, the quantitative PCR reaction systems for norovirus, rotavirus and adenovirus are described in the prior art (Standard A, et al (2012)) Molecular detection and generation of novirusviruses, food and environmental vision 4(4): 153-. When the virus is a new coronavirus pseudovirus, the invention preferably designs a fluorescent quantitative primer and a probe aiming at the luc gene of the new coronavirus pseudovirus, wherein the fluorescent quantitative primer is preferably selected from the group consisting of luc-F: 5'-ACCTACGCCGAGTACTTCGAGA-3', respectively; luc-R: 5'-ACACCGATGAACAGGGCACCCAA-3', respectively; the probe is preferably luc-TaqMan: 5 '-FAM-TCGCTGCACACCACGATCCGA-TAMRA-3'), and then establishing a new quantitative PCR system for coronavirus by referring to the above quantitative PCR system for norovirus.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Mixing 0.045g of graphene oxide and 45mL of ethylene glycol in a beaker, and placing the mixture in an ultrasonic cleaning instrument for dispersing for 30min to obtain graphene oxide suspension; adding 0.225g of ferric acetylacetonate into the graphene oxide suspension, and dispersing in an ultrasonic cleaning instrument for 30min to obtain a suspension; adding 1.5g ammonium acetate into the suspension, placing on a constant temperature magnetic stirrer, stirring for 30min, and transferring the obtained mixed liquid to polytetrafluoroethyleneIn a reaction kettle with a alkene lining, horizontally placing the reaction kettle in an electrothermal blowing dry box after the reaction kettle is sealed, setting the conditions of the dry box to 200 ℃, carrying out solvothermal reaction for 24 hours, after the reaction is finished, naturally cooling the reaction kettle to room temperature, transferring a product to a 50mL centrifugal tube, centrifuging for 3 minutes at 12000r/min, pouring off a supernatant, transferring an obtained black precipitate to a beaker, cleaning the black precipitate with ultrapure water, precipitating black particles with a magnet, pouring off the supernatant, repeatedly washing until the pH value of an obtained eluate is 7.0, and obtaining graphene ferroferric oxide particles, wherein the obtained graphene ferroferric oxide particles are marked as G-Fe3O4；
Dispersing the graphene ferroferric oxide particles (0.5G) in 100mL of ultrapure water, adding 3G of CTAB (cetyl trimethyl ammonium bromide) powder into the obtained dispersion, stirring for 3h on a constant-temperature magnetic stirrer, then cleaning the obtained black particles with excess ultrapure water, placing the black particles in a vacuum freeze dryer for drying for 48h, and obtaining a virus enrichment material (CTAB modified graphene ferroferric oxide, marked as CTAB-G-Fe) after drying3O4Composite material) and stored in a dryer at normal temperature for standby.
Performance testing
1) CTAB-G-Fe prepared in example 1 was subjected to high-resolution transmission electron microscopy (TEM, JEOL JEM-2100,200Kv)3O4The surface morphology and lattice structure of the composite material were characterized and elemental analysis was performed by an energy spectrum analyzer, and the results are shown in fig. 1. In FIG. 1 a) is CTAB-G-Fe3O420000 times magnified TEM image of the composite material with scale bar of 200 nm. From a) in FIG. 1, Fe is visualized3O4The nanoparticles are uniformly dispersed on the surface of the corrugated graphene, and the average diameter of the nanoparticles is 15 nm. In FIG. 1 b) is CTAB-G-Fe3O4Transmission electron microscopy at 600000 Xmagnification of the composite, 5nm scale. As can be clearly seen from b) in FIG. 1, Fe3O4Lattice fringes of nanoparticles (d ═ 0.27 nm). In FIG. 1 c) is CTAB-G-Fe3O4The spectrum analysis of the composite material, as can be seen from c) in FIG. 1, CTAB-G-Fe3O4The composite material has the element composition of C, N, O, Fe, Cu and Br, wherein Cu is used for preparing the composite materialFrom copper mesh in TEM testing; the C element is derived from CTAB-G-Fe3O4Carbon coating on the composite material and the copper mesh; n, O, Fe and Br from CTAB-G-Fe3O4A composite material.
