CN116298297A - Colloidal gold chromatographic test strip for simultaneously detecting syncytial virus and novel coronavirus antigen - Google Patents
Colloidal gold chromatographic test strip for simultaneously detecting syncytial virus and novel coronavirus antigen Download PDFInfo
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Abstract
The invention discloses a colloidal gold chromatographic test strip for simultaneously detecting syncytial virus and novel coronavirus antigens, which comprises a bottom plate, a sample pad, a combination pad, an NC film and a water absorption pad, wherein the sample pad, the combination pad, the NC film and the water absorption pad are sequentially lapped and stuck on the bottom plate according to the chromatographic direction; be provided with two detection lines and a matter control line on the NC membrane, two detection lines are coated with syncytial virus capture antibody and novel coronavirus capture antibody respectively, and the matter control line is coated with staphylococcal protein A, a colloidal gold chromatographic test strip of simultaneous detection syncytial virus and novel coronavirus antigen of this application only once samples, can detect syncytial virus antigen and novel coronavirus antigen simultaneously, can quick differential diagnosis, in time selects effective antiviral treatment to provide the basis.
Description
Technical Field
The invention relates to the technical field of biomedical detection, in particular to a colloidal gold chromatographic test strip for simultaneously detecting syncytial virus and novel coronavirus antigens.
Background
Respiratory syncytial virus is a highly contagious paramyxovirus, and along with the development of disease conditions, the syncytial virus not only can cause upper respiratory tract infection, but also can cause lower respiratory tract diseases such as pneumonia, bronchitis and the like, and can cause higher death rate; the rapid detection of syncytial virus is very important for the timely diagnosis of disease and prevention of nosocomial infections.
The novel coronaviruses responsible for the infection with the novel coronaviruses belong to the group of RNA viruses and to the group of beta coronaviruses; it has three major seriousness: firstly, infection is not classified into species; secondly, coronavirus proteins can be further replicated; thirdly, the mutation is fast; furthermore asymptomatic infected persons are difficult to identify. Thus, early diagnosis has been found to play a very important role.
The methods applied to clinical examination at present for respiratory syncytial virus and novel coronavirus mainly comprise a PCR method and a colloidal gold method, and the methods are single virus detection or combined detection with A and B flow. Such as respiratory syncytial virus nucleic acid detection kit (fluorescence PCR method), novel coronavirus nucleic acid detection kit (fluorescence PCR method), respiratory syncytial virus antigen detection kit (colloidal gold method), novel coronavirus antigen detection kit (colloidal gold method); however, all the current fluorescent PCR detection methods still have the defects of complicated sample treatment, long detection time, high requirements on the expertise of detection personnel and the like, and the colloidal gold method also has the blank of combined detection. The above shortcomings result in the limitation of the current simultaneous diagnosis of respiratory syncytial virus and novel coronavirus in clinical tests, which cannot meet the requirements of rapid differential diagnosis.
Disclosure of Invention
Aiming at the defects of the prior art, the application provides a colloidal gold chromatographic test strip for simultaneously detecting syncytial virus and novel coronavirus antigens.
In a first aspect, the present application provides a colloidal gold chromatographic test strip for simultaneously detecting syncytial virus and novel coronavirus antigens, which adopts the following technical scheme:
the colloidal gold chromatographic test strip comprises a bottom plate, a sample pad, a binding pad, an NC membrane and a water absorption pad, wherein the sample pad, the binding pad, the NC membrane and the water absorption pad are sequentially lapped and stuck on the bottom plate according to the chromatographic direction, and the binding pad is coated with a colloidal gold-labeled syncytial virus detection antibody and a novel coronavirus detection antibody; the NC film is provided with two detection lines and a quality control line, the two detection lines are respectively coated with a syncytial virus capture antibody and a novel coronavirus capture antibody, and the quality control line is coated with a staphylococcus A protein.
The two detection lines and one quality control line are arranged in parallel and are spaced 3.5mm apart from each other, and the two detection lines are respectively an R detection line, an N detection line and a C quality control line; the R detection line is close to the bonding pad, the C quality control line is close to the water absorption pad, and the N detection line is positioned between the R detection line and the C quality control line.
In a specific embodiment, the method for preparing the quality control line comprises the following steps:
(1) Diluting staphylococcus protein A with sodium dihydrogen phosphate-beta cyclodextrin solution to obtain quality control line working solution;
(2) Drawing a film on the NC film by using the quality control line working solution;
(3) And drying the NC film with the quality control line to obtain the quality control line.
By adopting the technical scheme, the beta cyclodextrin is a product generated by acid hydrolysis and cyclization of starch, and the beta cyclodextrin added in the method can embed partial pore diameters on the nitrocellulose membrane, so that the pore diameter difference of different batches of nitrocellulose membranes is reduced, and the precision of the kit is improved; meanwhile, due to the special cylindrical space structure, the beta cyclodextrin can build a stable bridge between nitrocellulose membrane and protein and protect antigen-antibody binding sites, so that the precision of the kit can be improved.
Staphylococcal protein a has the ability to bind to human and many animal proteins and is broad in scope; after the staphylococcus protein A is added, no additional mark quality control line protein is needed, and no specific requirement is imposed on animal sources (such as mouse resistance, rabbit resistance, sheep resistance and the like) of the marked antibody.
Preferably, the concentration of sodium dihydrogen phosphate-beta cyclodextrin in step (1) is 0.01M-0.05M; for example, 0.01M, 0.02M, 0.03M, 0.04M, 0.05M, etc.
Preferably, the concentration of staphylococcal protein A after dilution in step (1) is 0.25-0.5mg/mL, e.g. 0.25mg/mL, 0.35mg/mL, 0.45mg/mL, 0.5mg/mL etc.
Preferably, the drying temperature in step (3) is 2-30deg.C, such as 2deg.C, 5deg.C, 12deg.C, 14deg.C, 16deg.C, 18deg.C, 20deg.C, 22deg.C, 25deg.C, 28deg.C, 30deg.C, etc.
The low-temperature (2-30 ℃) drying is adopted in the method, the heat loss in the protein drying process can be reduced by the low-temperature drying, the physical combination rate of the protein and the nitrocellulose membrane is improved, and the protein combining site is protected.
Preferably, the humidity of the drying in step (3) is not more than 30%; for example, 5%, 10%, 15%, 20%, 25%, 30%, etc.
The application adopts low humidity (not more than 30%) drying, can shorten drying time, protects the surface hydrophilicity of the nitrocellulose membrane, improves the physical binding rate of protein and nitrocellulose membrane, and protects the protein binding site.
