CN111007251B - Method for detecting norovirus by nano enzyme test strip - Google Patents
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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Abstract
The application discloses a method for detecting norovirus by a nano enzyme test strip, which comprises the following steps: (1) synthesis of nano-enzyme; (2) preparation of nano enzyme-nori antibody probe; (3) assembling a nano enzyme immunochromatographic test strip; (4) And detecting the norovirus by the nano enzyme immunochromatography test strip. The sensitivity of the nano enzyme immunochromatographic test strip in detection can be improved by 10 times compared with that of a colloidal gold test strip, and the nano enzyme immunochromatographic test strip has high sensitivity and reliability in detection of norovirus and has specific recognition on the norovirus. The method for detecting the norovirus by using the nano enzyme test strip has the advantages of simplicity, convenience, rapidness and field application of a colloidal gold technology, has the characteristic of high sensitivity, and has wide development prospect.
Description
Technical Field
The application relates to the technical field of norovirus detection. In particular to a method for detecting norovirus by a nano enzyme test strip.
Background
Norovirus (also known as Norwalk virus), a member of the caliciviridae family, is one of the main causes of human viral gastroenteritis, accounting for about 18% of diarrhea cases worldwide. In developed countries, norovirus is considered the most common cause of outbreaks of non-bacterial diarrhea in children and adults, causing approximately 1900-2100 tens of thousands of patients to die in the united states alone each year, 56000-71000 in hospital, 570-800. Although rotavirus is considered to be the main cause of diarrhea in children in developing countries, norovirus is rapidly becoming the most common pathogen in severe gastroenteritis in children in countries where rotavirus vaccines have been incorporated into immune programs. A recent study considered that more than 20 tens of thousands of children died from norovirus in developing countries.
Therefore, the norovirus still has certain infectivity and harm, and at present, the method for detecting the norovirus mainly comprises the steps of nucleic acid detection, ELISA, colloidal gold and the like, and when the nucleic acid detection and ELISA method are complex in technology, the operation of professional technicians is required, and the sensitivity of the colloidal gold test strip is low, so that the rapid and sensitive detection method of the norovirus is very necessary to be developed.
The norovirus is a non-enveloped single-strand positive-strand RNA virus, has high mutation speed, can generate new variants causing global epidemic every 2-3 years, has strong environmental resistance, low infection dosage, short post-infection latency period, long toxin expelling time, short immune protection time, various transmission paths and general susceptibility of the whole population, and therefore, the norovirus has high infectivity and quick transmission capability. The onset of norovirus infection is mainly manifested by diarrhea and/or vomiting, commonly referred to internationally as acute gastroenteritis. The report management of other infectious diarrhea diseases in the class-C infectious diseases is always carried out in China, and the report of norovirus infection cases taking emesis as a main symptom and outbreaks of norovirus infection cases is affected to a certain extent. In addition, the norovirus has self-healing property, so that many hospitals do not check the norovirus, and thus the monitoring of the norovirus is ignored. Norovirus is the main causative agent of sporadic cases and outbreaks of acute gastroenteritis worldwide, and has serious disease burden. Thus, detection and control of norovirus should be enhanced.
In the aspect of detection, norovirus has no protein envelope, and in vitro culture is not performed at present, so that virus separation and serotyping identification are limited. The identification is only carried out by biochemical methods and molecular biological methods.
The main detection methods at present are as follows:
1. nucleic acid detection and genotyping mainly include Real-time RT-PCR and traditional PCR, which have relatively high accuracy, but complicated procedures, and require special instruments and equipment and specialized technicians.
2. Antigen detection
The antigen detection mainly comprises an ELISA method, and a large number of commercial kits have been developed, which can be suitable for screening a large number of samples in epidemic situations, but the ELISA kit has high cost and low sensitivity, needs special technicians to operate, and is not simple and rapid. Colloidal gold detection is the most common rapid detection in the market at present, but has relatively low sensitivity. Currently, there are also studies on immunochromatography methods such as nanogold. So as to seek a simple, convenient and high-sensitivity detection method.
Disclosure of Invention
Therefore, the technical problem to be solved by the application is to provide a method for detecting the norovirus by using the nano enzyme test strip with high detection sensitivity and simple and convenient operation.