2) CTAB-G-Fe prepared in example 13O4Composite material and G-Fe3O4XRD testing was performed, and the results are shown in FIG. 2; as can be seen from FIG. 2, CTAB-G-Fe3O4Composite material and G-Fe3O4Fe appears at 30.1 °, 35.5 °, 43.1 °, 53.4 °, 57.0 ° and 62.6 ° 2 θ3O4The characteristic peaks of the nanoparticles are consistent with those of the standard JCPDS (PDF # 89-0691). In addition, both composites have a hump at 25.5 ° 2 θ, which is attributed to the diffraction peak of graphene. The results show that graphene and Fe3O4The nanoparticles are fully bound. With G-Fe3O4Compared with the XRD curve of CTAB-G-Fe3O4The composite material has diffraction peaks at 16.9 °, 20.4 ° and 23.8 ° due to CTAB crystallization, which indicates CTAB and G-Fe3O4The composite material was successfully bonded.
3) CTAB-G-Fe prepared in example 1 was subjected to IR spectroscopy (FTIR, Nicolet 6700)3O4Composite material and G-Fe3O4Infrared tests were performed and the results are shown in figure 3; as can be seen in FIG. 3, the two materials are at 3450cm-1And 1580cm-1Has absorption peaks caused by tensile and bending vibration caused by adsorption of-OH and H-O-H bonds in water by the composite material. CTAB-G-Fe3O4Composite material and G-Fe3O4The composite material is at 1110cm-1Has an absorption peak, and is from the stretching vibration of the C-O-C bond. In addition, the two materials are at 582cm-1The absorption peak from Fe-O bond proves that Fe exists in the material3O4. With G-Fe3O4Composite material comparison, CTAB-G-Fe3O4The composite material is in 2926cm-1And 2855cm-1The absorption peak is shown, which is the stretching vibration of the C-H bond in the alkyl chain, and proves that the composite material contains CTAB.
4) CTAB-G-Fe prepared in example 1 was subjected to X-ray photoelectron spectroscopy (XPS, Thermo ESCALAB 250XI)3O4The composite was subjected to XPS test and the results are shown in FIG. 4. In FIG. 4, a is the full spectrum, b is Fe2p, C is N1s, d is O1s, and e is C1 s. As can be seen from a in FIG. 4, CTAB-G-Fe3O4The composite material mainly comprises four elements of Fe, O, C and N. As can be seen from b in FIG. 4, Fe2p has two peaks at binding energies 709.9eV and 723.5eV, and the corresponding orbitals are Fe2p1/2With Fe2p3/2. As can be seen in c in fig. 4, a peak N1s appears at a binding energy of 398.5eV, indicating that CTAB successfully modifies the composite. The result of the peak fitting to the O1s spectrum is shown as d in fig. 4. As can be seen from d in fig. 4, the oxygen element has Fe — O bond at 528.9eV and C ═ O bond at 531.6 eV. The results after peak fitting to the C1s spectrum (e in FIG. 4) show C-C, C-OH and C-N bonds at 283.2eV, 284.3eV and 285.4eV, respectively. This result is consistent with FTIR, indicating that the oxygen-containing functional groups are present on the reduced surface of GO, and CTAB successfully modified the composite.
5) CTAB-G-Fe prepared in example 1 was subjected to Zeval potentiostat (Malvern Zetasizer Nano ZS90)3O4Composite material and G-Fe3O4Zeta potential analysis was performed by adding CTAB-G-Fe to 1.6mL of PBS buffers (pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0, respectively) at different pH values3O4The composite materials were each made to have a concentration of 0.2mg/mL, and the resulting mixed solution was then tested for zeta potential. For comparison, 1.6mL PBS buffer solutions with different pH values were added with equal amounts of G-Fe3O4The zeta potential of the resulting mixture was measured for the composite material, and the results are shown in FIG. 5. As can be seen from FIG. 5, CTAB-G-Fe was observed in the system at different pH values3O4The zeta potential of the composite material is higher than that of G-Fe3O4Composite material and the difference is larger as the pH increases. G-Fe3O4The isoelectric point of the composite material is 9.3, and CTAB-G-Fe3O4The isoelectric point of the composite material is greater than 10. The results show that CTAB-G-Fe3O4The surface charge of the composite material is larger than G-Fe3O4The composite material shows that the former is more favorable for adsorbing virus.