In a specific embodiment, the method for preparing the quality control line comprises the following steps:
(1) Diluting staphylococcus protein A to 0.25-0.5mg/mL by using a sodium dihydrogen phosphate-beta cyclodextrin solution with the concentration of 0.01-0.05M to obtain a quality control line working solution;
(2) A quality control line working solution is filled, a scribing instrument is started, a spraying pen spraying amount of 1.0ul/cm, an X-axis position and a Y-axis position are set, film scribing is carried out, and a quality control line is coated;
(3) And (3) drying the NC film with the quality control line at the temperature of 2-30 ℃ and the humidity of not more than 30%, thereby obtaining the quality control line.
In a specific embodiment, the method for colloidal gold-labeled syncytial virus detection antibodies and novel coronavirus detection antibodies comprises the steps of:
s1, centrifuging a colloidal gold solution, and taking a precipitate;
s2, re-dissolving the precipitate with sodium tetraborate-sodium hydroxide buffer solution to the volume of the colloidal gold solution in the step S1;
s3, adding a syncytial virus detection antibody for stirring, adding a novel coronavirus detection antibody for stirring, and then adding BSA and PEG20000 for stirring;
s4, centrifuging, taking a precipitate, and redissolving the precipitate to 1/10 times of the volume of the colloidal gold solution by using a phosphoric acid preservation solution.
Through adopting above-mentioned technical scheme, this application is through carrying out the centrifugation to the colloidal gold solution earlier, and rethread buffer carries out pH adjustment, can get rid of the irregularly shaped granule in the colloidal gold preparation process for particle size distribution is more even in the colloidal gold solution.
Sodium tetraborate-sodium hydroxide buffer solution is added in the application, a weak alkaline environment is provided, colloidal gold is negatively charged in the weak alkaline environment, a stable system can be provided, and firm combination can be formed with positive charge groups of protein molecules.
Preferably, the volume of the colloidal gold solution in step S1 is 10mL-40mL, such as 10mL, 15mL, 20mL, 25mL, 30mL, 35mL, 40mL.
Preferably, the concentration of sodium tetraborate-sodium hydroxide buffer in step S2 is in the range of 0.1M to 0.5M, e.g. 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, etc.
Preferably, the amount of syncytial virus detection antibody added in step S3 is 200-300. Mu.g; for example 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290 μg, 300 μg, etc.
Preferably, the novel coronavirus detection antibody in step S3 is added in an amount of 200-300. Mu.g; for example 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290 μg, 300 μg, etc.
Preferably, the mass concentration of BSA added in the step S3 is 0.5-2%; the mass concentration of the added PEG20000 is 0.5-2%.
Preferably, in the step S3, a syncytial virus detection antibody is added, and the temperature of water bath stirring is 35-40 ℃ for 10-20min; adding a novel coronavirus detection antibody, and stirring in a water bath at 35-40deg.C for 10-20min; adding BSA and PEG20000, stirring in water bath at 35-40deg.C for 40-60min.
In the application, traditional separate marking or simultaneous sample adding marking is changed into 35-40 ℃ water bath separate sample adding and simultaneous marking, and different antibodies and blocking agents (BSA and PEG 20000) are sequentially added in the application, so that colloidal gold is fully utilized to have larger specific surface area and good surface effect under the water bath condition of 35-40 ℃ to perform physical adsorption, and the operation is simple.
Preferably, in step S4, the phosphoric acid preserving fluid comprises the following raw materials in percentage by mass: 0.5-1% sucrose, 0.25-0.5% casein, 0.3-0.5% tween-20, and water, the balance being pH7.4.
In a specific embodiment, the method for colloidal gold-labeled syncytial virus detection antibodies and novel coronavirus detection antibodies comprises the steps of:
s1, cleaning: placing 10-40 mL of colloidal gold solution into a centrifuge tube, centrifuging, and taking a precipitate;
s2, adjusting pH: re-dissolving the precipitate to the original volume (the volume of the colloidal gold solution in the step S1) by using sodium tetraborate-sodium hydroxide buffer solution with the concentration of 0.1M-0.5M; the observation is clear and transparent, and no suspended matters and sediment are generated;
s3, labeling an antibody: firstly, adding 200-300 mug of syncytial virus detection antibody, and stirring in a water bath at 35-40 ℃ for 10-20min; adding 200-300 μg of novel coronavirus detection antibody, stirring in 35-40deg.C water bath for 10-20min; adding 0.5-2% BSA and 0.5-2% PEG20000, and stirring in 35-40deg.C water bath for 40-60min;
s4, centrifuging, taking the precipitate, redissolving the precipitate to 1/10 of the original volume (1/10 of the volume of the colloidal gold solution) by using a phosphoric acid preservation solution, and preserving the precipitate at 2-8 ℃ for later use.
The phosphoric acid preservation solution comprises the following raw materials in percentage by mass: 0.5-1% sucrose, 0.25-0.5% casein, 0.3-0.5% tween-20, and the balance purified water, pH7.4.
In a second aspect, the present application provides a kit, which adopts the following technical scheme:
a kit comprises the test strip.
Through adopting above-mentioned technical scheme, the kit in this application only needs once to take a sample, can detect syncytial virus antigen and novel coronavirus antigen simultaneously, easy operation, the result is quick.
In summary, the present application includes at least one of the following beneficial technical effects:
the colloidal gold chromatographic test strip for simultaneously detecting the syncytial virus and the novel coronavirus antigen can simultaneously detect the syncytial virus antigen and the novel coronavirus antigen only by sampling once, can rapidly carry out differential diagnosis, and provides a basis for timely selecting effective antiviral treatment.
According to the method, the sensitivity of the test strip is effectively improved through improvement of a colloidal gold marking process and an NC film preparation process, so that a product with higher specificity is obtained.
Detailed Description
The raw materials used in the present application are commercially available products, and the present application will be further described in detail with reference to examples and comparative examples.
Preparation example: colloidal gold preparation
The preparation process of the colloidal gold comprises the following steps:
(1) 96mL of ultrapure water was added to the round-bottomed flask, followed by addition of 4mL of chloroauric acid at a mass concentration of 1%; heating the round-bottom flask in an oil bath, wherein the rotating speed is set to be 100rpm;
(2) After boiling, adding 1mL trisodium citrate with the mass concentration of 1%, keeping the rotating speed at 100rpm, and continuously boiling for 10 minutes to obtain a colloidal gold solution;
(3) Recovering the prepared colloidal gold solution to room temperature, and then using ultrapure water to fix the volume to 100mL; the maximum absorption peak wavelength is about 525nm and the particle size is about 40nm by using a full-wavelength spectrophotometer test; observing that the colloidal gold solution is clear and transparent, and has no suspended matters or sediment; and storing the colloidal gold solution at 2-8deg.C in dark.
