CN111007251A - Method for detecting norovirus by using 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
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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
- G01N33/558—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
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
Abstract
The invention discloses a method for detecting norovirus by using a nano enzyme test strip, which comprises the following steps: (1) synthesizing nano enzyme; (2) preparing a nano enzyme-nucleic acid antibody probe; (3) assembling the nano enzyme immunochromatographic test strip; (4) and (3) detecting the norovirus by using the nano enzyme immunochromatographic test strip. The detection sensitivity of the nano enzyme immunochromatographic test strip can be improved by 10 times compared with that of a colloidal gold test strip, and the nano enzyme immunochromatographic test strip is high in sensitivity and reliability for detecting norovirus and has specific recognition for norovirus. The method for detecting 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 a wide development prospect.
Description
Technical Field
The invention relates to the technical field of norovirus detection. In particular to a method for detecting norovirus by using a nano enzyme test strip.
Background
Norovirus (also known as Norwalk Viruses), a member of the caliciviridae family, is one of the leading causes of viral gastroenteritis in humans, accounting for approximately 18% of cases of diarrhea worldwide. In developed countries, norovirus is considered to be the most common cause of outbreaks of non-bacterial diarrhea in children and adults, causing about 1900-. Although rotavirus is considered to be the major cause of diarrhea in children in developing countries, norovirus is rapidly becoming the most common pathogen of severe gastroenteritis in children in countries where rotavirus vaccines have been incorporated into immune programs. A recent study suggests that more than 20 million children die of norovirus in developing countries.
Therefore, norovirus still has certain infectivity and harmfulness, and at present, the methods for detecting norovirus mainly comprise nucleic acid detection and ELISA, colloidal gold and the like, and when the nucleic acid detection and ELISA methods are complicated in technology and need to be operated by professional technicians, the colloidal gold test strip is low in sensitivity, so that the development of a rapid and sensitive detection method for norovirus is very necessary.
The norovirus is a non-enveloped single-stranded positive-strand RNA virus, has high mutation speed, can appear as a new variant strain which causes global epidemic every 2-3 years, has strong environmental resistance, low infection dosage, short incubation period after infection, long detoxification time, short immune protection time, various transmission ways and universal susceptibility of the whole population, and therefore, the norovirus has high infectivity and rapid transmission capability. The main manifestations of the onset of norovirus infection are diarrhea and/or vomiting, commonly referred to internationally as acute gastroenteritis. China always lists other infectious diarrhea diseases in the class-C infectious diseases for report management, which influences the report of norovirus infection cases with vomiting as the main symptom and outbreak thereof to a certain extent. In addition, norovirus has self-healing property, so that many hospitals do not check norovirus, and thus the monitoring of norovirus is neglected. Nore viruses are the main causative agents of acute gastroenteritis sporadic cases and outbreaks worldwide, and the burden of the disease is severe. 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 the separation and serotype typing identification of the virus are limited. Identification is only carried out by biochemical methods and molecular biological methods.
The current main detection methods are as follows:
1. nucleic acid detection and genotype identification mainly comprise Real-time RT-PCR and traditional PCR, the accuracy is relatively high, but the process is complicated, and special instruments and equipment and professional technicians are required.
2. Antigen detection
The antigen detection mainly comprises an ELISA method, a large number of commercial kits are developed, and the kit is suitable for screening a large number of samples in outbreak epidemic situations, but the ELISA kit is high in cost and low in sensitivity, needs to be operated by professional technicians, and is not simple, convenient and quick. Colloidal gold detection is the most common rapid detection in the market at present, but the sensitivity is relatively low. At present, immunochromatography methods such as nanogold have been gradually studied. So as to search for a simple, convenient and high-sensitivity detection method.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a method for detecting norovirus by using a nano-enzyme test strip, which has high detection sensitivity and simple and convenient operation.
In order to solve the technical problems, the invention provides the following technical scheme:
the method for detecting norovirus by using the nano enzyme test strip comprises the following steps:
(1) synthesizing nano enzyme;
(2) preparing a nano enzyme-nucleic acid antibody probe;
(3) assembling the nano enzyme immunochromatographic test strip;
(4) and (3) detecting the norovirus by using the nano enzyme immunochromatographic test strip.
