CN111426839A - 2019 novel coronavirus detection test paper and preparation process thereof - Google Patents
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Abstract
The invention provides a test paper for 2019 novel coronavirus, which comprises a sample pad, a combination pad, a chromatography microporous membrane and a water absorption pad, wherein the sample pad, the combination pad, the chromatography microporous membrane and the water absorption pad are sequentially overlapped on any one side of a substrate, the combination pad is combined with a compound for adsorbing goat anti-human IgA antibody by colloidal gold particles, the chromatography microporous membrane is sequentially provided with a detection line and a quality control line, the detection line is arranged at the position, close to the combination pad, of the chromatography microporous membrane, and the quality control line is arranged at the position, far away from the combination pad, of the chromatography microporous membrane; the detection line is a 2019 novel coronavirus antigen, and the quality control line is an antibody for resisting sheep species. According to the invention, the IgA subtype of the specific immunoglobulin of the 2019 novel coronavirus is used as a detection target point, and compared with other subtypes (IgD, IgE, IgM and IgG), the IgA specificity of the specific immunoglobulin of the 2019 novel coronavirus is stronger, the sensitivity and the stability are higher, and the detection efficiency is favorably improved.
Description
Technical Field
The invention relates to the field of biotechnology detection, in particular to a detection test paper for 2019 novel coronavirus and a preparation process thereof.
Background
Coronaviruses are a large family of viruses known to cause the common cold and more serious diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). 2019 the novel coronavirus is a novel strain of coronavirus that has not been previously found in humans. After people are infected with the coronavirus, the common signs of the person are respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death.
In the face of such severe prevention and control situation, the 2019-nCoV infected person is quickly identified, and isolation treatment is immediately carried out, so that the key point of winning the national battle is achieved. The detection of the novel coronavirus mainly relies on nucleic acid detection, and the nucleic acid detection method has high sensitivity and specificity, but the nucleic acid detection of the novel coronavirus has three main problems at present. (1) The nucleic acid detection of part of samples is negative, the sampling is the main reason, the sampling positive rate of the lower respiratory tract is high, but the large-scale popularization is not easy, the sampling operation of the upper respiratory tract is convenient, but the sampling is not standard and the false negative is easy to generate; (2) the nucleic acid detection operation is relatively complex and difficult to realize automation; (3) nucleic acid detection has certain requirements on laboratory environment, needs professional PCR laboratories, and is limited by clinical large-scale popularization. Above three deficiency lead to present novel coronavirus to detect and rely on nucleic acid detection alone and can't satisfy the demand that epidemic situation prevented and controlled, for improving novel coronavirus detection ability, need provide quick, accurate, safe detection means, uses in coordination with current nucleic acid detection method, further improves detection ability.
Disclosure of Invention
The invention aims to provide a test strip for detecting a 2019 novel coronavirus and a preparation process thereof, and aims to solve the problems of complex operation and low detection efficiency of nucleic acid detection of the 2019 novel coronavirus in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
the test paper for the 2019 novel coronavirus comprises a sample pad, a binding pad, a chromatography microporous membrane and a water absorption pad which are sequentially overlapped on any one side of a substrate, wherein the binding pad is bound with a compound with colloidal gold particles for adsorbing goat anti-human IgA antibodies; the chromatography microporous membrane is sequentially provided with a detection line and a quality control line, the detection line is arranged at the position of the chromatography microporous membrane close to the combination pad, and the quality control line is arranged at the position of the chromatography microporous membrane far away from the combination pad; the detection line is a 2019 novel coronavirus antigen, and the quality control line is an antibody for resisting sheep species.
And the detection device for the 2019 novel coronavirus comprises a sleeve and the detection test paper for the 2019 novel coronavirus.
And, a method for preparing a test strip for a 2019 novel coronavirus, the method comprising the steps of:
providing a compound with colloidal gold particles adsorbing goat anti-human IgA, and depositing the compound with colloidal gold particles adsorbing goat anti-human IgA to a combination pad to obtain the combination pad with the compound with colloidal gold particles adsorbing goat anti-human IgA;
providing a 2019 novel coronavirus antigen and an anti-sheep species antibody, and spraying the 2019 novel coronavirus antigen and the anti-sheep species antibody on the chromatography microporous membrane in sequence to obtain a chromatography microporous membrane with a detection line and a quality control line in sequence;
and sequentially arranging a sample pad, the combination pad combined with the compound for adsorbing the goat anti-human IgA by the colloidal gold particles, the chromatography microporous membrane and the water absorption pad which are sequentially provided with the detection line and the quality control line on any one side of the substrate to obtain the test paper for the 2019 novel coronavirus.
The test paper for detecting the 2019 novel coronavirus is used for detecting the 2019 novel coronavirus based on the immunochromatography principle of serological immunoglobulin, and has the following advantages that:
firstly, the IgA subtype of the specific immunoglobulin of the 2019 novel coronavirus is used as a detection target, and compared with other subtypes (IgD, IgE, IgM and IgG), the IgA specificity of the specific immunoglobulin of the 2019 novel coronavirus is stronger, the sensitivity and the stability are higher, and the detection efficiency is favorably improved;
secondly, colloidal gold particles are adopted to carry out adsorption coating on the detection target, so that the specific immunoglobulin IgA of the 2019 novel coronavirus with positive charge and the gold particles with negative charge are adsorbed to form a compound through charge action, the colloidal gold particles are utilized to mark the specific immunoglobulin IgA of the 2019 novel coronavirus, colloidal gold is used as a tracer, a color development phenomenon can occur after a sample to be detected carries out antigen-antibody specific reaction, the detection result can be quickly judged through the color development phenomenon, and the detection sensitivity and the detection efficiency are improved;
thirdly, a detection line and a quality control line are sequentially arranged on a chromatography microporous membrane in the test paper, the detection line is coated with a 2019 novel coronavirus antigen, the quality control line is coated with an anti-sheep species antibody, after the 2019 novel coronavirus is infected, a body can be immunized to generate specific immunoglobulin, the detection line is coated with the 2019 novel coronavirus antigen, the specific immunoglobulin in a sample to be detected and the 2019 novel coronavirus antigen of the detection line can generate immunoreaction with high specificity and high affinity, so that an immune complex is enriched, a marker colloidal gold can be directly observed to obtain an intuitive experimental phenomenon, and a detection result is rapidly obtained.