Example 1: marking of colloidal gold
The method for labeling the syncytial virus detection antibody by using the colloidal gold and the novel coronavirus detection antibody is as follows:
s1, cleaning: 10mL of the colloidal gold solution (obtained in preparation example) was placed in a centrifuge tube, and the precipitate was collected by centrifugation.
S2, adjusting pH: re-dissolving the precipitate to 10mL of original volume by using a sodium tetraborate-sodium hydroxide buffer solution with the concentration of 0.1M; the observation was clear and transparent, with no suspended matter and no precipitate.
S3, labeling an antibody: firstly, adding 200 mug of syncytial virus detection antibody, and stirring in a water bath at 37 ℃ for 10min; adding 300 mug of novel coronavirus detection antibody, and stirring in a water bath at 37 ℃ for 10min; 0.1g BSA and 0.1g PEG20000 were added, and the reaction was completed after stirring in a water bath at 37℃for 40 minutes.
S4, centrifuging, wherein a centrifuging program is set to 12000rpm/min for 10min; taking the precipitate, and redissolving the precipitate to 1/10 of the original volume, namely 1mL, by using a phosphoric acid preservation solution; preserving at 2-8deg.C;
wherein the phosphoric acid preservation solution comprises the following raw materials in percentage by mass: 1% sucrose, 0.5% casein, 0.3% tween-20, the balance purified water, pH7.4.
Example 2: marking of colloidal gold
The method for labeling the syncytial virus detection antibody by using the colloidal gold and the novel coronavirus detection antibody is as follows:
s1, cleaning: 10mL of the colloidal gold solution (obtained in preparation example) was placed in a centrifuge tube, and the precipitate was collected by centrifugation.
S2, adjusting pH: re-dissolving the precipitate to 10mL of original volume by using a sodium tetraborate-sodium hydroxide buffer solution with the concentration of 0.2M; the observation was clear and transparent, with no suspended matter and no precipitate.
S3, labeling an antibody: firstly, adding 250 mug of syncytial virus detection antibody, and stirring in a water bath at 37 ℃ for 10min; adding 250 mug of novel coronavirus detection antibody, and stirring in a water bath at 37 ℃ for 10min; 0.1g BSA and 0.1g PEG20000 were added, and the reaction was completed after stirring in a water bath at 37℃for 40 minutes.
S4, centrifuging, wherein a centrifuging program is set to 12000rpm/min for 10min; taking the precipitate, and redissolving the precipitate to 1/10 of the original volume, namely 1mL, by using a phosphoric acid preservation solution; preserving at 2-8deg.C;
wherein the phosphoric acid preservation solution comprises the following raw materials in percentage by mass: 1% sucrose, 0.5% casein, 0.3% tween-20, the balance purified water, PH7.4.
Example 3: marking of colloidal gold
The method for labeling the syncytial virus detection antibody by using the colloidal gold and the novel coronavirus detection antibody is as follows:
s1, cleaning: 10mL of the colloidal gold solution (obtained in preparation example) was placed in a centrifuge tube, and the precipitate was collected by centrifugation.
S2, adjusting pH: re-dissolving the precipitate to 10mL of original volume by using a sodium tetraborate-sodium hydroxide buffer solution with the concentration of 0.5M; the observation was clear and transparent, with no suspended matter and no precipitate.
S3, labeling an antibody: firstly, 300 mug of syncytial virus detection antibody is added, and stirred in a water bath at 37 ℃ for 10min; adding 200 mug of novel coronavirus detection antibody, and stirring in a water bath at 37 ℃ for 10min; 0.1g BSA and 0.1g PEG20000 were added, and the reaction was completed after stirring in a water bath at 37℃for 40 minutes.
S4, centrifuging, wherein a centrifuging program is set to 12000rpm/min for 10min; taking the precipitate, and redissolving the precipitate to 1/10 of the original volume, namely 1mL, by using a phosphoric acid preservation solution; preserving at 2-8deg.C;
wherein the phosphoric acid preservation solution comprises the following raw materials in percentage by mass: 1% sucrose, 0.5% casein, 0.3% tween-20, the balance purified water, PH7.4.
Example 4: preparation of quality control line
The preparation method of the quality control line comprises the following steps:
(1) Dilution of staphylococcal protein a with disodium hydrogen phosphate-beta cyclodextrin buffer: 1mL of disodium hydrogen phosphate-beta cyclodextrin buffer (with the concentration of 0.01M) is added with 0.25mg of staphylococcus A protein, and the mixture is uniformly mixed to be used as a working solution of a quality control line (C).
(2) And (3) filling a working solution of a quality control line (C), setting the spraying amount of a spraying pen to be 1ul/cm, and marking a film at an X-axis position and a Y-axis position, and coating the quality control line and a detection line.
(3) And (3) drying: the coated NC film was dried overnight at low temperature (2 ℃) and low humidity (10%).
Example 5: preparation of quality control line
The preparation method of the quality control line comprises the following steps:
(1) Dilution of staphylococcal protein a with disodium hydrogen phosphate-beta cyclodextrin buffer: 1mL of disodium hydrogen phosphate-beta cyclodextrin buffer (with the concentration of 0.02M) is added with 0.35mg of staphylococcus A protein, and the mixture is uniformly mixed to be used as a working solution of a quality control line (C).
(2) And (3) filling a working solution of a quality control line (C), setting the spraying amount of a spraying pen to be 1ul/cm, and marking a film at an X-axis position and a Y-axis position, and coating the quality control line and a detection line.
(3) And (3) drying: the coated NC film was dried overnight at low temperature (14 ℃) and low humidity (20%).
Example 6: preparation of quality control line
The preparation method of the quality control line comprises the following steps:
(1) Dilution of staphylococcal protein a with disodium hydrogen phosphate-beta cyclodextrin buffer: 1mL of disodium hydrogen phosphate-beta cyclodextrin buffer (with the concentration of 0.05M) is added with 0.5mg of staphylococcus A protein, and the mixture is uniformly mixed to be used as a working solution of a quality control line (C).
(2) And (3) filling a working solution of a quality control line (C), setting the spraying amount of a spraying pen to be 1ul/cm, and marking a film at an X-axis position and a Y-axis position, and coating the quality control line and a detection line.
(3) And (3) drying: the coated NC film was dried overnight at low temperature (30 ℃) and low humidity (30%).
Example 7: combined detection kit for syncytial virus antigen and novel coronavirus antigen
The preparation method of the kit for combined detection of syncytial virus and novel coronavirus antigen comprises the following steps:
step one, preparation of bond pad
(1) The formula of the bonding pad pretreatment liquid comprises the following components: 0.02M phosphate buffer containing 0.5% triton X100,0.4mg/mL blocker, 1% sucrose, 0.5% casein, the balance purified water, pH7.4.