In order to solve the technical problems, the application provides the following technical scheme:
the method for detecting the norovirus by the nano enzyme test strip comprises the following steps:
(1) Synthesizing nano enzyme;
(2) Preparing nano enzyme-nori antibody probes;
(3) Assembling a nano enzyme immunochromatography test strip;
(4) And detecting the norovirus by the nano enzyme immunochromatography test strip.
In the method for detecting the norovirus by using the nano-enzyme test strip, in the step (1), a hydrothermal method is adopted to synthesize nano-enzyme: dissolving FeCl3.6H2O in ethylene glycol, stirring for dissolving, adding sodium acetate, sealing in a high-pressure reaction kettle after the mixture is completely dissolved, performing reaction, magnetically separating the obtained product, discarding the supernatant, washing the precipitate with ethanol, and drying and preserving with an electric heating constant-temperature drying oven to obtain the nano enzyme.
In the method for detecting the norovirus by using the nano-enzyme test strip, in the step (1), a hydrothermal method is adopted to synthesize nano-enzyme: dissolving FeCl3.6H2O in ethylene glycol, stirring for dissolving, adding a certain amount of sodium acetate, sealing in a high-pressure reaction kettle after the mixture is completely dissolved, reacting at 200 ℃ for 14 hours, magnetically separating the obtained product, discarding the supernatant, washing the precipitate with ethanol for 3 times, and then drying and preserving the precipitate with an electric heating constant-temperature drying oven to obtain the nano enzyme.
In the method for detecting the norovirus by the nano-enzyme test strip, in the step (2),
(2-1) taking the nano-enzyme prepared in the step (1), and washing with deionized water;
(2-2) centrifuging, and discarding the supernatant; the sediment is resuspended by MES buffer solution, EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) are added at the same time, and after being evenly mixed, the mixture is placed in a shaking table for activation;
(2-3) after activation, washing with an MES buffer solution, and then resuspending with the MES buffer solution to obtain an activated nano-enzyme;
(2-4) incubation of norovirus murine monoclonal antibody with activated nanoenzyme solution overnight;
(2-5) adding Tris buffer to terminate the reaction;
(2-6) after magnetic separation, re-suspending with 5% BSA-Tris buffer to obtain the nano enzyme-nore antibody probe.
In the method for detecting the norovirus by the nano-enzyme test strip, in the step (2),
(2-1) taking 500. Mu.L of the nano-enzyme prepared in the step (1), wherein the concentration of the nano-enzyme is 5mg/mL, and washing with 1mL of deionized water three times;
(2-2) centrifuging, and discarding the supernatant; the pellet was resuspended in 1mL of MES buffer, pH 6.0, with 20. Mu.L of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 50. Mu.L of NHS (N-hydroxysuccinimide) added, mixed and placed in a shaker for 30min; EDC concentration is 0.4mol/L, NHS concentration is 0.1mol/L; the rotation speed of the shaking table is 70rpm;
(2-3) after activation, washing once with 1mL of MES buffer solution, and then resuspension with 500 μl of MES buffer solution, to obtain activated nanoenzyme;
(2-4) taking 50 mug of norovirus mouse monoclonal antibody and incubating the activated nano-enzyme solution overnight, wherein the incubation temperature is 4 ℃;
(2-5) incubating with Tris buffer at room temperature for 30min, and terminating the reaction; the concentration of the Tris buffer is 0.05mol/L and the pH is 7.2;
(2-6) after magnetic separation, re-suspending with 0.5mL of 5% BSA-Tris buffer to obtain the nano enzyme-nore antibody probe.