The test paper for the 2019 novel coronavirus has the advantages of high sensitivity, standardized sampling, simple and automatic operation and unlimited site, is used for detecting based on the serology principle, a detected sample is body blood, the blood sample collection safety is far higher than that of a throat swab, and the risk of infection of medical and health care personnel is greatly reduced; the test paper is easy to operate, high in sensitivity, strong in specificity, good in repeatability, easy to interpret, wide in applicability, free of other instruments and equipment, and suitable for clinical examination, epidemiological investigation, field quarantine and the like of all levels of medical detection mechanisms.
The invention provides a detection device for a 2019 novel coronavirus, which comprises a plastic card and detection test paper for the 2019 novel coronavirus. The detection device is a closed detection device, is used for detecting the 2019 novel coronavirus, is convenient and efficient to detect, has high sensitivity, is not interfered by the outside world, and is suitable for clinical examination, epidemiological investigation, on-site quarantine and the like of various medical detection mechanisms.
The invention also provides a preparation method of the test paper for the 2019 novel coronavirus, the preparation method is simple, convenient and quick, and the test paper for the 2019 novel coronavirus prepared by the preparation method has the advantages and is suitable for wide application.
Drawings
Fig. 1 is a schematic structural diagram of a test strip for 2019 novel coronavirus provided by the embodiment of the invention.
FIG. 2 shows the crystal nucleus structure of the colloidal gold particles bound with the antibody according to the embodiment of the present invention.
Fig. 3 is a schematic diagram of a specificity verification result of the test strip provided in the embodiment of the present invention.
Fig. 4 is a schematic diagram illustrating the result determination of the test strip provided in the embodiment of the present invention.
Fig. 5 is a structure diagram of a ferrule of the detection apparatus according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and technical effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments obtained by a person of ordinary skill in the art without any inventive step in connection with the embodiments of the present invention are within the scope of the present invention.
In the description of the present invention, it is to be understood that the terms "first", "second" and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
The embodiment of the invention provides a test paper for 2019 novel coronavirus, which comprises a sample pad, a combination pad, a chromatography microporous membrane and a water absorption pad, wherein the sample pad, the combination pad, the chromatography microporous membrane and the water absorption pad are sequentially overlapped on any one side of a substrate, and the combination pad is combined with a compound that colloidal gold particles adsorb goat anti-human IgA antibodies; the chromatography microporous membrane is sequentially provided with a detection line and a quality control line, the detection line is arranged at the position of the chromatography microporous membrane close to the binding pad, and the quality control line is arranged at the position of the chromatography microporous membrane far away from the binding pad; the detection line is a 2019 novel coronavirus antigen, and the quality control line is an anti-caprine antibody. The test paper for detecting the 2019 novel coronavirus is used for detecting the 2019 novel coronavirus based on the immunochromatography principle of the serological immunoglobulin, and has the following advantages that:
firstly, the IgA subtype of the specific immunoglobulin of the 2019 novel coronavirus is used as a detection target point, and compared with other subtypes (IgD, IgE, IgM and IgG), the IgA specificity of the specific immunoglobulin of the 2019 novel coronavirus is stronger, the sensitivity and the stability are higher, and the detection efficiency is favorably improved;
secondly, colloidal gold particles are adopted to adsorb and coat the detection target, so that the specific immunoglobulin IgA of the 2019 novel coronavirus with positive charge and the gold particles with negative charge are adsorbed to form a compound through charge action, the specific immunoglobulin IgA of the 2019 novel coronavirus is marked by the colloidal gold particles, the colloidal gold is used as a tracer, a color development phenomenon can occur after the antigen-antibody specific reaction of a sample to be detected, the detection result can be quickly judged through the color development phenomenon, and the detection sensitivity and the detection efficiency are improved;
thirdly, a detection line and a quality control line are sequentially arranged on a chromatography microporous membrane in the test paper, the detection line is coated with a 2019 novel coronavirus antigen, the quality control line is coated with an antibody for resisting sheep species, after the 2019 novel coronavirus is infected, a body can be immunized to generate specific immunoglobulin, the detection line is coated with the 2019 novel coronavirus antigen, the specific immunoglobulin in a sample to be detected and the 2019 novel coronavirus antigen of the detection line can generate immunoreaction with high specificity and high affinity, so that an immune complex is enriched, a marker colloidal gold can be directly observed to obtain an intuitive experimental phenomenon, and a detection result is rapidly obtained. And the anti-sheep species antibody coated in the quality control line can be specifically combined with the sheep anti-human IgA antibody to serve as a control group, so that the accuracy of the detection test paper is further ensured.