Spreading the bonding pad pretreatment liquid on a glass fiber film, adding 200mm 300mm glass fiber film into 40mL bonding pad pretreatment liquid, drying in an oven, cutting into 8mm 300mm glass fiber film, and preserving at room temperature and low humidity.
Step two, metal spraying
And (3) starting a metal spraying instrument to set the spraying amount to be 1-3ul/cm, spraying the marked metal solution prepared in the embodiment 1 into the bonding pad prepared in the step one at the X-axis position and the Y-axis position, and then placing the bonding pad into an oven for drying and preserving at a low humidity at room temperature.
Step three, preparation of sample pad
Sample pad pretreatment liquid formula: 0.02M phosphate buffer containing 0.5% triton X100,5% sucrose, 1% BSA,1% PEG20000,1% PVP10,0.3% sulfur Liu Gongna, the balance purified water, pH8.0.
The sample pad pretreatment liquid was spread on a glass fiber film, and 200mm by 300mm specification glass fiber film was added to 40mL of the sample pad pretreatment liquid. Oven drying, cutting into 17mm 300mm, and storing at room temperature and low humidity.
Step four, NC film preparation
(1) Sticking an NC film on a PVC bottom plate;
(2) And (3) preparing a detection line (N):
dilution of novel coronavirus capture antibody with 0.01M disodium hydrogen phosphate-beta cyclodextrin buffer solution: 1mL of disodium hydrogen phosphate-beta cyclodextrin buffer solution (the concentration is 0.01M) is added with 0.5mg of novel coronavirus capture antibody, and the mixture is uniformly mixed to be used as a detection line (N) working solution.
(3) And (3) preparing a detection line (R):
syncytial virus capture antibody was diluted with 0.01M disodium hydrogen phosphate-beta cyclodextrin buffer: 1mL disodium hydrogen phosphate-beta cyclodextrin buffer (with the concentration of 0.01M) is added with 0.5mg of syncytial virus capture antibody, and the mixture is uniformly mixed to be used as a detection line (R) working solution.
(4) The working solution (prepared in example 4) of the quality control line (C) is filled, a detection line (N, R) is used, a scribing instrument is started, the spraying amount of the spraying pen is set to be 1ul/cm, the X-axis position and the Y-axis position are set, the film scribing is carried out, and the quality control line and the detection line are coated.
(5) And (3) drying: the coated NC film was dried overnight at low temperature (2 ℃) and low humidity (10%) (drying as in example 4).
Step five, assembling test paper strips
Sequentially sticking the sample pad, the bonding pad, the NC film and the water absorption pad to prepare a large plate, and then cutting the large plate into test strips with the diameters of 3mm and 60 mm.
Example 8: the difference between this example and example 7 is that in step two, the labeled gold solution used was prepared as in example 2.
Example 9: the difference between this example and example 7 is that in the second step, the labeled gold solution used was prepared as in example 3.
Example 10: combined detection kit for syncytial virus antigen and novel coronavirus antigen
The difference between this embodiment and embodiment 7 is that in the fourth step, the quality control line working solution is prepared in embodiment 5, and the drying temperature and humidity are the drying modes in embodiment 5.
Example 11: the difference between the present example and example 7 of the kit for combined detection of syncytial virus antigen and novel coronavirus antigen is that in the fourth step, the working solution of quality control line is prepared in example 6, and the drying temperature and humidity are the drying modes in example 6.
Comparative example
Comparative example 1:
step one, preparation of colloidal gold
(1) 96mL of ultrapure water was added to the round-bottomed flask, followed by addition of 4mL of chloroauric acid at a mass concentration of 1%; heating the round-bottom flask in an oil bath, wherein the rotating speed is set to be 100rpm;
(2) After boiling, adding 1mL trisodium citrate with the mass concentration of 1%, keeping the rotating speed at 100rpm, and continuously boiling for 10 minutes to obtain a colloidal gold solution;
(3) And (3) recovering the prepared colloidal gold to room temperature, and then using ultrapure water to fix the volume to 100mL. The maximum absorption peak wavelength is about 525nm and the particle size is about 40nm by using a full-wavelength spectrophotometer test; observing that the colloidal gold solution is clear and transparent, and has no suspended matters or sediment; the colloid Jin Chu is stored at 2-8deg.C in dark place.
Step two, marking with colloidal gold
(1) Cleaning: 10mL of the colloidal gold solution was placed in a centrifuge tube, and the precipitate was collected by centrifugation.
(2) Adjusting pH: re-dissolving the precipitate to 10mL of original volume by using a sodium tetraborate-sodium hydroxide buffer solution with the concentration of 0.1M; the observation was clear and transparent, with no suspended matter and no precipitate.
(3) Antibody labeling: firstly, 200 mug of syncytial virus detection antibody is added, and the mixture is stirred in a water bath at 37 ℃ for 10min; step two, adding 300 mug of novel coronavirus detection antibody, and stirring in a water bath at 37 ℃ for 10min; thirdly, adding 500 mug of chicken IgY antibody, and stirring in a water bath at 37 ℃ for 10min; and step four, 0.1g of BSA and 0.1g of PEG20000 are synchronously added, and the reaction is finished after the water bath stirring at 37 ℃ is continued for 30 min.
(4) Centrifuging, and setting a centrifuging program to 12000rpm/min for 10min; taking the precipitate, and redissolving the precipitate to 1/10 of the original volume, namely 1mL, by using a phosphoric acid preservation solution; preserving at 2-8deg.C;
the phosphoric acid preservation solution comprises the following raw materials in percentage by mass: 1% sucrose, 0.5% casein, 0.3% tween 20, the balance purified water, PH7.4.
Step three, preparation of the bonding pad
(1) The formula of the bonding pad pretreatment liquid comprises the following components: 0.02M phosphate buffer containing 0.5% triton X100,0.4mg/mL blocker, 1% sucrose, 0.5% casein, the balance purified water, pH7.4.
(2) Spreading the treatment liquid on a glass fiber membrane, adding the glass fiber membrane with the specification of 200 mm-300 mm into 40mL of the bonding pad pretreatment liquid, drying in an oven, cutting into the specification of 8 mm-300 mm, and preserving at room temperature and low humidity.
Step four, metal spraying
Spraying the marked gold solution prepared in the second step into the bonding pad prepared in the third step. Then put into an oven for drying, and stored at room temperature and low humidity.
Step five, preparation of sample pad
Sample pad pretreatment liquid formula: 0.02M phosphate buffer containing 0.5% triton X100,5% sucrose, 1% BSA,1% PEG20000,1% PVP10,0.3% sulfur Liu Gongna, the balance purified water, pH8.0.
The sample pad pretreatment liquid was spread on a glass fiber film, and 200mm by 300mm specification glass fiber film was added to 40mL of the sample pad pretreatment liquid. Oven drying, cutting into 17mm 300mm, and storing at room temperature and low humidity.