The method for detecting the norovirus by the nano-enzyme test strip comprises the following steps in the step (3):
(3-1) drawing a quality control line C and a detection line T on the nitrocellulose membrane (4): marking a line on a nitrocellulose membrane by using a goat anti-mouse antibody with the concentration of 1mg/mL as a quality control line; marking a line on a nitrocellulose membrane by using the murine norubicin with the concentration of 2mg/mL as a detection line, wherein marking parameters are 0.1 mu L/mm, and the distance between a quality control line and the detection line is 7mm; after scribing, placing the nitrocellulose membrane (4) in an incubator for drying for 1h at a drying temperature of 40 ℃;
(3-2): spraying nano enzyme-nore antibody probes on the bonding pad (3): diluting the coupled nano enzyme-nore probe with 1% BSA-Tris buffer solution to prepare the nano enzyme-nore probe with the concentration of 1mg/mL; spraying the diluted nano enzyme-nori probe onto the bonding pad (3) with a spraying parameter of 0.5 mu L/mm; then the bonding pad (3) is placed in an incubator for drying for 1h, and the drying temperature is 40 ℃;
(3-3): assembling a test strip: sequentially attaching a sample pad (2), a bonding pad (3), a nitrocellulose membrane (4) and an absorption pad (5) to a bottom card (1), enabling a detection line on the nitrocellulose membrane (4) to be close to the bonding pad (3), and enabling a quality control line on the nitrocellulose membrane (4) to be close to the absorption pad (5); the sample pad (2) and the bonding pad (3) are both glass fiber membranes, and the absorption pad (5) is water-absorbing filter paper; assembling into a complete nano enzyme immunochromatographic test strip, and then cutting into test strips with the width of 4mm by a strip cutting machine;
(3-4) covering a clamping shell (6) on each nano enzyme immunochromatographic test strip, wherein a sample adding hole (6-1) and a color development hole (6-2) are formed in the clamping shell (6); the sample adding hole (6-2) is covered on the sample pad (2), the color development hole (6-2) is covered on the nitrocellulose membrane (4), and the quality control line and the detection line are positioned in the color development hole (6-2), after the assembly of the nano enzyme immunochromatographic test strip is completed, the assembled nano enzyme immunochromatographic test strip is placed in a sealing bag and dried and stored at room temperature.
The method for detecting the norovirus by the nano-enzyme test strip in the step (4) comprises the following steps:
(4-1) taking out the nano enzyme immunochromatographic test strip, adding 100 mu L of nore antigen liquid into the sample adding hole (6-2), starting chromatographic detection, and timing for 10 minutes; after chromatography, adding 1mL of nano enzyme substrate color development liquid into a color development hole (6-2) of the nano enzyme immunochromatographic test strip, so that the color development hole (6-2) is filled with the nano enzyme substrate color development liquid, and developing for 5-10 minutes in a dark place; the nano enzyme substrate color development liquid is diaminobenzidine DAB and hydrogen peroxide;
and (4-2) when the color development is carried out for 5-10 minutes in a dark place, absorbing the nano enzyme substrate color development liquid from the sample adding hole (6-2) by using a liquid transferring gun, then observing color signals of a quality control line and a detection line on the nano enzyme immunochromatographic test strip, and judging that the detection result is invalid after exceeding 15 minutes.
The method for detecting the norovirus by the nano enzyme test strip comprises the following steps of: if the nano enzyme immunochromatography test strip simultaneously shows a quality control line and a detection line, the detection result is positive, namely the sample to be detected contains the norovirus; if the norovirus nano-enzyme immunochromatographic test strip only has a quality control line, the detection result is negative, namely the sample to be detected has no norovirus; if the norovirus nano-enzyme immunoassay test strip only shows a detection line, the detection result is invalid and detection needs to be repeated.
The technical scheme of the application has the following beneficial technical effects:
the application uses immunochromatography technology, takes norovirus as a detection sample, replaces traditional colloidal gold with nano-enzyme by utilizing the characteristic that nano-enzyme has catalytic activity of peroxidase, researches the application of nano-enzyme immunochromatography test strips in norovirus detection, and can remarkably enhance detection signals and achieve the effect of higher detection sensitivity by adding catalytic substrate DAB of enzyme after immunochromatography. The sensitivity of the nano enzyme immunochromatographic test strip can be improved by 10 times compared with that of a colloidal gold method, and the nano enzyme immunochromatographic test strip has high sensitivity and reliability for detecting norovirus and has specific recognition for the norovirus. The method for detecting the norovirus by using the nano enzyme test strip has the advantages of simplicity, convenience, rapidness and field application of a colloidal gold technology, has the characteristic of high sensitivity, and has wide development prospect.