The method for detecting norovirus by using the nano-enzyme test strip comprises the following steps of (1) synthesizing nano-enzyme by a hydrothermal method: 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, carrying out reaction, carrying out magnetic separation on the obtained product, discarding the supernatant, washing the precipitate with ethanol, and drying and storing in an electrothermal constant-temperature drying oven to obtain the nano enzyme.
The method for detecting norovirus by using the nano-enzyme test strip comprises the following steps of (1) synthesizing nano-enzyme by a hydrothermal method: 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 for 14 hours at 200 ℃, magnetically separating the obtained product, discarding the supernatant, washing the precipitate with ethanol for 3 times, and drying and storing in an electrothermal constant-temperature drying oven to obtain the nano enzyme.
The method for detecting norovirus by using the nano-enzyme test strip comprises the following steps of (2),
(2-1) washing the nano enzyme prepared in the step (1) by using deionized water;
(2-2) centrifuging, and then removing a supernatant; resuspending the precipitate with MES buffer solution, adding EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), mixing, and activating in shaking table;
(2-3) after activation, washing with MES buffer solution, and then re-suspending with MES buffer solution to obtain activated nano enzyme;
(2-4) incubating the norovirus mouse monoclonal antibody with the activated nano enzyme solution overnight;
(2-5) adding a Tris buffer solution to terminate the reaction;
(2-6) after magnetic separation, using 5% BSA-Tris buffer solution to carry out heavy suspension, thus obtaining the nano enzyme-nucleic acid antibody probe.
The method for detecting norovirus by using the nano-enzyme test strip comprises the following steps of (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 the nano enzyme with 1mL of deionized water for three times;
(2-2) centrifuging, and then removing a supernatant; the precipitate was resuspended in 1mL MES buffer, pH 6.0, 20. mu.L EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 50. mu.L NHS (N-hydroxysuccinimide) were added, mixed well and activated in a shaker for 30 min; the concentration of EDC is 0.4mol/L, and the concentration of NHS is 0.1 mol/L; the rotating speed of the shaking table is 70 rpm;
(2-3) after activation, washing once by using 1mL of MES buffer solution, and then re-suspending by using 500 mu L of MES buffer solution to obtain activated nano enzyme;
(2-4) taking 50 mu g of norovirus mouse monoclonal antibody and incubating with the activated nano enzyme solution overnight at the incubation temperature of 4 ℃;
(2-5) incubating for 30min at room temperature by using a Tris buffer solution, and stopping 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, using 0.5mL of 5% BSA-Tris buffer solution to carry out heavy suspension, thus obtaining the nano enzyme-nucleic acid antibody probe.
The method for detecting norovirus by using the nano-enzyme test strip comprises the following steps in step (3):
(3-1) scribing a quality control line C line and a detection line T line on the nitrocellulose membrane (4): drawing a line on the nitrocellulose membrane by using a goat anti-mouse antibody with the concentration of 1mg/mL as a quality control line; scribing on a nitrocellulose membrane by using the murine norovirus monoclonal antibody with the concentration of 2mg/mL as a detection line, wherein the scribing parameter is 0.1 muL/mm, and the distance between a quality control line and the detection line is 7 mm; after scribing, putting the nitrocellulose membrane (4) in a thermostat for drying for 1h, wherein the drying temperature is 40 ℃;
(3-2): spraying nano enzyme-noro antibody probes on the binding pad (3): diluting the coupled nano enzyme-nucleic acid probe with 1% BSA-Tris buffer solution to prepare the nano enzyme-nucleic acid probe with the concentration of 1 mg/mL; spraying the diluted nano enzyme-noe probe on the bonding pad (3) with the spraying parameter of 0.5 muL/mm; then, the bonding pad (3) is placed in a thermostat and dried for 1h, and the drying temperature is 40 ℃;
(3-3): assembling the test strip: sequentially attaching the sample pad (2), the combination pad (3), the nitrocellulose membrane (4) and the absorption pad (5) to the bottom card (1), enabling a detection line on the nitrocellulose membrane (4) to be close to the combination 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 combination pad (3) are both glass fiber membranes, and the absorption pad (5) is absorbent filter paper; assembling the test paper into a complete nano enzyme immunochromatographic test strip, and then cutting the test paper into test paper strips with the width of 4mm by adopting a slitter;
(3-4) covering a clamping shell (6) on each nano enzyme immunochromatographic test strip, wherein the clamping shell (6) is provided with a sample adding hole (6-1) and a color developing hole (6-2); 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), 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 immunochromatographic test strip is completed, the assembled nano enzyme immunochromatographic test strip is placed in a sealing bag for dry storage at room temperature.