Step six, NC film preparation
(1) Sticking an NC film on a PVC bottom plate;
(2) The novel coronavirus capture antibody, syncytial virus capture antibody and goat anti-chicken IgY antibody were then diluted to 0.5mg/mL with 0.01M phosphate buffer containing 3% sucrose by mass, respectively.
(3) The novel coronavirus capture antibody with the concentration of 0.5mg/mL is used as a detection line N, the syncytial virus capture antibody with the concentration of 0.5mg/mL is used as a detection line R, the goat anti-chicken IgY antibody with the concentration of 0.5mg/mL is used as a quality control line C, and the antibodies are respectively fixed on an NC film, wherein the coating amount is 1ul/cm.
(4) And (3) placing the NC film in the step (3) into an oven to dry for 6 hours, and storing the NC film in a room temperature and low humidity condition for standby.
Step seven, assembling test paper strips
Sequentially sticking the sample pad, the bonding pad, the NC film and the water absorption pad to prepare a large plate, and then cutting the large plate into test strips with the diameters of 3mm and 60 mm.
Comparative example 2:
step one, preparation of colloidal gold
(1) 96mL of ultrapure water was added to the round-bottomed flask, followed by addition of 4mL of chloroauric acid at a mass concentration of 1%; heating the round-bottom flask in an oil bath, wherein the rotating speed is set to be 100rpm;
(2) After boiling, adding 1mL trisodium citrate with the mass concentration of 1%, keeping the rotating speed at 100rpm, and continuously boiling for 10 minutes to obtain a colloidal gold solution;
(3) And (3) recovering the prepared colloidal gold to room temperature, and then using ultrapure water to fix the volume to 100mL. The maximum absorption peak wavelength is about 525nm and the particle size is about 40nm by using a full-wavelength spectrophotometer test; observing that the colloidal gold solution is clear and transparent, and has no suspended matters or sediment; the colloid Jin Chu is stored at 2-8deg.C in dark place.
Step two, marking with colloidal gold
(1) Taking 10mL of colloidal gold solution, then adding 20 mu L of 0.2M potassium carbonate solution, and uniformly mixing;
(2) Then, 200 mug of syncytial virus detection antibody is rapidly added for reaction for 15 minutes, and then 100ul of 10% BSA solution with mass concentration is added for continuous reaction;
(3) After the reaction is finished for 30 minutes, the syncytial virus detection antibody colloidal gold preservation solution is obtained and preserved at 2-8 ℃ in a dark place for standby.
(4) Taking 10mL of colloidal gold solution, then adding 20 mu L of 0.2M potassium carbonate solution, and uniformly mixing;
(5) Then, 300 mug of novel coronavirus detection antibody is rapidly added for reaction for 15 minutes, and then 100ul of 10% BSA solution with mass concentration is added for continuous reaction;
(6) After the reaction is finished for 30 minutes, the novel coronavirus detection antibody colloidal gold preservation solution is obtained and preserved at 2-8 ℃ in a dark place for standby.
(7) Taking 10mL of colloidal gold solution, then adding 20 mu L of 0.2M potassium carbonate solution, and uniformly mixing;
(8) The syncytial virus detection antibody colloidal gold solution and the novel coronavirus detection antibody colloidal gold solution were used as a mixture with 300. Mu.g each.
Step three, preparation of the bonding pad
The formula of the bonding pad pretreatment liquid comprises the following components: 0.02M phosphate buffer containing 0.5% triton X100,0.4mg/mL blocker, 1% sucrose, 0.5% casein, the balance purified water, pH7.4.
Spreading the bonding pad pretreatment liquid on a glass fiber film, adding 200mm 300mm glass fiber film into 40mL bonding pad pretreatment liquid, drying in an oven, cutting into 8mm 300mm glass fiber film, and preserving at room temperature and low humidity.
Step four, metal spraying
Spraying the marked gold solution prepared in the second step into the bonding pad prepared in the third step. Then put into an oven for drying, and stored at room temperature and low humidity.
Step five, preparation of sample pad
Sample pad pretreatment liquid formula: 0.02M phosphate buffer containing 0.5% triton X100,5% sucrose, 1% BSA,1% PEG20000,1% PVP10,0.3% sulfur Liu Gongna, the balance purified water, pH8.0.
The sample pad pretreatment liquid was spread on a glass fiber film, and 200mm by 300mm specification glass fiber film was added to 40mL of the sample pad pretreatment liquid. Oven drying, cutting into 17mm 300mm, and storing at room temperature and low humidity.
Step six, NC film preparation
(1) Sticking an NC film on a PVC bottom plate;
(2) Preparing a quality control line (C):
staphylococcal protein a was diluted with 0.01M disodium hydrogen phosphate-beta cyclodextrin buffer: 1mL of disodium hydrogen phosphate-beta cyclodextrin buffer solution (the concentration is 0.01M) is added with 0.5mg of staphylococcus A protein, and the mixture is uniformly mixed to be used as a working solution of a quality control line (C).
(3) And (3) preparing a detection line (N):
dilution of novel coronavirus capture antibody with disodium hydrogen phosphate-beta cyclodextrin buffer solution at concentration 0.01M: 1mL of disodium hydrogen phosphate-beta cyclodextrin buffer solution (the concentration is 0.01M) is added with 0.5mg of novel coronavirus capture antibody, and the mixture is uniformly mixed to be used as a detection line (N) working solution.
(4) And (3) preparing a detection line (R):
the syncytial virus capture antibody was diluted with disodium hydrogen phosphate-beta cyclodextrin buffer at a concentration of 0.01M: 1mL disodium hydrogen phosphate-beta cyclodextrin buffer (with the concentration of 0.01M) is added with 0.5mg of syncytial virus capture antibody, and the mixture is uniformly mixed to be used as a detection line (R) working solution.
(4) And (3) a quality control line (C) working solution, a detection line (N, R), a scribing instrument is started, a spraying pen spraying amount of 1ul/cm is set, the X-axis position and the Y-axis position are set, film scribing is carried out, and the quality control line and the detection line are coated.
(5) And (3) drying: the coated NC film was dried overnight (as in example 4) at low temperature (2 ℃) and low humidity (10%).
Step seven, assembling test paper strips
Sequentially sticking the sample pad, the bonding pad, the NC film and the water absorption pad to prepare a large plate, and then cutting the large plate into test strips with the diameters of 3mm and 60 mm.