6) CTAB-G-Fe prepared in example 1 was subjected to degassing for 5 hours by a specific surface area analyzer (Quantachrome Autosorb iQ Station 1, 150 ℃ C.)3O4Composite material for N2The results of the adsorption-desorption analysis are shown in FIG. 6. As can be seen from FIG. 6, CTAB-G-Fe3O4N of composite material2Adsorption and desorption are typical IV curves. A hysteresis loop is arranged between the relative pressure of 0.6 and 1.0, which indicates that the composite material has a porous structure. Furthermore, CTAB-G-Fe3O4The specific surface area of the composite material was 41.1m2/g。
7) CTAB-G-Fe prepared in example 13O4The magnetic saturation analysis of the composite material was performed to analyze the magnetic properties of the composite material, and the results are shown in fig. 7. As can be seen from FIG. 7, CTAB-G-Fe3O4The composite material has an S-shaped magnetization curve, does not have a hysteresis loop, almost has no hysteresis phenomenon, and has good superparamagnetism. CTAB-G-Fe3O4The magnetic saturation strength of the composite material was 49.7 emu/g. The result shows that CTAB-G-Fe can be quickly and conveniently applied after the magnetic field is applied3O4The composite material is separated from the liquid phase.
Example 2
CTAB-G-Fe prepared in example 13O4Composite materials a 1.6mL virus enrichment system for different viruses was established comprising 1.4mL PBS buffer (concentration 0.01mM, pH 7.4), 100 μ L virus suspension (concentration see test example 1) and 100 μ L CTAB-G-Fe3O4PBS Dispersion of composite (0.2mg/mL), 1.4mL PBS buffer, 100. mu.L virus suspension and 100. mu.L CTAB-G-Fe3O4Mixing PBS dispersion liquid of the composite material to obtain 1.6mL of virus enrichment system; the viruses in the virus suspension are norovirus, rotavirus, adenovirus and new coronavirus pseudovirus respectively.
In the following test examples, the quantitative PCR reaction systems for norovirus, rotavirus and adenovirus are, in order, Stals A, et al (2012), Molecular detection and genetic of norviruses, food and environmental virology 4(4): 153-; (Jin M, et al (2014) Development of a novel filter card system with electrophoretic gain medium to concentration viruses from large volumes of natural surface water & engineering 48(12): 6947. about. 6956; Xagoraraki I, et al (2007) occupancy of human adonovation languages. applied and environmental microbiology 73(24): 7874. about. 7881). The fluorescent quantitative primer of the luc gene of the new coronavirus pseudovirus is luc-F: 5'-ACCTACGCCGAGTACTTCGAGA-3', respectively; luc-R: 5'-ACACCGATGAACAGGGCACCCAA-3', respectively; the probe is luc-TaqMan: 5 '-FAM-TCGCTGCACACCACGATCCGA-TAMRA-3'), and then establishing a new quantitative PCR system for coronavirus by referring to the above quantitative PCR system for norovirus.
Test example 1
Sources of virus samples used in this test example: the GII.4 type norovirus is a sample collected by a hospital, and a norovirus positive specimen is preserved after extraction); human rotaviruses (Human rotaviruses strain wa, HRVWA) (ATCC VR-2018) and Human adenovirus type 41 (Human adenovirus type 41, HAdV41) (ATCCVR-930) were purchased from american type Culture Collection, ATCC (american type Collection, ATCC), and neocoronavirus pseudoviruses were purchased from gibbon biotechnology (shanghai) ltd. The virus samples were stored in a freezer at-80 ℃.