The method for detecting norovirus by using the nano-enzyme test strip comprises the following steps in step (4):
(4-1) taking out the nano enzyme immunochromatographic test strip, adding 100 mu L of nori antigen solution into the sample adding hole (6-2), starting chromatographic detection, and timing for 10 minutes; after chromatography, 1mL of nano enzyme substrate color development liquid is added into a color development hole (6-2) of the nano enzyme immunochromatography test strip, so that the color development hole (6-2) is filled with the nano enzyme substrate color development liquid, and color development is carried out in a dark place for 5-10 minutes; the nano enzyme substrate color developing solution is diaminobenzidine DAB and hydrogen peroxide;
(4-2) when the color is developed in a dark place for 5-10 minutes, absorbing the color developing solution of the nano enzyme substrate from the sample adding hole (6-2) by using a liquid transferring gun, observing color signals of a quality control line and a detection line on the nano enzyme immunochromatography test strip, judging the detection result, and making the result invalid after more than 15 minutes.
The method for detecting norovirus by using the nano enzyme test strip comprises the following steps: if the nano enzyme immunochromatographic test strip has a quality control line and a detection line at the same time, the detection result is positive, namely the sample to be detected has norovirus; if only the quality control line appears on the norovirus nanoenzyme immunochromatographic test strip, the detection result is negative, namely no norovirus exists in the sample to be detected; if only a detection line appears on the norovirus nanoenzyme immune chromatography test strip, the detection result is invalid, and the detection needs to be carried out again.
The technical scheme of the invention achieves the following beneficial technical effects:
according to the application, the immunochromatography technology is applied, norovirus is taken as a detection sample, the characteristic that nano-enzyme has peroxidase catalytic activity is utilized, traditional colloidal gold is replaced by the nano-enzyme, the application of the nano-enzyme immunochromatography test strip in norovirus detection is researched, and the detection signal can be obviously enhanced by adding a catalytic substrate DAB of the enzyme after the immunochromatography, so that the effect of higher detection sensitivity is achieved. The detection 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 on norovirus detection and has specific recognition on norovirus. The method for detecting 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 a wide development prospect.
Drawings
FIG. 1 is a schematic structural diagram of a nanoenzyme immunochromatographic test strip for use in the method of detecting norovirus by using the nanoenzyme test strip of the present invention;
FIG. 2 is a schematic structural diagram of a nano-enzyme immunochromatographic test strip covered with a card shell according to the method for detecting norovirus by using the nano-enzyme test strip of the present invention;
FIG. 3 is a schematic diagram of a detection principle of the nanoenzyme immunochromatographic test strip of the method for detecting norovirus by using the nanoenzyme test strip of the present invention;
FIG. 4 is a representation diagram of a transmission electron microscope of nanoenzyme in the method for detecting norovirus by using the nanoenzyme test strip of the invention;
FIG. 5 is a graph showing the dynamic light scattering characteristics of nanoenzymes in the method for detecting norovirus by using the nanoenzyme test strip of the present invention;
FIG. 6 is a representation of the enzymatic activity of nanoenzyme material in the method of detecting norovirus by nanoenzyme test strip of the invention;
FIG. 7 is a detection diagram of the method for detecting norovirus by using the nanoenzyme test strip of the invention after chromatography for 10 min;
FIG. 8 is a detection diagram after 5min of color development of the method for detecting norovirus by using the nanoenzyme test strip;
FIG. 9 is a graph showing the detection results of norovirus antigen at different concentrations according to the method of detecting norovirus by using the nanoenzyme test strip of the present invention;
FIG. 10 is a graph showing the results of a colloidal gold test strip detecting various concentrations of norogan;
FIG. 11 is a specific test result diagram of the method for detecting norovirus by using the nanoenzyme test strip of the present invention, wherein 1 is rotavirus, 2 is filovirus, 3 is helicobacter pylori, and 4 is norovirus.