Comparative example 3:
step one, preparation of colloidal gold
(1) 96mL of ultrapure water was added to the round-bottomed flask, followed by addition of 4mL of chloroauric acid at a mass concentration of 1%; heating the round-bottom flask in an oil bath, wherein the rotating speed is set to be 100rpm;
(2) After boiling, adding 1mL trisodium citrate with the mass concentration of 1%, keeping the rotating speed at 100rpm, and continuously boiling for 10 minutes to obtain a colloidal gold solution;
(3) And (3) recovering the prepared colloidal gold to room temperature, and then using ultrapure water to fix the volume to 100mL. The maximum absorption peak wavelength is about 525nm and the particle size is about 40nm by using a full-wavelength spectrophotometer test; observing that the colloidal gold solution is clear and transparent, and has no suspended matters or sediment; the colloid Jin Chu is stored at 2-8deg.C in dark place.
Step two, marking with colloidal gold
(1) Taking 10mL of colloidal gold solution, then adding 20 mu L of 0.2M potassium carbonate solution, and uniformly mixing;
(2) Then, 200 mug of syncytial virus detection antibody is rapidly added for reaction for 15 minutes, and then 100ul of 10% BSA solution with mass concentration is added for continuous reaction;
(3) After the reaction is finished for 30 minutes, the syncytial virus detection antibody colloidal gold preservation solution is obtained and preserved at 2-8 ℃ in a dark place for standby.
(4) Taking 10mL of colloidal gold solution, then adding 20 mu L of 0.2M potassium carbonate solution, and uniformly mixing;
(5) Then, 300 mug of novel coronavirus detection antibody is rapidly added for reaction for 15 minutes, and then 100ul of 10% BSA solution with mass concentration is added for continuous reaction;
(6) After the reaction is finished for 30 minutes, the novel coronavirus detection antibody colloidal gold preservation solution is obtained and preserved at 2-8 ℃ in a dark place for standby.
(7) Taking 10mL of colloidal gold solution, then adding 20 mu L of 0.2M potassium carbonate solution, and uniformly mixing;
(8) Then, 500 mug of chicken IgY antibody is added rapidly to react for 15 minutes, and then 100 mug of 10% BSA solution is added to continue the reaction;
(9) After the reaction is finished for 30 minutes, the chicken IgY antibody colloidal gold preservation solution is obtained and is preserved for standby at 2-8 ℃ in a dark place.
(10) The syncytial virus detection antibody colloidal gold solution, the novel coronavirus detection antibody colloidal gold solution and the chicken IgY colloidal gold solution were mixed and used at 300 mug each.
Step three, preparation of the bonding pad
The formula of the bonding pad pretreatment liquid comprises the following components: 0.02M phosphate buffer containing 0.5% triton X100,0.4mg/mL blocker, 1% sucrose, 0.5% casein, the balance purified water, pH7.4.
Spreading the bonding pad pretreatment liquid on a glass fiber film, adding 200mm 300mm glass fiber film into 40mL bonding pad pretreatment liquid, drying in an oven, cutting into 8mm 300mm glass fiber film, and preserving at room temperature and low humidity.
Step four, metal spraying
Spraying the marked gold solution prepared in the second step into the bonding pad prepared in the third step. Then put into an oven for drying, and stored at room temperature and low humidity.
Step five, preparation of sample pad
Sample pad pretreatment liquid formula: 0.02M phosphate buffer containing 0.5% triton X100,5% sucrose, 1% BSA,1% PEG20000,1% PVP10,0.3% sulfur Liu Gongna, the balance purified water, pH8.0.
The sample pad pretreatment liquid was spread on a glass fiber film, and 200mm by 300mm specification glass fiber film was added to 40mL of the sample pad pretreatment liquid. Oven drying, cutting into 17mm 300mm, and storing at room temperature and low humidity.
Step six, NC film preparation
(1) Sticking an NC film on a PVC bottom plate;
(2) The novel coronavirus capture antibody, syncytial virus capture antibody and goat anti-chicken IgY antibody were then diluted to 0.5mg/mL with 0.01M phosphate buffer containing 3% sucrose by mass, respectively.
(3) The novel coronavirus capture antibody with the concentration of 0.5mg/mL is used as a detection line N, the syncytial virus capture antibody with the concentration of 0.5mg/mL is used as a detection line R, the goat anti-chicken IgY antibody with the concentration of 0.5mg/mL is used as a quality control line C, and the antibodies are respectively fixed on an NC film, wherein the coating amount is 1ul/cm.
(4) And (3) placing the NC film in the step (3) into an oven to dry for 6 hours, and storing the NC film in a room temperature and low humidity condition for standby.
Step seven, assembling test paper strips
Sequentially sticking the sample pad, the bonding pad, the NC film and the water absorption pad to prepare a large plate, and then cutting the large plate into test strips with the diameters of 3mm and 60 mm.
Comparative example 4:
this comparative example differs from example 7 in that beta cyclodextrin was not added in the preparation of NC film.
Comparative example 5:
this comparative example differs from example 7 in that no staphylococcal protein A was added in the preparation of NC membrane.
Comparative example 6:
this comparative example is different from example 7 in that the NC film with the quality control line was dried at 31 ℃.
Comparative example 7:
the present comparative example is different from example 7 in that the NC film with the quality control line was set to have a humidity of 31% at the time of preparing the NC film.
Comparative example 8:
this comparative example differs from example 7 in that the sodium tetraborate-sodium hydroxide buffer solution was replaced with a potassium carbonate solution when the colloidal gold-labeled syncytial virus detection antibody and the novel coronavirus detection antibody were used.
Comparative example 9:
this comparative example differs from example 8 in that, when the colloidal gold-labeled syncytial virus-detecting antibody and the novel coronavirus-detecting antibody were added simultaneously, the syncytial virus-detecting antibody, the novel coronavirus-detecting antibody, 1% BSA and 1% PEG20000 were stirred in a water bath at 37℃for 60 minutes.
Comparative example 10:
respiratory syncytial virus detection kit, commercially available, manufacturer: hangzhou Innovative biological detection and control technologies Co.
Novel coronavirus antigen detection kit, commercially available, manufacturer: nanjinouzan Biotechnology Co., ltd.
Performance detection
1. Sensitivity comparison
Collecting a sample of the cytovirus swab, then carrying out 10-time gradient dilution on the sample by using a sample preservation solution to obtain P1-P5 and 5 gradient samples, wherein P6 is the sample preservation solution, and then detecting the six samples, wherein the detection results are shown in table 1.
TABLE 1 syncytial Virus sensitivity detection Table
Collecting a novel coronavirus swab sample, then carrying out 10-time gradient dilution on the sample by using a sample preservation solution to obtain P1-P5 and 5 gradient samples, wherein P6 is the sample preservation solution, and then detecting the six samples, wherein the detection results are shown in Table 2.