Drawings
FIG. 1 is a schematic diagram of a nano-enzyme immunochromatographic test strip structure of a method for detecting norovirus by using the nano-enzyme test strip of the present application;
FIG. 2 is a schematic diagram of a structure of a nano-enzyme immunochromatographic test strip capped with a cartridge in a method for detecting norovirus by using the nano-enzyme test strip of the present application;
FIG. 3 is a schematic diagram of the detection of the nano-enzyme immunochromatographic strip of the method for detecting norovirus by the nano-enzyme strip of the present application;
FIG. 4 is a diagram showing a transmission electron microscope characterization of a nanoenzyme in a method for detecting a norovirus by using the nanoenzyme test strip of the application;
FIG. 5 is a graph showing dynamic light scattering characterization of nanoenzymes of the method for detecting norovirus using the nanoenzyme test strip of the present application;
FIG. 6 is a graph depicting the enzymatic activity of a nanoenzyme material of the method for detecting norovirus using a nanoenzyme test strip of the present application;
FIG. 7 is a detection chart of the nano-enzyme test strip of the application after 10min chromatography;
FIG. 8 is a detection chart of the method for detecting the norovirus by using the nano-enzyme test strip of the application after 5min of color development;
FIG. 9 is a graph showing the detection results of different concentrations of norovirus by the nanoenzyme test strip of the application;
FIG. 10 is a graph showing the detection results of different concentrations of norantigen by using a colloidal gold test strip;
FIG. 11 is a diagram showing the result of specific detection of norovirus by the nanoenzyme test strip of the present application, wherein 1 is rotavirus, 2 is filovirus, 3 is helicobacter pylori, and 4 is norovirus.
The reference numerals in the drawings are as follows: 1-a bottom card; 2-sample pad; 3-a bonding pad; 4-nitrocellulose membrane; 5-an absorbent pad; 6-clamping the shell; 6-1-sample addition hole; 6-2-color development holes.
Detailed Description
The method for detecting the norovirus by the nano enzyme test strip of the embodiment comprises the following steps of;
1. synthesizing nano enzyme;
the nano enzyme is synthesized by adopting a hydrothermal method: 0.3g FeCl 3 ·6H 2 O is dissolved in 20mL of glycol, stirred and dissolved, then 1.5g of sodium acetate is added, after the mixture is completely dissolved, the mixture is sealed in a high-pressure reaction kettle and reacts for 14 hours at 200 ℃, the obtained product is magnetically separated, the supernatant is discarded, the precipitate is washed with ethanol for 3 times, and then the nano-enzyme is obtained by drying and preserving in an electrothermal constant-temperature drying oven at 50-60 ℃.
Wherein: sodium acetate, feCl 3 ·6H 2 O, ethanol and ethylene glycol are purchased from Beijing chemical reagent plant; high temperature ovens were purchased from Tianjin Test.
Characterization of nanoenzyme: the nanoenzymes prepared were characterized by transmission electron microscopy (JEOL 2000fx 200kv, japan electronics company) and dynamic light scattering. The prepared nano enzyme material has very high particle size uniformity: as shown in FIG. 4, the particle size of the nano enzyme material is 110+ -10 nm as characterized by a transmission electron microscope, and as shown in FIG. 5, the particle size of the nano enzyme material is about 130nm as characterized by dynamic light scattering, because a hydration layer is formed on the periphery of the nano enzyme material, and the particle size of the nano enzyme material is slightly increased.
Enzyme activity measurement of nano enzyme: and testing the enzyme catalytic activity of the prepared nano enzyme material by using an enzyme-labeled instrument, and measuring the enzyme activity of the prepared nano enzyme material by using a spectrophotometry.
As shown in fig. 6: the enzyme catalytic activity of the nano enzyme material is tested and analyzed by using an enzyme-labeled instrument, and the prepared nano enzyme material has good peroxidase catalytic activity, and can reach the highest activity value when the catalytic reaction is carried out for 10 min.
The specific activity of the prepared nano enzyme material was measured by a spectrophotometer, and the specific activity of the prepared nano enzyme material was calculated to be 9.33U/mg according to the specific activity standardization article (Standardized assays for determining the catalytic activity and kinetics of peroxidase-like nanozymes, nat Protoc,2018.13 (7): p.1506-1520) published by Jiang et al.