The test method comprises the following steps:
to a 5mL centrifuge tube were added 1.4mL of the PBS buffer (concentration 0.01mM, pH 7.4) of example 2 and 100 μ L of the virus suspension (gene copy number 10)4/mL), mixing well, adding 100 μ L CTAB-G-Fe3O4PBS dispersion of composite material (concentration 0.2mg/mL), then placing 5mL centrifuge tube in constant temperature shaking table, incubating at 150r/min for 20min (i.e. adsorption time), and then incubating CTAB-G-Fe in the obtained system by magnet3O4Separating the composite material from the liquid phase to obtain a supernatant;
taking 140. mu.L of supernatant, and using it against RNA virus
RNA of RNA virus was extracted using Viral RNAMini Kit, and PrimeScript from TaKaRa was usedTM1st Strand cDNA Synthesis Kit for reverse transcription of viral RNA; aiming at DNA virus, the DNA of the DNA virus is extracted by using a UNlQ-10 column type virus genome DNA extraction kit of Shanghai biological engineering Co., Ltd, and then the residual content of the virus in the supernatant is determined by an established quantitative PCR reaction system. Meanwhile, a control group is set, namely only 1.5mL of PBS buffer solution and 100 mu L of virus suspension are added into a virus enrichment system, and CTAB-G-Fe is not added3O4And (3) after incubation of the PBS dispersion liquid of the composite material, determining the residual content of the virus in the supernatant through the quantitative PCR reaction system.
The enrichment efficiency calculation formula is as follows:
wherein the total virus copy number is CTAB-G-Fe3O4Viral copy number in supernatant when composite dispersion.
1) At 104Measuring CTAB-G-Fe under different conditions according to the above test method for gene copy number norovirus3O4The effect of the composite on the virus enrichment efficiency, the results are shown in fig. 8:
with graphene (G) and G-Fe3O4In comparison, a in FIG. 8 is a graph showing the enrichment efficiency of different enrichment materials for viruses at different concentrations (PBS buffer pH 7.4, adsorption time 20 min). As can be seen from a in FIG. 8, CTAB-G-Fe increases with the concentration of the enrichment material3O4Composite material, G-Fe3O4The removal efficiency of the composite material and graphene (G) against norovirus tends to increase. This is due to the increased concentration of the enrichment material, with a concomitant increase in the active sites in the enrichment system. When CTAB-G-Fe3O4When the concentration of the composite material is 0.2mg/mL, the enrichment efficiency of the composite material on viruses reaches the maximum value of 99.38%. At the same concentration level, CTAB-G-Fe3O4The enrichment efficiency of the composite material to the virus is larger than that of G-Fe3O4Composite material and graphene. As can also be seen from a in FIG. 8, when the concentration of the enrichment material is higher than 0.125mg/mL, the virus enrichment efficiency of the graphene is higher than that of G-Fe3O4Composite material, which may be due to the same quality of graphene and G-Fe3O4Compared with the composite material, the graphene component of the former is larger than that of the latter, which is more beneficial to adsorbing viruses. When the concentration of the enrichment material is lower than 0.125mg/mL, the virus enrichment efficiency of the graphene is lower than that of G-Fe3O4Composite materials, which may be due to unnecessary losses due to easy agglomeration of graphene, and difficult recycling of graphene. The above results show that CTAB-G-Fe is in the system3O4When the composite material is 0.2mg/mL, the high virus enrichment efficiency and the rapid separation performance can be simultaneously ensured, and the composite material is suitable for adsorbing viruses.
In FIG. 8 b is CTAB-G-Fe3O4Enrichment efficiency curve chart (CTAB-G-Fe) of composite material on virus at different adsorption time (namely incubation time)3O4The composite concentration was 0.2mg/mL, PBS buffer pH 7.4). As can be seen from b in FIG. 8, CTAB-G-Fe increases with the adsorption time3O4The enrichment efficiency of the composite material on the virus gradually increases and reaches a maximum value of 99.62 percent at 20 min. Thereafter, the enrichment efficiency remained unchanged with increasing time. This indicates that CTAB-G-Fe was present at an adsorption time of 20min3O4The composite material can sufficiently adsorb the norovirus in the system.