The test paper for the 2019 novel coronavirus has the advantages of high sensitivity, standardized sampling, simple and automatic operation and unlimited site, is used for detecting based on the serology principle, a detected sample is body blood, the blood sample collection safety is far higher than that of a throat swab, and the risk of infection of medical and health care personnel is greatly reduced; the test paper is easy to operate, high in sensitivity, strong in specificity, good in repeatability, easy to interpret, wide in applicability, free of other instruments and equipment, and suitable for clinical examination, epidemiological investigation, field quarantine and the like of all levels of medical detection mechanisms.
Specifically, the combination pad is combined with a compound that colloidal gold particles adsorb goat anti-human IgA antibodies. In the embodiment of the invention, the IgA subtype of the specific immunoglobulin of the 2019 novel coronavirus is used as a detection target, and compared with other subtypes (IgD, IgE, IgM and IgG), the IgA specificity of the specific immunoglobulin of the 2019 novel coronavirus is stronger, the sensitivity and the stability are higher, and the detection efficiency is favorably improved.
Further, colloidal gold particles are adopted to adsorb and coat the detection target spots, as shown in the attached drawing 2, the specific immunoglobulin IgA of the 2019 novel coronavirus with positive charges and the gold particles with negative charges are adsorbed to form a compound through charge action, the specific immunoglobulin IgA of the 2019 novel coronavirus is marked by the colloidal gold particles, the colloidal gold is used as a tracer, a color development phenomenon can occur after the antigen-antibody specificity reaction is carried out on a sample to be detected, the detection result can be quickly judged through the color development phenomenon, and the detection sensitivity and the detection efficiency are improved. Wherein the colloidal gold is a suspension emulsion composed of a plurality of single gold particles reduced by gold ions, the colloidal gold particles are composed of a basic crystal nucleus (an icosahedron with 11 gold atoms around the atomic gold) and a double ion layer surrounding the atomic gold, and the inner layer negative ions (AuCl) are tightly connected to the surface of the gold nucleus2-) Outer ionic layer H+The dispersion in the inter-colloidal solution, in which the negatively charged layer surrounded by the gold particle surface, called the Zata potential, makes it possible to keep the suspension stable by repelling the colloidal gold particles from each other.
Preferably, the particle size of the gold particles is 15-25 nm. If the particle size of the gold particles is too large, the specific surface area of the particles is too small, so that the amount of the antibody loaded on the particles is reduced, the enrichment of the antibody is influenced, and the detection sensitivity is further reduced; if the particle size of the gold particles is too small, aggregation can be formed among the gold particles, so that smaller steric hindrance is caused, and the antibody loaded on each gold particle is too small, so that the enrichment of the antibody is influenced, and the detection sensitivity is further reduced. In a preferred embodiment of the invention, the gold particles have a particle size of 20 nm. The gold particles with the particle size are selected, so that the enrichment amount of the antibody is moderate, and the high sensitivity is ensured.
Specifically, the test paper for the 2019 novel coronavirus also comprises a chromatography microporous membrane, wherein the chromatography microporous membrane is sequentially provided with a detection line and a quality control line, the detection line is a 2019 novel coronavirus antigen, and the quality control line is an antibody against sheep species. After 2019 new coronavirus infection, some specific immunoglobulin is produced in the body through immunization. According to the embodiment of the invention, the detection line of the 2019 novel coronavirus antigen is set, so that the immunoreaction with high specificity and high affinity between the specific immunoglobulin in the sample to be detected and the 2019 novel coronavirus antigen in the detection line can be initiated, the immune complex is enriched, the colloidal gold of the marker can be directly observed to obtain an intuitive experimental phenomenon, and the detection result can be quickly obtained. And the quality control line is an anti-sheep species antibody, can be specifically combined with a sheep anti-human IgA antibody, and is used as the quality control line to further ensure the accuracy of the detection test paper.
Preferably, the concentration of the 2019 novel coronavirus antigen in the detection line is 0.8-1.2 mg/m L, if the concentration of the 2019 novel coronavirus antigen in the detection line is too high, false positive can be formed in a test process, and antigen waste is caused, and if the concentration is too low, the antigen quantity is insufficient, the detection sensitivity is influenced, false negative is caused, and the detection accuracy is influenced.
Preferably, the line width of the detection line is 0.7-0.8 mm. If the line width of the sprayed detection line is too wide, the use amount of the antigen is too much, the antigen is too much combined with non-specific immunoglobulin in blood, the false positive result is easy to occur, and the antigen waste is caused; if the line width of the sprayed detection line is too narrow, the observation of the test result is not facilitated.
Preferably, the concentration of the anti-sheep species antibody of the quality control line is 1.0-1.5 mg/m L, if the concentration of the anti-sheep species antibody of the quality control line is too high, false positive can be formed in the test process, and antibody waste is caused, and if the concentration is too low, the antibody quantity is insufficient, the quality control line is not clear, and the quality control is not closed.
Preferably, the line width of the quality control line is 0.7-0.8 mm. If the line width of the sprayed quality control line is too wide, the use amount of the antigen is excessive, the antigen can be combined with non-specific immunoglobulin in blood too much, the false positive result is easy to occur, and the antigen waste is caused; if the line width of the sprayed quality control line is too narrow, the observation of the test result is not facilitated.
Preferably, the distance between the detection line and the quality control line is 0.5-0.6 cm; if the distance between the two is too small, the judgment of the test result is influenced, and the observation of the test result is not facilitated; if the distance between the two is too large, the test paper is easy to cause overlarge volume and inconvenient to use.