2. Preparation of nano enzyme-nore antibody probe
(2-1) taking 500. Mu.L of the nano-enzyme prepared in the step (1), wherein the concentration of the nano-enzyme is 5mg/mL, and washing with 1mL of deionized water three times;
(2-2) centrifuging, and discarding the supernatant; the precipitate was resuspended in 1mL of MES (2- (N-morpholinoethanesulfonic acid) buffer, pH 6.0, with 20. Mu.L of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 50. Mu.L of NHS (N-hydroxysuccinimide) added, mixed and placed in a shaker for 30min; EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) at a concentration of 0.4mol/L and NHS (N-hydroxysuccinimide) at a concentration of 0.1mol/L; the rotation speed of the shaking table is 70rpm;
(2-3) after activation, washing once with 1mL of MES buffer solution, and then resuspension with 500 μl of MES buffer solution, to obtain activated nanoenzyme;
(2-4) taking 50 mug of norovirus mouse monoclonal antibody and incubating the activated nano-enzyme solution overnight, wherein the incubation temperature is 4 ℃;
(2-5) incubating with Tris buffer at room temperature for 30min, and terminating the reaction; the concentration of the Tris buffer solution is 0.05mol/L, and the pH value is 7.2;
(2-6) after magnetic separation, re-suspending with 0.5mL of 5% BSA-Tris buffer to obtain the nano enzyme-nore antibody probe.
Wherein: EDC is 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 1- (3-dimethylaminoopyl) -3-ethylcarbodiimide hydrochloride, available from Sigma-Aldrich;
NHS is N-Hydroxysuccinimide, N-hydroxycicinimide, available from Sigma-Aldrich
The norovirus murine monoclonal antibody was purchased from BIOCare, and the ELISA was purchased from Bio-Rad.
3. Assembling a nano enzyme immunochromatography test strip; as shown in fig. 1 and 2:
(3-1) scribing a quality control line C and a detection line T on the nitrocellulose membrane 4: marking a line on a nitrocellulose membrane by using a goat anti-mouse antibody with the concentration of 1mg/mL as a quality control line; marking a line on a nitrocellulose membrane by using the murine norubicin with the concentration of 2mg/mL as a detection line, wherein marking parameters are 0.1 mu L/mm, and the distance between a quality control line and the detection line is 7mm; after scribing, placing the nitrocellulose membrane 4 in an incubator for drying for 1h at a drying temperature of 40 ℃;
(3-2): spraying nano enzyme-nore antibody probes on the binding pad 3: diluting the coupled nano enzyme-nori probe into 1% BSA-Tris buffer solution, and preparing to obtain the nano enzyme-nori probe with the concentration of 1mg/mL; spraying the diluted nano enzyme-nori probe onto the bonding pad 3 with a spraying parameter of 0.5 mu L/mm; then placing the bonding pad 3 in an incubator for drying for 1h at 40 ℃;
(3-3): assembling a test strip: sequentially attaching a sample pad 2, a bonding pad 3, a nitrocellulose membrane 4 and an absorption pad 5 to the bottom card 1, enabling a detection line on the nitrocellulose membrane 4 to be close to the bonding pad 3, and enabling a quality control line on the nitrocellulose membrane 4 to be close to the absorption pad 5; the sample pad 2 and the bonding pad 3 are both glass fiber membranes, and the absorption pad 5 is water-absorbing filter paper; assembling into a complete nano enzyme immunochromatographic test strip, and then cutting into test strips with the width of 4mm by a strip cutting machine;
(3-4) covering a clamping shell 6 on each nano enzyme immunochromatographic test strip, wherein a sample adding hole 6-1 and a color development hole 6-2 are formed in the clamping shell 6; the sample adding hole 6-2 is covered on the sample pad 2, the color developing hole 6-2 is covered on the nitrocellulose membrane 4, and the quality control line and the detection line are positioned in the color developing hole 6-2, and after the assembly of the nano enzyme immunochromatography test strip is completed, the assembled nano enzyme immunochromatography test strip is placed in a sealed bag and dried and stored at room temperature.
Wherein: PVDF film (Millipore), cut flow film-jet streaking machine (Arista, USA), slitter (Tianjin Corp. Torch organism), XYZ three-dimensional film-jet gold-spraying machine (Shanghai gold mark).
4. Detection of norovirus by nano enzyme immunochromatography test strip:
4.1, detection principle: as shown in fig. 3, the nano enzyme-antibody probe was constructed by coupling the nano enzyme magnetic particles instead of colloidal gold with norovirus labeled antibodies. When the nano enzyme-antibody probe is combined with the norovirus in the sample, and then the detection line (T line, coated with the capture antibody of the norovirus) and the control line (C line, coated with the goat anti-mouse antibody) are chromatographed to the test strip, the complex of the nano enzyme-antibody probe and the norovirus is combined with the capture antibody at the T line and the goat anti-mouse antibody at the C line, so that the aggregation of nano enzyme particles is formed at the T line and the C line, and then the substrate of peroxidase such as DAB is added, and the enzyme activity of the nano enzyme is utilized to catalyze DAB to generate a large amount of brown precipitate by chemical reaction, and amplify the detection signal, thereby realizing the rapid sensitive detection of the norovirus.