In FIG. 8 c is CTAB-G-Fe3O4Enrichment efficiency curve diagram (CTAB-G-Fe) of composite material under different pH values3O4The composite concentration is 0.2mg/mL, and the adsorption time is 20 min). As can be seen from c in FIG. 8, the enrichment efficiency did not change much as the pH of the enrichment system increased. This may be in comparison with CTAB-G-Fe3O4The isoelectric point of the composite material is greater than 10. Thus, for ease of handling, the experiment determined the pH of the enrichment system to be 7.4 consistent with the PBS buffer used. The results show that CTAB-G-Fe in the enrichment system3O4The composite material is 0.2mg/mL, the adsorption time is 20min, and when the pH value of the enrichment system is 7.4, the enrichment efficiency of the virus enrichment system is optimal.
2) Investigation of CTAB-G-Fe3O4Enrichment efficiency of composite materials for different viruses (norovirus, rotavirus, adenovirus and new coronavirus pseudoviruses):
when the virus in the 1.6mL virus-enriched system established in example 2 was norovirus (HuNoV GII), the gene copy numbers of the virus suspensions were 1.42X 10, respectively6、1.08×105And 1.20X 104The enrichment efficiencies were calculated according to the above test method, and the corresponding enrichment efficiencies were 93.77%, 97.73% and 100%, respectively, as shown in fig. 9;
when the virus in the 1.6mL virus-enriched system established in example 2 was rotavirus (HAdV), the gene copy numbers of the virus suspensions were 1.51X 10, respectively6、1.55×105And 1.13X 104The enrichment efficiencies were calculated according to the above test method, and the corresponding enrichment efficiencies were 87.22%, 100%, and 100%, respectively, as shown in fig. 9;
when the virus in the 1.6mL virus-enriched system established in example 2 was adenovirus (HRV), the gene copy numbers of the virus suspensions were 1.11X 10, respectively6、1.49×105And 1.31X 104The enrichment efficiencies were calculated according to the above test method, and the corresponding enrichment efficiencies were 74.01%, 100%, and 100%, respectively, as shown in fig. 9;
when the virus in the 1.6mL virus-enriched system established in example 2 was a novel coronavirus Pseudovirus (SARS-CoV-2Spike Pseudovirus), the gene copy numbers of the virus suspensions were 2.37X 10, respectively6、3.65×105And 9.13X 104The enrichment efficiency was calculated according to the above test method, and the corresponding enrichment efficiencies were 92.2%, 100%, and 100%, respectively, as shown in fig. 9.
As can be seen from FIG. 9, the virus enrichment system established by the virus enrichment material of the present invention has high enrichment efficiency for norovirus, rotavirus, adenovirus and new coronavirus pseudoviruses.
Example 3
CTAB-G-Fe prepared in example 13O4Composite materials four 1.6mL virus enrichment systems were established, comprising 1.4mL pbs buffer (concentration 0.01mM, pH 7.4), 100 μ L virus suspension (concentration see test example 2) and 100 μ L CTAB-G-Fe3O4PBS Dispersion of composite (0.2mg/mL), 1.4mL of tap water/sea river water/sea water/PBS buffer, 100. mu.L of virus suspension and 100. mu.L of CTAB-G-Fe3O4Mixing PBS dispersion liquid of the composite material to obtain 1.6mL of virus enrichment system; the viruses in the virus suspension are norovirus, rotavirus, adenovirus and new coronavirus pseudovirus respectively. Meanwhile, tap water, sea river water and sea water are used for replacing the PBS buffer solution respectively, and virus enrichment systems of different water samples are obtained.
Test example 2
The virus samples used in this test example were from the same sources as in test example 1.