In a specific embodiment of the invention, the test paper for the 2019 novel coronavirus comprises a sample pad, a combination pad, a chromatography microporous membrane and a water absorption pad which are sequentially overlapped on any one side of a substrate, wherein the combination pad is combined with a compound for adsorbing goat anti-human IgA antibodies by colloidal gold particles, the chromatography microporous membrane is sequentially provided with a detection line and a quality control line, the detection line is 2019 novel coronavirus antigens, the quality control line is rabbit anti-goat IgG antibodies, the concentration of the 2019 novel coronavirus antigens is 0.8-1.2 mg/m L, and the concentration of the rabbit anti-goat IgG antibodies is 1.0-1.5 mg/m L.
The test paper for detecting the 2019 novel coronavirus adopts a compound with colloidal gold particles adsorbing a sheep anti-human IgA antibody as an indicator, and uses IgA for detection, so that the specificity is high, and the sensitivity is high; the test paper is used for detection based on the serology principle, a detection sample is body blood, the collection safety of the blood sample is far higher than that of a throat swab, and the infection risk of medical personnel is greatly reduced; the method has the advantages of simple and automatic operation, no restriction on sites, good repeatability, easy interpretation, wide applicability, no need of other instruments and equipment, and suitability for clinical examination, epidemiological investigation, on-site quarantine and the like of various medical detection mechanisms.
Further, the embodiment of the invention also provides a detection device for the 2019 novel coronavirus, which comprises a sleeve and the detection test paper for the 2019 novel coronavirus.
The detection device is a closed detection device, is used for detecting the 2019 novel coronavirus, is convenient and efficient to detect, has high sensitivity, is not interfered by the outside world, and is suitable for clinical examination, epidemiological investigation, field quarantine inspection and the like of various medical detection mechanisms.
Preferably, the cutting sleeve is as shown in fig. 5, and the cutting sleeve comprises a base and a cover surface, wherein a fixing groove for placing the test paper and a latch embedded with the cover surface are arranged on one side of the base; the cover surface is provided with a sample adding hole (S), an observation window and a latch embedded with the base; letters T and C are printed beside the observation window respectively, and a letter S is printed beside the sampling hole; wherein, T corresponds to a detection line of the detection test paper and represents the detection line; c, representing a control line corresponding to the quality control line of the test paper; t is the end near the loading hole.
In a preferred embodiment of the invention, the test paper for the 2019 novel coronavirus is placed in a fixing groove of a plastic card base, the surface cover is embedded with a latch of the base through a card pool to obtain a closed detection device, the detection device is used for detecting the 2019 novel coronavirus, is convenient and efficient to detect, is high in sensitivity, is not interfered by the outside, and is suitable for clinical examination, epidemiological investigation, field quarantine and the like of various medical detection mechanisms.
In the specific embodiment of the invention, the detection device is used for detecting the 2019 novel coronavirus, and the result is judged as follows, as shown in fig. 4, if C, T lines appear simultaneously, the sample contains the antigen to be detected, and the sample is judged to be positive; if the C line appears and the T line does not appear, the sample does not contain the antigen to be detected, and the sample is judged to be negative; if the C, T line does not appear or the T line does not appear, the detection is invalid, which indicates that the quality of the test strip is poor or the test strip is not operated properly, and the test strip should be re-detected or replaced.
Correspondingly, the embodiment of the invention also provides a preparation method of the test paper for the 2019 novel coronavirus. The method comprises the following steps:
s01, providing a compound with colloidal gold particles adsorbing goat anti-human IgA, and depositing the compound with colloidal gold particles adsorbing goat anti-human IgA to a combination pad to obtain the combination pad with the compound with colloidal gold particles adsorbing goat anti-human IgA;
s02, providing a 2019 novel coronavirus antigen and an anti-sheep species antibody, and spraying the 2019 novel coronavirus antigen and the anti-sheep species antibody on the chromatography microporous membrane in sequence to obtain a chromatography microporous membrane with a detection line and a quality control line arranged in sequence;
s03, sequentially arranging a sample pad, the combination pad combined with the compound for adsorbing the goat anti-human IgA through the colloidal gold particles, the chromatography microporous membrane and the water absorption pad which are sequentially provided with the detection line and the quality control line on any one side of the substrate to obtain the test paper for the 2019 novel coronavirus.
The preparation method of the test paper for the 2019 novel coronavirus is simple, convenient and quick, and low in price, and the test paper for the 2019 novel coronavirus prepared by the preparation method has the advantages and is suitable for wide application.
In step S01, a sheep anti-human IgA complex is adsorbed onto a colloidal gold particle, and preferably, the method for preparing the sheep anti-human IgA complex comprises the steps of:
s011, mixing colloidal gold solution and goat anti-human IgA, adding bovine serum albumin, mixing, and standing at 4-6 ℃ to obtain a goat anti-human IgA mixture;
s012, carrying out first centrifugal treatment on the sheep anti-human IgA mixture, collecting sheep anti-human IgA mixture supernatant, carrying out second centrifugal treatment on the sheep anti-human IgA mixture supernatant, and collecting sheep anti-human IgA mixture precipitate;
s013, providing a first buffer solution, adding the first buffer solution into the sheep anti-human IgA mixture precipitate, and mixing to obtain the compound with the sheep anti-human IgA adsorbed by the colloidal gold particles.
In the above step S011, a colloidal gold solution prepared by mixing chloroauric acid (HAuCl) at a concentration of 100m L and 0.01% with goat anti-human IgA is preferably prepared4) And after the solution is heated to boil, quickly adding 1% trisodium citrate water solution, and keeping boiling for 10-15 min to prepare the colloidal gold solution with the gold particle size of 20 nm. The prepared colloidal gold is particles with negative charges and can mutually absorb antigens with positive chargesForming a complex; meanwhile, the colloidal gold can be used as a tracer, and the color development phenomenon can occur after the antigen combined with the colloidal gold and the sample to be detected have antigen-antibody reaction, so that the rapid judgment of the detection result is facilitated, and the detection sensitivity is improved.