4.2, the specific detection method is as follows:
(4-1) the concentration of the norantigen was diluted to 0ng/ml, 5ng/ml, 10ng/ml, 25ng/ml, 50ng/ml, 100ng/ml, 250ng/ml, 500ng/ml.
(4-2) taking out the nano enzyme immunochromatographic test strip, adding 100 mu L of the norovirus antigen liquid with different concentrations in the step (4-1) into the sample adding hole 6-2, starting chromatographic detection, and timing for 10 minutes; after chromatography, adding 1mL of nano enzyme substrate color development liquid into the color development hole 6-2 of the nano enzyme immunochromatographic test strip to enable the color development hole 6-2 of the nano enzyme substrate color development liquid to be filled up and develop color for 5-10 minutes in a dark place; the nano enzyme substrate color development liquid is diaminobenzidine DAB and hydrogen peroxide;
and (4-3) sucking the nano enzyme substrate color development liquid from the sample adding hole 6-2 by using a liquid-transferring gun when the color development is carried out for 5-10 minutes in a dark place, then observing color signals of a quality control line and a detection line on the nano enzyme immunochromatographic test strip, and judging that the detection result is invalid after exceeding 15 minutes.
As shown in fig. 7 and 8: after the peroxidase substrate DAB is added into the nano enzyme immunochromatography test strip, the color development effect is obviously improved, the macroscopic detection sensitivity is 50ng/mL before color development (chromatography for 10 min), and the macroscopic detection sensitivity is 5ng/mL after color development. The nano enzyme can actually catalyze the peroxidase substrate DAB to generate a color reaction and generate a visual signal, so that the detection effect of the chromatographic test strip is enhanced.
4.3, comparing the detection sensitivity of the nano enzyme test strip and the colloidal gold test strip
The detection method of the colloidal gold test strip is the same as that of the nano enzyme test strip. As shown in fig. 9 and 10: in the eight norovirus antigen concentration detection gradients, the lowest antigen detection concentration of the nano-enzyme immunochromatographic test strip is 5ng/mL, and the lowest antigen detection concentration of the commercial colloidal gold test strip is 50ng/mL, so that the detection sensitivity of the nano-enzyme immunochromatographic test strip to the norovirus antigen is improved by about 10 times.
4.4, specific detection of nano enzyme immunochromatographic test strip
In order to verify the specificity of the nano-enzyme immunochromatography test strip for norovirus on norovirus detection, rotavirus, filovirus and helicobacter pylori are detected in the embodiment, as shown in fig. 11, the prepared nano-enzyme immunochromatography test strip for norovirus can only specifically detect the norovirus, which indicates that the prepared nano-enzyme test strip has very good detection specificity for the norovirus.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While the obvious variations or modifications which are extended therefrom remain within the scope of the claims of this patent application.