TABLE 2 novel coronavirus sensitivity test meter
| P1 | P2 | P3 | P4 | P5 | P6 | |
| Example 7 | + | + | + | + | + | - |
| Example 8 | + | + | + | + | + | - |
| Example 9 | + | + | + | + | + | - |
| Example 10 | + | + | + | + | + | - |
| Example 11 | + | + | + | + | + | - |
| Comparative example 1 | + | + | + | - | - | - |
| Comparative example 2 | + | + | + | - | - | - |
| Comparative example 3 | + | + | - | - | - | - |
| Comparative example 4 | + | + | + | - | - | - |
| Comparative example 5 | + | + | - | - | - | - |
| Comparative example 6 | + | + | + | - | - | - |
| Comparative example 7 | + | + | + | - | - | - |
| Comparative example 8 | - | - | - | - | - | - |
| Comparative example 9 | + | + | + | - | - | - |
| Comparative example 10 | + | + | + | + | - | - |
By comparison, the sensitivity of the kit prepared in comparative example 1 was 100-fold lower than that of example 7, as can be seen from the combination of tables 1 and 2, example 7, and comparative example 1. The control of NC membrane low-temperature low-humidity drying by using beta cyclodextrin as coating liquid improves the binding rate of protein and NC membrane and improves the sensitivity of the kit.
By comparison, the sensitivity of the kit prepared in comparative example 2 was 100-fold lower than that in example 7, as can be seen from the combination of tables 1 and 2, example 7, and comparative example 2. The method has the advantages that the sample is added in a 37 ℃ water bath, and is labeled simultaneously, colloidal gold is fully utilized, the specific surface area is large, the surface effect is good, physical adsorption is carried out, the labeling efficiency is greatly improved, and the color development of the kit is enhanced.
By comparison, the sensitivity of the kit prepared in comparative example 3 was 1000-fold lower than that in example 7, as can be seen from the combination of tables 1 and 2, example 7, and comparative example 3. The improvement of the colloidal gold marking process and the NC film preparation process in the application is illustrated, and the sensitivity of the test strip is effectively improved.
By comparison, as can be seen from the combination of tables 1 and 2, example 7 and comparative example 4, the kit prepared in comparative example 4 has a sensitivity 100-fold lower than that in example 7. The addition of beta cyclodextrin in the application is proved to protect the binding site of antigen and antibody and avoid the false negative condition.
By comparison, the kit obtained in comparative example 5 showed 1000-fold lower sensitivity and reddening of the test strip compared with example 7, as can be seen from the combination of tables 1, 2, example 7 and comparative example 5. The staphylococcus A protein is used as a quality control line, so that the problem of background redness caused by high colloidal gold spraying is solved, the range of detection antibodies is wider, and the detection antibodies can be used as detection antibodies no matter sheep antibodies, rabbit antibodies or mouse antibodies, so that the sensitivity of the kit is improved.
By comparison, the sensitivity of the kit obtained in comparative example 6 was 100-fold lower than that of example 7, as can be seen from the combination of tables 1 and 2, example 7, and comparative example 6. The low temperature in the application is more favorable for reducing the heat loss in the protein drying process, protecting the antigen-antibody binding site and enhancing the color development of the kit.
By comparison, the sensitivity of the kit obtained in comparative example 7 was 100-fold lower than that of example 7, as can be seen from the combination of tables 1 and 2, example 7, and comparative example 7. The low humidity in the application is more beneficial to protecting the hydrophilicity of the nitrocellulose membrane and improving the binding rate of protein and the nitrocellulose membrane.
As can be seen from the results of Table 1, table 2, example 7 and comparative example 8, the sensitivity of the kit obtained in comparative example 8 was not measured by comparison with that of example 7. The sodium tetraborate-sodium hydroxide buffer solution provides a softer and more stable weak alkaline environment, so that the combination of the colloidal gold and positive charge groups of protein molecules can be firmer.
By comparison, the sensitivity of the kit obtained in comparative example 9 was 100-fold lower than that of example 7, as can be seen from the combination of tables 1 and 2, example 7, and comparative example 9. The method has the advantages that the method can be used for carrying out separate sample adding and simultaneous labeling in a 37 ℃ water bath, so that the labeling efficiency can be effectively improved, and the sensitivity of the kit can be improved.
By comparison, the sensitivity of the kit of comparative example 10 was 10-fold lower than that of example 7, as can be seen from the combination of tables 1 and 2, example 7, and comparative example 10. The performance of the kit is superior to that of the commercial respiratory syncytial virus antigen detection kit and the novel coronavirus antibody detection kit.
Therefore, the test shows that the sensitivity of the kit prepared by the method is 100-1000 times higher than that of the kit prepared by the technical scheme of comparative examples 1-9. The sensitivity was 10 times higher than that of comparative example 10. Therefore, the kit prepared by the method has better sensitivity performance.
1. Specific detection
2. 100 negative samples and 100 healthy person swab samples are collected, 300ul of preservation solution is added into each swab sample, and then test strips are carried out on the 100 samples, and the detection results are shown in table 3.
TABLE 3 specificity detection Table
| Negative coincidence rate | |
| Example 7 | 100％(100/100) |
| Example 8 | 100％(100/100) |
| Example 9 | 100％(100/100) |
| Example 10 | 100％(100/100) |
| Example 11 | 100％(100/100) |
| Comparative example 1 | 90％(90/100) |
| Comparative example 2 | 90％(90/100) |
| Comparative example 3 | 80％(80/100) |
| Comparative example 4 | 95％(95/100) |
| Comparative example 5 | 80％(80/100) |
| Comparative example 6 | 99％(99/100) |
| Comparative example 7 | 98％(98/100) |
| Comparative example 8 | 95％(95/100) |
| Comparative example 9 | 97％(97/100) |
As can be seen from Table 3, the specificity detection rate of the kit prepared by the method is 100%.
Comparative examples 1-9 tested negative samples, and the detection rate did not reach 100%.
As can be seen from the combination of Table 3, the specificity of the kit prepared in comparative example 1 was 90%; the method has the advantages that beta cyclodextrin is used as coating liquid, NC film low-temperature low-humidity drying is controlled, the binding rate of protein and NC film is improved, and the sensitivity of the kit is improved, so that a product with higher specificity is obtained.
As can be seen from table 3, the specificity of the kit prepared in comparative example 2 was 90%; the method has the advantages that the sample is added in a 37 ℃ water bath, and is labeled simultaneously, colloidal gold is fully utilized, the specific surface area is large, the surface effect is good, physical adsorption is carried out, the labeling efficiency is greatly improved, the color development of the kit is enhanced, and therefore the product with higher specificity is obtained.
As can be seen from the combination of Table 3, the specificity of the kit prepared in comparative example 3 was 80%; the improvement of the colloidal gold marking process and the NC film preparation process in the application is illustrated, and the sensitivity of the test strip is effectively improved, so that a product with higher specificity is obtained.