Preferably, the sheep anti-human IgA is a purchased sheep species anti-human IgA antibody, the antibody only aims at the reaction of human IgA immunoglobulin, the antibody is used for marking colloidal gold to prepare a gold-labeled antibody which can be combined with the IgA immunoglobulin in a sample to be detected, the application adopts the IgA subtype of the specific immune globulin of the 2019 novel coronavirus as a detection target spot, and compared with other subtypes (IgD, IgE, IgM and IgG), the specificity of the specific immunoglobulin IgA of the 2019 novel coronavirus is stronger, the sensitivity and the stability are higher, and the detection efficiency is favorably improved.
Preferably, the colloidal gold solution is pretreated, wherein the pretreatment comprises the steps of adjusting the pH of the colloidal gold solution to 8.0-8.2, and adjusting the solution to be alkaline, so that the subsequent reaction is facilitated.
Preferably, the colloidal gold solution and the goat anti-human IgA are mixed, wherein the addition amount of 2-18 mu g of goat anti-human IgA is added into every 1m L colloidal gold solution for mixing, if the addition amount of the goat anti-human IgA is too small, the load capacity is too low, and the detection sensitivity is further influenced, if the addition amount of the goat anti-human IgA is too large, the antibody cannot be completely loaded due to too much antibody, and the antibody is wasted.
In step S012, the sheep anti-human IgA mixture is subjected to a first centrifugation process, and a sheep anti-human IgA mixture supernatant is collected, and the sheep anti-human IgA mixture precipitate is collected by subjecting the sheep anti-human IgA mixture supernatant to a second centrifugation process.
Preferably, the centrifugation conditions of the first centrifugation treatment are: centrifuging at 2000r/min for 20 min, and collecting supernatant of sheep anti-human IgA mixture after the first centrifugation. Preferably, the supernatant of the sheep anti-human IgA mixture is subjected to a second centrifugation under the following conditions: centrifuging at 10000r/min for 40 min, and collecting precipitate of goat anti-human IgA mixture after second centrifugation treatment.
In step S013, a first buffer is provided, and the first buffer is added to the sheep anti-human IgA mixture precipitate and mixed to obtain a complex in which the colloidal gold particles adsorb sheep anti-human IgA.
Preferably, the first buffer solution comprises a PBS solution with the concentration of 10mM and the pH value of 7.0, a BSA solution with the concentration of 1%, a sucrose buffer solution with the concentration of 1%, and sodium azide with the concentration of 0.01-0.06%. And adding the first buffer solution into the sheep anti-human IgA mixture precipitate, mixing, and carrying out heavy suspension to obtain the compound with the sheep anti-human IgA adsorbed by the colloidal gold particles. Preferably, the mass ratio of the sheep anti-human IgA mixture precipitate to the first buffer solution is 10-1.
The compound with the colloidal gold particles adsorbing the goat anti-human IgA is prepared, the colloidal gold particles are adopted to adsorb and coat the detection target, so that the specific immunoglobulin IgA of the 2019 novel coronavirus with positive charges and the gold particles with negative charges are adsorbed to form the compound through charge action, the colloidal gold particles are used for marking the specific immunoglobulin IgA of the 2019 novel coronavirus, the colloidal gold is used as a tracer, a color development phenomenon can occur after the sample to be detected performs antigen-antibody specific reaction, the detection result can be quickly judged through the color development phenomenon, and the detection sensitivity and the detection efficiency are improved.
Further, the complex of the colloidal gold particles adsorbing goat anti-human IgA is deposited on a binding pad, preferably, the binding pad is pretreated with a second buffer solution. Wherein the second buffer solution is a mixed solution of 10mM PBS solution with pH8.2 concentration, 3% BSA solution, 0.1% TritonX-100 solution and 1% PVP-40000 solution. Pre-treating the conjugate pad with the second buffer solution, the second buffer solution having a primary function of blocking non-specific but non-specific binding epitopes on the conjugate pad, thereby facilitating the gold particles to leave the conjugate pad by chromatography; and secondly, removing other pollutants such as dust and the like on the bonding pad to ensure that the bonding pad is clean. Preferably, the pretreatment step is: and (3) placing the combined pad in a second buffer solution for soaking for 30 minutes, and then carrying out vacuum drying to obtain the pretreated combined pad. The pretreated combined pad can be used for carrying out deposition treatment on the sheep anti-human IgA compound adsorbed by the colloidal gold particles. In a particular embodiment of the invention, the conjugate pad is selected from fiberglass membranes.
Preferably, in terms of per 1cm2And depositing the compound with the colloidal gold particles adsorbing the goat anti-human IgA to the bonding pad by the addition amount of the compound with the colloidal gold particles adsorbing the goat anti-human IgA, wherein the addition amount of the compound with the colloidal gold particles adsorbing the goat anti-human IgA is 1-1.2 mu L, and depositing the compound with the colloidal gold particles adsorbing the goat anti-human IgA to the bonding pad to obtain the bonding pad with the compound with the colloidal gold particles adsorbing the goat anti.