The reference numbers in the figures denote: 1-bottom card; 2-sample pad; 3-a conjugate pad; 4-nitrocellulose membrane; 5-an absorbent pad; 6-clamping the shell; 6-1-wells; 6-2-color wells.
Detailed Description
The method for detecting norovirus by using the nanoenzyme test strip comprises the following steps;
1. synthesizing nano enzyme;
synthesizing the nano enzyme by a hydrothermal method: 0.3g FeCl3·6H2Dissolving O in 20mL of glycol, stirring for dissolving, adding 1.5g of sodium acetate, sealing the mixture in a high-pressure reaction kettle after the mixture is completely dissolved, reacting for 14 hours at 200 ℃, performing magnetic separation on the obtained product, removing supernatant, washing the precipitate with ethanol for 3 times, and drying and storing at 50-60 ℃ by using an electrothermal constant-temperature drying oven to obtain the nano enzyme.
Wherein: sodium acetate, FeCl3·6H2O, ethanol and ethylene glycol are purchased from Beijing chemical reagent factory; high temperature ovens were purchased from Tianjin Tester.
Characterization of the nanoenzyme: the prepared nanoenzyme was characterized by transmission electron microscope (JEOL 2000FX 200KV, Japan Electron Co., Ltd.) and dynamic light scattering apparatus. The prepared nano enzyme material has very high uniformity of particle size: as shown in FIG. 4, the particle size was 110. + -.10 nm in the transmission electron microscope, and as shown in FIG. 5, the hydrated particle size was about 130nm in the dynamic light scattering, which was slightly increased due to the formation of the hydrated layer on the periphery of the nanoenzyme material.
And (3) measuring the enzyme activity of the nano enzyme: enzyme catalytic activity of the prepared nano enzyme material is tested by an enzyme-labeling instrument, and enzyme activity of the prepared nano enzyme material is tested by a spectrophotometer.
As shown in fig. 6: the enzyme-labeling instrument is used for testing and analyzing the enzyme catalytic activity of the nano enzyme material, and the peroxidase catalytic activity of the prepared nano enzyme material is found to be good, and the highest activity value can be reached when the catalytic reaction is carried out for 10 min.
The specific activity of the prepared nano-enzyme material is measured by a spectrophotometer, and the detection and calculation method is obtained according to the nano-enzyme specific activity standardization article published by Jiang and the like (standard analyzed for determining the catalytic activity and kinetics of peroxidase-like enzymes, Nat Protoc,2018.13(7): p.1506-1520.), and the specific activity of the prepared nano-enzyme material is calculated to be 9.33U/mg.
2. Preparation of nano enzyme-nucleic acid 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 the nano enzyme with 1mL of deionized water for three times;
(2-2) centrifuging, and then removing a supernatant; the precipitate was resuspended in 1mL MES (2- (N-morpholine) ethanesulfonic acid) buffer, pH 6.0, 20. mu.L EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 50. mu.L NHS (N-hydroxysuccinimide) were added, mixed well and activated in a shaker for 30 min; the concentration of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) was 0.4mol/L and the concentration of NHS (N-hydroxysuccinimide) was 0.1 mol/L; the rotating speed of the shaking table is 70 rpm;
(2-3) after activation, washing once by using 1mL of MES buffer solution, and then re-suspending by using 500 mu L of MES buffer solution to obtain activated nano enzyme;
(2-4) taking 50 mu g of norovirus mouse monoclonal antibody and incubating with the activated nano enzyme solution overnight at the incubation temperature of 4 ℃;
(2-5) incubating for 30min at room temperature by using a Tris buffer solution, and stopping 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, using 0.5mL of 5% BSA-Tris buffer solution to carry out heavy suspension, thus obtaining the nano enzyme-nucleic acid antibody probe.