The test method comprises the following steps:
1.4mL of PBS buffer (or tap water, sea river water or sea water) and 100. mu.L of the virus suspension of example 3 were added to a 5mL centrifuge tube, mixed, and then 100. mu.L of CTAB-G-Fe was added3O4PBS dispersion of composite material (0.2mg/mL), then placing 5mL centrifuge tube in constant temperature shaking table, incubating at 150r/min for 20min, then incubating CTAB-G-Fe in the obtained system by magnet3O4Separating the composite material from the liquid phase to obtain a supernatant;
the virus nucleic acid extraction, quantification and virus enrichment efficiency calculation were the same as in test example 1;
when the number of gene copies of viruses in the virus suspension in 1.6mL of the virus-enriched system (PBS buffer) established in example 3 was 2.75X 105Has a norovirus gene copy number of 8.57X 104The rotavirus and gene copy number is 1.58 multiplied by 105Has a gene copy number of 3.65X 105When the coronavirus pseudovirus is new, the enrichment efficiency is calculated according to the test method of test example 1, and the corresponding enrichment efficiencies are 69.2%, 100%, 66.0% and 96.6%, respectively, as shown in fig. 10.
1.6mL of virus established in example 3The virus in the virus suspension in the enrichment system (tap water) has the gene copy number of 2.75 multiplied by 105Has a norovirus gene copy number of 8.57X 104The rotavirus and gene copy number is 1.58 multiplied by 105Has a gene copy number of 3.65X 105When the coronavirus pseudovirus is new, the enrichment efficiency is calculated according to the test method of test example 1, and the corresponding enrichment efficiencies are 76.27%, 81.98%, 100% and 100%, respectively, as shown in fig. 10.
When the number of gene copies of the viruses in the virus suspension in the 1.6mL virus enrichment system (sea and river) established in example 3 was 2.75X 105Has a norovirus gene copy number of 8.57X 104The rotavirus and gene copy number is 1.58 multiplied by 105Has a gene copy number of 3.65X 105When the coronavirus pseudovirus is new, the enrichment efficiency is calculated according to the test method of test example 1, and the corresponding enrichment efficiencies are 72.36%, 82.70%, 100% and 100%, respectively, as shown in fig. 10.
When the virus in the virus suspension in the 1.6mL virus enrichment system (seawater) established in example 3 has the gene copy number of 2.75 multiplied by 105Has a norovirus gene copy number of 8.57X 104The rotavirus and gene copy number is 1.58 multiplied by 105Has a gene copy number of 3.65X 105When the coronavirus pseudovirus is new, the enrichment efficiency is calculated according to the test method of test example 1, and the corresponding enrichment efficiencies are 81.79%, 100% and 90.5%, respectively, as shown in fig. 10.
Example 4
CTAB-G-Fe prepared in example 13O4Composite materials a 40mL virus enrichment system was established for five different virus suspension concentrations, comprising 39.4mL PBS buffer (concentration 0.01mM, pH 7.4), 100 μ L virus suspension (concentration see test example 3) and 500 μ L CTAB-G-Fe3O4PBS Dispersion of composite (0.04mg/mL), 39.4mL PBS buffer, 100. mu.L virus suspension and 500. mu.L CTAB-G-Fe3O4Mixing PBS dispersion liquid of the composite material to obtain a 40mL virus enrichment system; the viruses in the virus suspension are respectivelyNorovirus, rotavirus, adenovirus and neocoronavirus pseudoviruses.
Test example 3
The virus samples used in this test example were from the same sources as in test example 1.
The test method comprises the following steps:
39.4mL of PBS buffer (pH 7.4) from example 4 and 100. mu.L of the virus suspension were added to a 50mL centrifuge tube, mixed, and 500. mu.L of CTAB-G-Fe was added3O4PBS dispersion of composite material (concentration 0.01mM), then placing 50mL centrifuge tube in constant temperature shaking table, incubating at 150r/min for 60min, then incubating CTAB-G-Fe in the obtained system by magnet3O4Separating the composite material from the liquid phase to obtain CTAB-G-Fe3O4A composite material;
taking the obtained CTAB-G-Fe3O4Composite material, use of
RNA of RNA virus was extracted using Viral RNAMini Kit, and PrimeScript from TaKaRa was usedTM1st Strand cDNA Synthesis Kit for reverse transcription of viral RNA; extracting DNA of the DNA virus by using a UNlQ-10 columnar virus genome DNA extraction kit of Shanghai biological engineering Co., Ltd; and then determining the content of the virus adsorbed by the composite material through an established quantitative PCR reaction system. Meanwhile, a control group is set, namely only 39.9mL of PBS buffer solution and 100 mu L of virus suspension are added into a virus enrichment system, and CTAB-G-Fe is not added3O4And (3) after incubation of the PBS dispersion liquid of the composite material, detecting the virus in the system through a quantitative PCR reaction system.