Further, the method also comprises the steps of placing the bonding pad attached with the compound with the colloidal gold particles for absorbing the goat anti-human IgA in a third buffer solution for soaking and drying for later use. Preferably, the third buffer solution is a 2% BSA blocking solution, and the soaking is performed to block blank epitopes on the surfaces of the colloidal gold particles, reduce non-specific binding of antibodies, improve detection specificity and reduce false positive of detection. Further preferably, the soaking condition is heat preservation at 37 ℃ for 1 hour. And (3) drying after soaking, preferably, the drying condition is vacuum pumping at room temperature or drying at 37 ℃ for 2 hours. Drying, and storing at 4 deg.C.
Specifically, in the step S02, a 2019 novel coronavirus antigen is provided, and preferably, the 2019 novel coronavirus antigen is prepared by infecting a single cell with a virus to obtain a virus infection solution, purifying to obtain a virus antigen or expressing the 2019-nCoV virus glycoprotein or nucleoprotein antigen by using a yeast expression system/mammalian cell expression system through a base sequence of the virus. In the test process, the virus antigen is prepared by selecting a method of infecting single cells by using virus to obtain virus infection liquid and then purifying to obtain the virus antigen, and the property representation and the protein loading condition of the virus antigen are close to the performance of the novel coronavirus antigen of the organism 2019. And the virus antigen is prepared by selecting a base sequence of the virus and adopting a method of expressing the 2019-nCoV virus glycoprotein or nucleoprotein antigen by a yeast expression system/mammalian cell expression system, the property characterization of the virus antigen is similar to that of the novel coronavirus antigen of the organism 2019, but the loading capacity of the protein and other conditions are inconsistent.
In the preferred embodiment of the invention, the 2019 novel coronavirus antigen is prepared by a method of selecting a virus to infect a single cell to obtain a virus infection solution and then purifying to obtain a virus antigen, and the method comprises the following steps:
s021, providing a 2019 novel coronavirus strain, infecting a monolayer host cell with the virus strain to perform culture treatment, and collecting a first generation of venom;
s022, carrying out secondary infection and culture treatment on the single-layer host cells by the first generation of venom to obtain a second generation of venom;
s023, performing third centrifugation treatment on the second-generation venom, collecting culture solution supernatant, and purifying the culture solution supernatant to obtain the 2019 novel coronavirus antigen.
In the above step S021, a virus strain of the novel coronavirus is provided 2019, a monolayer of host cells is infected with the virus strain, and a first generation of venom is collected.
In step S022, the first generation venom is cultured after secondary infection of the monolayer host cells, thereby obtaining a second generation venom. Preferably, the culture treatment condition is to culture for 24-48 hours at 35 ℃, wash for 3 times by a serum-free culture solution, replace the serum-free culture solution, and culture for 4-5 days at 35 ℃.
In the step S023, the second-generation venom is centrifuged for 15min at 5000r/min, preferably for a third time.
And further preferably, the purification step comprises the steps of inactivating the culture solution supernatant by adopting β propiolactone, removing impurities in the supernatant by adopting a zinc acetate precipitation method and an ultrafiltration concentration method, and purifying by adopting a Sepharose 4FF chromatographic column to obtain the 2019 novel coronavirus antigen.
Furthermore, an antibody against the ovine species is provided, preferably, the antibody against the ovine species can be any one of a rabbit antibody against the ovine, a mouse antibody against the ovine, a dog antibody against the ovine, and a donkey antibody against the ovine. Further preferably, the immunoglobulin type of the antibody is any one selected from IgA, IgM, IgE and IgG. In a preferred embodiment of the invention, the antibodies against ovine species are selected from rabbit anti-ovine IgG antibodies. The anti-sheep IgG antibody from the rabbit species is purchased, and is prepared by taking sheep immunoglobulin as an antigen to immunize rabbits and extracting antibody globulin in immune rabbit serum. Preferably, the quality control line is coated with an anti-sheep species antibody, and the anti-sheep species antibody is used as the quality control line of the test paper, so that the test paper can be simultaneously combined with a compound which is combined with colloidal gold particles and adsorbs sheep anti-human IgA, and the effectiveness of the test paper can be further detected.
Further, the 2019 novel coronavirus antigen and the anti-sheep species antibody are sequentially sprayed on the chromatography microporous membrane to obtain the chromatography microporous membrane with a detection line and a quality control line sequentially arranged. Preferably, the chromatography microporous membrane is selected from nitrocellulose membrane.
Specifically, 2019 novel coronavirus antigens are coated in the detection line, and antibodies for sheep species are coated in the quality control line. After the 2019 novel coronavirus is infected, a body can be immunized to generate a certain of specific immunoglobulin, the detection line is coated with the 2019 novel coronavirus antigen, the specific immunoglobulin in a sample to be detected and the 2019 novel coronavirus antigen in the detection line can generate immunoreaction with high specificity and high affinity, so that an immune compound is enriched, the colloidal gold of a marker can be directly observed to obtain an intuitive experimental phenomenon, and a detection result is quickly obtained. And the anti-sheep species antibody coated in the quality control line can be specifically combined with the sheep anti-human IgA antibody of the combination pad to be used as a quality control group, so that the accuracy of the detection test paper is further ensured.
Preferably, the concentration of the 2019 novel coronavirus antigen coated in the detection line is 0.8-1.2 mg/m L, if the concentration of the 2019 novel coronavirus antigen coated in the detection line is too high, false positive can be formed in a test process, antigen waste is caused, and if the concentration is too low, the antigen quantity is insufficient, the detection sensitivity is influenced, false negative is caused, and the detection accuracy is influenced.