As can be seen from table 3, the specificity of the kit prepared in comparative example 4 was 95%; the beta cyclodextrin is added in the method, so that the binding site of the antigen and the antibody is protected, the false negative condition is avoided, and a product with higher specificity is obtained.
As can be seen from the combination of Table 3, the specificity of the kit prepared in comparative example 5 was 80%; the staphylococcus A protein is used as a quality control line, the problem of background redness caused by high colloidal gold spraying is solved, the range of detection antibodies is wider, and the detection antibodies can be used as detection antibodies no matter sheep antibodies, rabbit antibodies or mouse antibodies, so that the sensitivity of the kit is improved, and a product with higher specificity is obtained.
As can be seen from the combination of Table 3, the specificity of the kit prepared in comparative example 6 was 99%; the low temperature in the application is more favorable for reducing the heat loss in the protein drying process, protecting the antigen-antibody binding site and enhancing the color development of the kit, so that a product with higher specificity is obtained.
As can be seen from the combination of Table 3, the specificity of the kit prepared in comparative example 7 was 98%; the low humidity in the application is more beneficial to protecting the hydrophilicity of the nitrocellulose membrane, and improving the binding rate of protein and the nitrocellulose membrane, so that a product with higher specificity is obtained.
As can be seen from table 3, the specificity of the kit prepared in comparative example 8 was 95%; the sodium tetraborate-sodium hydroxide buffer solution provides a softer and more stable weak alkaline environment, so that the combination of the colloidal gold and positive charge groups of protein molecules is firmer, and a product with higher specificity is obtained.
As can be seen from the combination of Table 3, the specificity of the kit prepared in comparative example 9 was 97%; the method shows that the water bath at 37 ℃ in the application is used for separate sample adding and simultaneous labeling, so that the labeling efficiency can be effectively improved, the sensitivity of the kit is improved, and a product with higher specificity is obtained.
Therefore, the kit prepared by the method is obviously superior to the detection scheme of the comparative example in sensitivity and specificity.
The present embodiment is merely illustrative of the present application and is not intended to be limiting, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as required, but is protected by patent laws within the scope of the claims of the present application.
Claims (10)
1. A colloidal gold chromatographic test strip for simultaneously detecting syncytial virus and novel coronavirus antigen is characterized in that: the test strip comprises a bottom plate, a sample pad, a binding pad, an NC film and a water absorption pad, wherein the sample pad, the binding pad, the NC film and the water absorption pad are sequentially lapped and stuck on the bottom plate according to the chromatographic direction, and the binding pad is coated with a syncytial virus detection antibody marked by colloidal gold and a novel coronavirus detection antibody; the NC film is provided with two detection lines and a quality control line, the two detection lines are respectively coated with a syncytial virus capture antibody and a novel coronavirus capture antibody, and the quality control line is coated with a staphylococcus A protein.
2. The colloidal gold chromatographic test strip for simultaneously detecting syncytial virus and novel coronavirus antigen according to claim 1, wherein the preparation method of the quality control line comprises the following steps:
(1) Diluting staphylococcus protein A with sodium dihydrogen phosphate-beta cyclodextrin solution to obtain quality control line working solution;
(2) Drawing a film on the NC film by using the quality control line working solution;
(3) And drying the NC film with the quality control line to obtain the quality control line.
3. The colloidal gold chromatographic test strip for simultaneously detecting syncytial virus and novel coronavirus antigen according to claim 2, wherein the colloidal gold chromatographic test strip is characterized in that: the concentration of the sodium dihydrogen phosphate-beta cyclodextrin in the step (1) is 0.01M-0.05M; the concentration of staphylococcal protein A after dilution is 0.25-0.5mg/mL.
4. The colloidal gold chromatographic test strip for simultaneously detecting syncytial virus and novel coronavirus antigen according to claim 2, wherein the colloidal gold chromatographic test strip is characterized in that: the drying temperature in the step (3) is 2-30 ℃ and the humidity is not more than 30%.
5. The colloidal gold chromatographic test strip for simultaneously detecting syncytial virus and novel coronavirus antigen according to claim 1, wherein the preparation method of the colloidal gold labeled syncytial virus detection antibody and novel coronavirus detection antibody comprises the following steps:
s1, centrifuging a colloidal gold solution, and taking a precipitate;
s2, re-dissolving the precipitate with sodium tetraborate-sodium hydroxide buffer solution to the volume of the colloidal gold solution in the step S1;
s3, adding a syncytial virus detection antibody for stirring, adding a novel coronavirus detection antibody for stirring, and then adding BSA and PEG20000 for stirring;
s4, centrifuging, taking a precipitate, and redissolving the precipitate to 1/10 times of the volume of the colloidal gold solution by using a phosphoric acid preservation solution.
6. The colloidal gold chromatographic test strip for simultaneously detecting syncytial virus and novel coronavirus antigen according to claim 5, wherein the colloidal gold chromatographic test strip is characterized in that: the volume of the colloidal gold solution in the step S1 is 10mL-40mL.
7. The colloidal gold chromatographic test strip for simultaneously detecting syncytial virus and novel coronavirus antigen according to claim 5, wherein the colloidal gold chromatographic test strip is characterized in that: the concentration of sodium tetraborate-sodium hydroxide buffer in step S2 is 0.1M-0.5M.
8. The colloidal gold chromatographic test strip for simultaneously detecting syncytial virus and novel coronavirus antigen according to claim 5, wherein the colloidal gold chromatographic test strip is characterized in that: the addition amount of the syncytial virus detection antibody in the step S3 is 200-300 mug; the addition amount of the novel coronavirus detection antibody is 200-300 mug; the mass concentration of added BSA is 0.5-2%; the mass concentration of the added PEG20000 is 0.5-2%.
9. The colloidal gold chromatographic test strip for simultaneously detecting syncytial virus and novel coronavirus antigen according to claim 5, wherein the colloidal gold chromatographic test strip is characterized in that: in the step S3, a syncytial virus detection antibody is added, and the temperature of water bath stirring is 35-40 ℃ for 10-20min; adding a novel coronavirus detection antibody, and stirring in a water bath at 35-40deg.C for 10-20min; after adding BSA and PEG20000, stirring in water bath at 35-40deg.C for 40-60min.
10. A kit, characterized in that: a test strip comprising the test strip of any one of claims 1-9.
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| CN116718771A (en) * | 2023-08-08 | 2023-09-08 | 北京库尔科技有限公司 | Influenza A virus, influenza B virus, respiratory syncytial virus and novel coronavirus antigen detection kit |
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| CN116718771B (en) * | 2023-08-08 | 2023-11-03 | 北京库尔科技有限公司 | Influenza A virus, influenza B virus, respiratory syncytial virus and novel coronavirus antigen detection kit |
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