When the virus in the 40mL virus enrichment system established in example 4 has a gene copy number of 3.6X 104、2.0×103、2.2×102、9.9×101And 4.3 norovirus (HuNoV GII), virus detection was calculated according to the above test method, and blank control (i.e., no CTAB-G-Fe was added)3O4Enriched material) results are shown in table 1;
40mL of virus established in example 4The virus in the virus suspension in the enrichment system has the gene copy number of 1.2 multiplied by 104、1.0×103、1.7×102、3.2×101And 1.1 rotavirus (HAdV), virus detection was calculated according to the above test method, and blank control (i.e., no CTAB-G-Fe was added) was performed3O4Enriched material) results are shown in table 1;
when the virus in the 40mL virus enrichment system established in example 4 has a gene copy number of 1.1X 104、1.3×103、1.6×102、1.0×101And adenovirus (HRV) of 1.3, virus detection was calculated according to the above test method, and blank control (i.e., no CTAB-G-Fe was added)3O4Enriched material) results are shown in table 1;
when the virus in the 40mL virus enrichment system established in example 4 has a gene copy number of 2.1X 104、2.1×103、4.2×102、4.2×101And 4.2 New coronavirus Pseudovirus (SARS-CoV-2Spike Pseudovirus), detection of the virus was calculated according to the above-described test method, and a blank control was performed (i.e., CTAB-G-Fe was not added)3O4Enriched material) results are shown in table 1.
Table 1 shows the virus detection in the 40mL virus enrichment system established in example 4
As can be seen from Table 1, no virus could be detected without adding enrichment material, and CTAB-G-Fe was used in the established 40mL virus enrichment system3O4After the composite material is enriched, the virus detection condition is better than that of a detection method without enrichment.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. The virus enrichment material is characterized by comprising graphene ferroferric oxide particles and CTAB attached to the surfaces of the graphene ferroferric oxide particles;
the mass ratio of CTAB to graphene ferroferric oxide particles is (5-6): 1;
the virus is food-borne virus or new corona virus.
2. The method for preparing a virus-enriched material according to claim 1, comprising the steps of:
and dispersing the graphene ferroferric oxide particles in ultrapure water, mixing the obtained dispersion liquid with CTAB, and freeze-drying to obtain the virus enrichment material.
3. Use of the virus-enriched material according to claim 1 or the virus-enriched material prepared by the preparation method according to claim 2 for enriching food-borne viruses or neocoronaviruses.
4. A virus enrichment system is characterized by comprising a PBS buffer solution, a virus suspension and a virus enrichment material dispersion liquid; the virus-enriched material in the virus-enriched material dispersion is the virus-enriched material according to claim 1 or the virus-enriched material prepared by the preparation method according to claim 2.
5. The virus enrichment system of claim 4, wherein the volume ratio of the PBS buffer solution to the virus suspension to the virus enrichment material dispersion is (1-100): (0-0.5): (0-1), and the volumes of the virus suspension and the virus-enriched material dispersion liquid are not 0.
6. The virus enrichment system of claim 4 or 5, wherein the PBS buffer has a concentration of 0.01mM and a pH of 7.4.
7. The virus enrichment system of claim 4 or 5, wherein the concentration of the virus-enriched material dispersion is 0-0.25 mg/mL and is not 0.
8. Use of the virus enrichment system of any one of claims 4 to 7 for the enrichment of viruses.
9. The use according to claim 8, wherein the virus is a food-borne virus or a neocoronavirus.
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