Wherein: EDC is 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 1- (3-methylenepropyl) -3-ethylcarbodiimide hydrochloride, available from Sigma-Aldrich;
NHS is N-Hydroxysuccinimide, N-Hydroxysuccinimide, available from (Sigma-Aldrich)
Murine monoclonal antibodies to norovirus were purchased from BIOCare, and enzyme-linked immunosorbent assay was purchased from Bio-Rad.
3. Assembling the nano enzyme immunochromatographic test strip; as shown in fig. 1 and 2:
(3-1) scribing a quality control line C line and a detection line T line on the nitrocellulose membrane 4: drawing a line on the nitrocellulose membrane by using a goat anti-mouse antibody with the concentration of 1mg/mL as a quality control line; scribing on a nitrocellulose membrane by using the murine norovirus monoclonal antibody with the concentration of 2mg/mL as a detection line, wherein the scribing parameter is 0.1 muL/mm, and the distance between a quality control line and the detection line is 7 mm; after scribing, placing the nitrocellulose membrane 4 in a constant temperature box for drying for 1h, wherein the drying temperature is 40 ℃;
(3-2): spraying nanoenzyme-nucleic acid antibody probes on the conjugate pad 3: diluting the coupled nano enzyme-noe probe into 1% BSA-Tris buffer solution, and preparing to obtain the nano enzyme-noe probe with the concentration of 1 mg/mL; spraying the diluted nanoenzyme-noe probe onto the conjugate pad 3 with a spraying parameter of 0.5 μ L/mm; then, the bonding pad 3 is placed in a constant temperature box to be dried for 1h, and the drying temperature is 40 ℃;
(3-3): assembling the test strip: sequentially attaching a sample pad 2, a combination pad 3, a nitrocellulose membrane 4 and an absorption pad 5 on a bottom card 1, enabling a detection line on the nitrocellulose membrane 4 to be close to the combination 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 combination pad 3 are both glass fiber membranes, and the absorption pad 5 is absorbent filter paper; assembling the test paper strip into a complete nano enzyme immunochromatographic test paper strip, and then cutting the test paper strip into test paper strips with the width of 4mm by adopting a slitter;
(3-4) covering a card shell 6 on each nano enzyme immunochromatographic test strip, wherein the card shell 6 is provided with a sample adding hole 6-1 and a color developing hole 6-2; 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, 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 for dry storage at room temperature.
Wherein: PVDF membrane (Millipore), jet flow membrane-spraying scriber (Arista, USA), slitting machine (bio torch of tianjin), XYZ three-dimensional membrane-spraying gold instrument (shanghai gold standard).
4. The nano enzyme immunochromatographic test strip is used for detecting norovirus:
4.1, detection principle: as shown in fig. 3, the nanoenzyme magnetic particles were used instead of colloidal gold, and were coupled with norovirus-labeled antibodies, thereby constructing nanoenzyme-antibody probes. When the nanoenzyme-antibody probe is combined with norovirus in a sample, the sample is chromatographed to a detection line (a T line, a capture antibody sprayed with norovirus) and a control line (a C line, a goat anti-mouse antibody sprayed with norovirus) of a test strip, a compound of the nanoenzyme-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 aggregation of nanoenzyme particles is formed at the T line and the C line, at the moment, a substrate of peroxidase such as DAB is added, and the enzymatic activity of the nanoenzyme is utilized, so that DAB is catalyzed to generate a large amount of tan precipitates through chemical reaction, and a detection signal is amplified, thereby realizing the rapid and sensitive detection of the norovirus.
4.2, the specific detection method comprises the following steps:
(4-1) diluting norathyriol antigen concentration to 0ng/ml, 5ng/ml, 10ng/ml, 25ng/ml, 50ng/ml, 100ng/ml, 250ng/ml, 500 ng/ml.
(4-2) taking out the nano enzyme immunochromatographic test strip, adding 100 mu L of nore antigen solution 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, 1mL of nano enzyme substrate color developing solution is added into the color developing holes 6-2 of the nano enzyme immunochromatographic test strip, so that the color developing holes 6-2 are filled with the nano enzyme substrate color developing solution, and light-shielding color development is carried out for 5-10 minutes; the nano enzyme substrate chromogenic solution is DAB and hydrogen peroxide;
(4-3) when the color is developed in a dark place for 5-10 minutes, absorbing the color developing solution of the nano-enzyme substrate from the sample adding hole 6-2 by using a liquid transfer gun, observing color signals of a quality control line and a detection line on the nano-enzyme immunochromatographic test strip, judging the detection result, and invalidating the result after more than 15 minutes.