Preferably, the line width of the detection line is 0.7-0.8 mm. If the line width of the sprayed detection line is too wide, the use amount of the antigen is too large, false positive results are easy to appear, and meanwhile, the antigen is wasted; if the line width of the sprayed detection line is too narrow, the observation of the test result is not facilitated.
Preferably, the concentration of the anti-sheep species antibody coated in the quality control line is 1.0-1.5 mg/m L, if the concentration of the anti-sheep species antibody coated in the quality control line is too high, false positive can be formed in the test process, and antibody waste is caused, and if the concentration is too low, the antibody quantity is insufficient, the quality control line is not clear, and the quality control is not closed.
Preferably, the line width of the quality control line is 0.7-0.8 mm. If the line width of the sprayed quality control line is too wide, the antigen consumption is excessive, false positive results are easy to occur, and the antigen waste is caused; if the line width of the sprayed quality control line is too narrow, the observation of the test result is not facilitated.
Preferably, the distance between the detection line and the quality control line is 0.5-0.6 cm; if the distance between the two is too small, the judgment of the test result is influenced, and the observation of the test result is not facilitated; if the distance between the two is too large, the test paper is easy to cause overlarge volume and inconvenient to use.
Preferably, in the step of sequentially spraying the 2019 novel coronavirus antigen and the anti-ovine species antibody on the chromatography microporous membrane to obtain the chromatography microporous membrane with a detection line and a quality control line, the spraying method comprises the following steps:
the method comprises the steps of providing the 2019 novel coronavirus antigen and the anti-sheep species antibody, spraying a detection line on a chromatography microporous membrane by using a membrane spraying machine, wherein the concentration of the 2019 novel coronavirus antigen coated in the detection line is 0.8-1.2 mg/m L, the line width of the detection line is 0.8mm, controlling the distance between the detection line and a quality control line to be 0.5cm, spraying the quality control line, wherein the concentration of the anti-sheep species antibody coated in the quality control line is 1.0-1.5 mg/m L, the line width of the quality control line is 0.8mm, drying for 2 hours at 37 ℃ after spraying is finished, sealing for 30 minutes at 37 ℃ in a PBS (phosphate buffer solution) with the concentration of 10 mmol/L pH7.0 and containing 5% BSA, rinsing by using a PBS solution with the concentration of 10 mmol/L pH7.0, and drying at 37 ℃, so that the chromatography microporous membrane with the detection line and the quality control line sequentially arranged is obtained.
In step S03, the sample pad, the binding pad to which the complex of colloidal gold particles adsorbing goat anti-human IgA is bound, the chromatography microporous membrane and the water absorbent pad, in which the detection line and the quality control line are sequentially disposed, are sequentially disposed on any one side of the substrate, so that the test paper for detecting the 2019 novel coronavirus is obtained.
Preferably, the sample pad is a thick filter paper with good water absorption capacity, plays a role in filtering samples such as blood cells and the like in the test paper strip, is positioned on the combination pad, and is attached to the substrate. Preferably, the absorbent pad is thick filter paper, and is attached to the tail end of the substrate to play a role in chromatography. Preferably, the base offset sheet material functions as a support sample pad, a conjugate pad, an NC membrane and a wicking pad.
Preferably, the detection test paper is prepared by sequentially arranging a sample pad, the binding pad combined with the compound for adsorbing goat anti-human IgA through colloidal gold particles, the chromatography microporous membrane and the water absorption pad, wherein the chromatography microporous membrane and the water absorption pad are sequentially provided with a detection line and a quality control line, on any one side of the substrate. In a preferred embodiment of the invention, the test paper for the 2019 novel coronavirus consists of a sample pad, a combination pad, a water absorption pad, a backing and a nitrocellulose membrane 5, wherein the sample pad is made of common filter paper, the combination pad is made of a glass fiber membrane, a colloidal gold-labeled goat anti-human IgA is sprayed on the combination pad, the nitrocellulose membrane is an NC membrane, a quality control line on the nitrocellulose membrane is rabbit anti-goat IgG, and a detection line 2019-nCoV virus antigen is detected.
The following further describes specific examples.
Example 1
Preparation of 2019 novel coronavirus antigen
The 2019 novel coronavirus antigen is prepared by a method of infecting a single cell with a virus to obtain a virus infection solution and then purifying to obtain a virus antigen, and comprises the following steps:
providing 2019 a novel coronavirus strain, infecting a monolayer African green monkey kidney cell (Vero) with the strain, culturing, and collecting a first generation of venom;
carrying out secondary infection and culture treatment on the single-layer African green monkey kidney cells (Vero) by the primary venom, wherein the culture treatment condition is that the cells are cultured for 24-48 hours at 35 ℃, the cells are washed for 3 times by serum-free culture solution, then the cells are replaced by serum-free culture solution, and the cells are cultured for 4-5 days at 35 ℃ to obtain secondary venom;
s023, centrifuging the second-generation venom at 5000r/min for 15min, collecting the supernatant of the culture solution, adding 1/10000 β propiolactone into the supernatant of the culture solution for inactivation, treating the supernatant by a zinc acetate precipitation method and an ultrafiltration concentration method, and purifying the supernatant by adopting a Sepharose 4FF chromatographic column to obtain the 2019 novel coronavirus antigen.
Example 2
Immunoglobulin target detection by selecting 2019-nCoV virus antigen
Antibodies from the same genus were purchased (1) anti-human IgA antibody derived from sheep species and directed only to human IgA immunoglobulin reaction, (2) anti-human IgD antibody derived from sheep species and directed only to human IgD immunoglobulin reaction, (3) anti-human IgE antibody derived from sheep species and directed only to human IgE immunoglobulin reaction, (4) anti-human IgG antibody derived from sheep species and directed only to human IgG immunoglobulin reaction, and the sensitivity of the five immunoglobulins to 2019-nCoV viral antigen was compared by enzyme linked immunosorbent assay (E L SA), and immunoglobulin targets were preferably detected.