Claims (1)
1. The method for detecting the norovirus by using the nano enzyme test strip is characterized by comprising the following steps of:
(1) Synthesizing nano enzyme;
(2) Preparing nano enzyme-nori antibody probes;
(3) Assembling a nano enzyme immunochromatography test strip;
(4) Detecting the norovirus by the nano enzyme immunochromatography test strip;
in the step (1), a hydrothermal method is adopted to synthesize nano enzyme: feCl is added 3 ·6H 2 Dissolving O in glycol, stirring to dissolve, adding a certain amount of sodium acetate, sealing in a high-pressure reaction kettle after the mixture is completely dissolved, reacting at 200deg.C for 14 hr, magnetically separating the obtained product, discarding supernatant, washing the precipitate with ethanol for 3 times, and then electrically heatingDrying and preserving in a constant temperature drying oven to obtain nano enzyme;
in the step (2) of the process,
(2-1) taking 500. Mu.L of the nano-enzyme prepared in the step (1), wherein the concentration of the nano-enzyme is 5mg/mL, and washing with 1mL of deionized water three times;
(2-2) centrifuging, and discarding the supernatant; the pellet was resuspended in 1mL of MES buffer, pH 6.0, with 20. Mu.L of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 50. Mu.L of NHS (N-hydroxysuccinimide) added, mixed and placed in a shaker for 30min; EDC concentration is 0.4mol/L, NHS concentration is 0.1mol/L; the rotation speed of the shaking table is 70rpm;
(2-3) after activation, washing once with 1mL of MES buffer solution, and then resuspension with 500 μl of MES buffer solution, to obtain activated nanoenzyme;
(2-4) taking 50 mug of norovirus mouse monoclonal antibody and incubating the activated nano-enzyme solution overnight, wherein the incubation temperature is 4 ℃;
(2-5) incubating with Tris buffer at room temperature for 30min, and terminating the reaction; the concentration of the Tris buffer is 0.05mol/L and the pH is 7.2;
(2-6) after magnetic separation, re-suspending with 0.5mL of 5% BSA-Tris buffer solution to obtain the nano enzyme-nore antibody probe;
in step (3), the method comprises the following steps:
(3-1) scribing a quality control line C and a detection line T on the nitrocellulose membrane: marking a line on a nitrocellulose membrane by using a goat anti-mouse antibody with the concentration of 1mg/mL as a quality control line; marking a line on a nitrocellulose membrane by using the murine norubicin with the concentration of 2mg/mL as a detection line, wherein marking parameters are 0.1 mu L/mm, and the distance between a quality control line and the detection line is 7mm; after scribing, placing the nitrocellulose membrane in an incubator for drying for 1h at a drying temperature of 40 ℃;
(3-2): spraying nano enzyme-nori antibody probes on the binding pad: diluting the coupled nano enzyme-nori probe with 1% BSA-Tris buffer solution to prepare nano enzyme-nori probe with concentration of 1mg/mL; spraying the diluted nano enzyme-nori probe onto the bonding pad with a spraying parameter of 0.5 mu L/mm; then the bonding pad is placed in a constant temperature box for drying for 1h, and the drying temperature is 40 ℃;
(3-3): assembling a test strip: sequentially attaching a sample pad, a bonding pad, a nitrocellulose membrane and an absorption pad to a bottom card, enabling a detection line on the nitrocellulose membrane to be close to the bonding pad, and enabling a quality control line on the nitrocellulose membrane to be close to the absorption pad; the sample pad and the bonding pad are both glass fiber membranes, and the absorption pad is water-absorbing filter paper; assembling into a complete nano enzyme immunochromatographic test strip, and then cutting into test strips with the width of 4mm by a strip cutting machine;
(3-4) covering a clamping shell on each nano enzyme immunochromatographic test strip, wherein a sample adding hole and a color development hole are formed in the clamping shell; the sample adding hole cover is arranged on the sample pad, the color developing hole cover is arranged on the nitrocellulose membrane, the quality control line and the detection line are positioned in the color developing hole, and after the assembly of the nano enzyme immunochromatography test strip is completed, the assembled nano enzyme immunochromatography test strip is placed in a sealed bag for drying and storage at room temperature;
in step (4), the method comprises the following steps:
(4-1) taking out the nano enzyme immunochromatographic test strip, adding 100 mu L of norovirus antigen liquid into the sample adding hole, starting chromatographic detection, and timing for 10 minutes; after chromatography, adding 1mL of nano enzyme substrate color development liquid into the color development hole of the nano enzyme immunochromatographic test strip to enable the color development hole of the nano enzyme substrate color development liquid to be filled up, and developing for 5-10 minutes in a dark place; the nano enzyme substrate color development liquid is diaminobenzidine DAB and hydrogen peroxide;
(4-2) sucking the nano enzyme substrate color development liquid from the sample adding hole by using a liquid-transferring gun when the color development is carried out for 5-10 minutes in a dark place, then observing color signals of a quality control line and a detection line on the nano enzyme immunochromatography test strip, and judging that the detection result is invalid after exceeding 15 minutes;
the method for judging the detection result comprises the following steps: if the nano enzyme immunochromatography test strip simultaneously shows a quality control line and a detection line, the detection result is positive, namely the sample to be detected contains the norovirus; if the norovirus nano-enzyme immunochromatographic test strip only has a quality control line, the detection result is negative, namely the sample to be detected has no norovirus; if the norovirus nano-enzyme immunochromatographic test strip only shows a detection line, the detection result is invalid and detection is needed again.
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