As shown in fig. 7 and 8: after the nano enzyme immunochromatographic test strip is added with the peroxidase substrate DAB, the color development effect is obviously increased, the visual detection sensitivity before color development (chromatography for 10min) is 50ng/mL, and the visual detection sensitivity after color development is 5 ng/mL. The nano enzyme can really catalyze the DAB substrate to generate color reaction and generate a visible signal, thereby enhancing the detection effect of the chromatographic test strip.
4.3, comparing the detection sensitivity of the nanoenzyme 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 detection gradient of the concentration of the eight norovirus antigens, the lowest antigen detection concentration of the nanoenzyme immunochromatographic strip is 5ng/mL, the lowest antigen detection concentration of the commercial colloidal gold strip is 50ng/mL, and the detection sensitivity of the nanoenzyme immunochromatographic strip to the norovirus antigens is improved by about 10 times.
4.4 detection of specificity of the test strip by nano enzyme immunochromatography
In order to verify the specificity of the norovirus nanoenzyme immunochromatographic test strip for detecting norovirus, rotavirus, filovirus and helicobacter pylori are detected in the embodiment, as shown in fig. 11, it is found that the prepared norovirus nanoenzyme immunochromatographic test strip can only detect norovirus specifically, which indicates that the prepared nanoenzyme test strip has very good detection specificity for norovirus.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications are possible which remain within the protective scope of the claims of the patent.
Claims (8)
1. The method for detecting norovirus by using the nano enzyme test strip is characterized by comprising the following steps of:
(1) synthesizing nano enzyme;
(2) preparing a nano enzyme-nucleic acid antibody probe;
(3) assembling the nano enzyme immunochromatographic test strip;
(4) and (3) detecting the norovirus by using the nano enzyme immunochromatographic test strip.
2. The method for detecting norovirus by using the nanoenzyme test strip as claimed in claim 1, wherein in the step (1), nanoenzyme is synthesized by a hydrothermal method: FeCl is added3·6H2Dissolving O in ethylene glycol, stirring for dissolving, adding sodium acetate, sealing in a high-pressure reaction kettle after the mixture is completely dissolved, carrying out reaction, carrying out magnetic separation on the obtained product, discarding the supernatant, washing the precipitate with ethanol, and drying and storing in an electrothermal constant-temperature drying oven to obtain the nano enzyme.
3. The method for detecting norovirus by using the nanoenzyme test strip according to claim 2, wherein in the step (1), nanoenzyme is synthesized by a hydrothermal method: FeCl is added3·6H2Dissolving O 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 for 14 hours at 200 ℃, magnetically separating the obtained product, discarding the supernatant, washing the precipitate with ethanol for 3 times, and drying and storing in an electrothermal constant-temperature drying oven to obtain the nano enzyme.
4. The method for detecting norovirus by using nanoenzyme test strips according to claim 1, wherein in the step (2),
(2-1) washing the nano enzyme prepared in the step (1) by using deionized water;
(2-2) centrifuging, and then removing a supernatant; resuspending the precipitate with MES buffer solution, adding EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), mixing, and activating in shaking table;
(2-3) after activation, washing with MES buffer solution, and then re-suspending with MES buffer solution to obtain activated nano enzyme;
(2-4) incubating the norovirus mouse monoclonal antibody with the activated nano enzyme solution overnight;
(2-5) adding a Tris buffer solution to terminate the reaction;
(2-6) after magnetic separation, using 5% BSA-Tris buffer solution to carry out heavy suspension, thus obtaining the nano enzyme-nucleic acid antibody probe.
5. The method for detecting norovirus by using the nanoenzyme test strip of claim 4, wherein 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 the nano enzyme with 1mL of deionized water for three times;
(2-2) centrifuging, and then removing a supernatant; the precipitate was resuspended in 1mL MES buffer, pH 6.0, 20. mu.L EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 50. mu.L NHS (N-hydroxysuccinimide) were added, mixed well and activated in a shaker for 30 min; the concentration of EDC is 0.4mol/L, and the concentration of NHS is 0.1 mol/L; the rotating speed of the shaking table is 70 rpm;
(2-3) after activation, washing once by using 1mL of MES buffer solution, and then re-suspending by using 500 mu L of MES buffer solution to obtain activated nano enzyme;
(2-4) taking 50 mu g of norovirus mouse monoclonal antibody and incubating with the activated nano enzyme solution overnight at the incubation temperature of 4 ℃;
(2-5) incubating for 30min at room temperature by using a Tris buffer solution, and stopping 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, using 0.5mL of 5% BSA-Tris buffer solution to carry out heavy suspension, thus obtaining the nano enzyme-nucleic acid antibody probe.
6. The method for detecting norovirus by using the nanoenzyme test strip of claim 1, wherein the step (3) comprises the following steps:
(3-1) scribing a quality control line C line and a detection line T line on the nitrocellulose membrane (4): drawing a line on the nitrocellulose membrane by using a goat anti-mouse antibody with the concentration of 1mg/mL as a quality control line; scribing on a nitrocellulose membrane by using the murine norovirus monoclonal antibody with the concentration of 2mg/mL as a detection line, wherein the scribing parameter is 0.1 muL/mm, and the distance between a quality control line and the detection line is 7 mm; after scribing, putting the nitrocellulose membrane (4) in a thermostat for drying for 1h, wherein the drying temperature is 40 ℃;
(3-2): spraying nano enzyme-noro antibody probes on the binding pad (3): diluting the coupled nano enzyme-noe probe with 1% BSA-Tris buffer solution to prepare the nano enzyme-noe probe with the concentration of 1 mg/mL; spraying the diluted nano enzyme-noe probe on the bonding pad (3) with the spraying parameter of 0.5 muL/mm; then, the bonding pad (3) is placed in a thermostat and dried for 1h, and the drying temperature is 40 ℃;
(3-3): assembling the test strip: sequentially attaching the sample pad (2), the combination pad (3), the nitrocellulose membrane (4) and the absorption pad (5) to the bottom card (1), enabling a detection line on the nitrocellulose membrane (4) to be close to the combination 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 combination pad (3) are both glass fiber membranes, and the absorption pad (5) is absorbent filter paper; assembling the test paper strip into a complete nano enzyme immunochromatographic test paper strip, and then cutting the test paper strip into test paper strips with the width of 4mm by adopting a slitter;
(3-4) covering a clamping shell (6) on each nano enzyme immunochromatographic test strip, wherein the clamping shell (6) is provided with a sample adding hole (6-1) and a color developing hole (6-2); 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), 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 immunochromatographic test strip is completed, the assembled nano enzyme immunochromatographic test strip is placed in a sealing bag for dry storage at room temperature.
7. The method for detecting norovirus by using the nanoenzyme test strip of claim 1, wherein the step (4) comprises the following steps:
(4-1) taking out the nano enzyme immunochromatographic test strip, adding 100 mu L of nori antigen solution into the sample adding hole (6-2), starting chromatographic detection, and timing for 10 minutes; after chromatography, 1mL of nano enzyme substrate color development liquid is added 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 color development is carried out in a dark place for 5-10 minutes; the color developing solution of the nano enzyme substrate is DAB and hydrogen peroxide;
(4-2) when the sample is developed for 5-10 minutes in a dark state, absorbing the developing solution of the nano enzyme substrate from the sample adding hole (6-2) by using a liquid transfer gun, observing color signals of a quality control line and a detection line on the nano enzyme immunochromatographic test strip, judging the detection result, and invalidating the result after more than 15 minutes.
8. The method for detecting norovirus by using the nanoenzyme test strip of claim 7, wherein the method for determining the detection result comprises: if the nano enzyme immunochromatographic test strip has a quality control line and a detection line at the same time, the detection result is positive, namely norovirus exists in the sample to be detected; if only the quality control line appears on the norovirus nanoenzyme immunochromatographic test strip, the detection result is negative, namely no norovirus exists in the sample to be detected; if only the detection line appears on the norovirus nanoenzyme immunochromatographic test strip, the detection result is invalid, and the detection needs to be carried out again.
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