The 2019-nCoV virus antigen coated plate prepared in example 1 is used, blank control and negative control are set in each experiment, 50 mu L serum diluted samples containing 2019-nCoV virus antibodies (stock solution; 1: 2; 1: 5; 1: 10; 1: 20; 1: 40; 1: 50; 1: 100; 1:1501: 200; 1: 250; 1:500) are added into each hole except blank control holes, 5 detections are repeated at each dilution, 50 mu L of the five (1) - (5) immunoglobulin solutions (10 mu g/m L) are added, a sealing film is attached, the plate is incubated at 37 ℃ for 30 minutes, samples in each hole are removed, the plate is dried, washed by washing liquor for 6 times, the blank holes are added into each hole, 50 mu L of a blank enzyme-labeled rabbit anti-sheep working solution is added, the sealing film is attached, the plate is placed at 37 ℃ for 30 minutes, after color development is finished, 50 mu L of each hole is added, the plate is incubated with light beating is carried out, the plate is mixed evenly, and the hole is measured by an enzyme-labeled rabbit anti-sheep working solution (OD) at zero wavelength of 630 nm.
And analyzing the sensitivity and stability of the five different types of immunoglobulin targets on the detection of the 2019-nCoV viral antigen, and further judging the immunoglobulin target for the detection of the 2019-nCoV viral antigen.
Example 3
Compound prepared by adsorbing sheep anti-human IgA through colloidal gold particles
By the assay of example 2, the immunoglobulin target for detection of 2019-nCoV virus antigen was selected as sheep anti-human IgA, which was detected as sheep anti-human IgA.
The preparation method of the compound for adsorbing the goat anti-human IgA by the colloidal gold particles comprises the following steps:
(1) preparing a colloidal gold solution, namely heating a chloroauric acid (HAuCl4) solution with the concentration of 100m L and the concentration of 0.01% to boil, quickly adding a 1% trisodium citrate water solution, keeping boiling for 10-15 min, and preparing the colloidal gold solution with the gold particle size of 20nm, and meanwhile, pretreating the colloidal gold solution by using a potassium carbonate solution with the concentration of 0.1 mol/L to obtain the colloidal gold solution with the pH value of 8.0-8.2.
(2) Mixing the colloidal gold solution and the goat anti-human IgA, adding 2-18 mu g of goat anti-human IgA into every 1m of L colloidal gold solution for mixing, adding bovine serum albumin for mixing treatment, adding 1-1.2 g of bovine serum albumin into every 1m of L colloidal gold solution for mixing, standing at 4-6 ℃ for 2-4 hours for treatment after mixing treatment to obtain the goat anti-human IgA mixture.
(3) And centrifuging the sheep anti-human IgA mixture for 20 minutes at the rotating speed of 2000r/min, collecting the supernatant of the sheep anti-human IgA mixture, centrifuging the supernatant of the sheep anti-human IgA mixture for 40 minutes at the rotating speed of 10000r/min, and collecting the precipitate of the sheep anti-human IgA mixture by second centrifugation.
(4) Providing a first buffer solution, wherein the first buffer solution comprises a PBS solution with the concentration of 10mM and the pH7.0, a BSA solution with the concentration of 1%, a sucrose buffer solution with the concentration of 1%, and sodium azide with the concentration of 0.01-0.06%. And adding the first buffer solution into the sheep anti-human IgA mixture precipitate for mixing, wherein the mass ratio of the sheep anti-human IgA mixture precipitate to the first buffer solution is 10-1, and obtaining the compound with the colloidal gold particles adsorbing sheep anti-human IgA.
Example 4
Preparation of test paper for 2019 novel coronavirus
(1) A conjugate pad, which is a glass fiber membrane and manufactured by Millipore, USA, was treated with 10mM pH8.2 PBS + 3% BSA + 0.1% Triton X-100+ 1% PVP-40000 pretreatment solution, soaked for 30 minutes and then vacuum-dried at 37 ℃. The chromatography microporous membrane is an NC membrane, is a material specially used for preparing the colloidal gold test strip and is produced by Millipore company in America. (3) The sample pad is provided, the sample pad is thick filter paper with good water absorption capacity, is produced by Millipore company in America, and plays a role in filtering samples such as blood cells and the like in a test strip. (4) Absorbent pads, which are thick filter papers manufactured by Millipore corporation of usa, are provided to perform the chromatography. (5) A substrate is provided, which is a flexographic plate material capable of supporting a sample pad, a conjugate pad, an NC film, and a bibulous pad.
(2) Providing the compound of sheep anti-human IgA adsorbed by the colloidal gold particles prepared in example 3, and mixing the colloidThe gold particles adsorb the sheep anti-human IgA compound according to the ratio of per 1cm2Deposition of 1-1.2 mu L on the binding pad, deposition of the goat anti-human IgA adsorbed complex by the colloidal gold particles to the binding pad obtained by pretreatment in example 4(1), obtaining the binding pad attached with the goat anti-human IgA adsorbed complex by the colloidal gold particles, placing in 2% BSA confining liquid, keeping the temperature for 1 hour at 37 ℃, drying, and placing at 4 ℃ for storage.
(3) Providing the 2019 novel coronavirus antigen prepared in the example 1, purchasing an antibody of anti-ovine species, spraying the 2019 novel coronavirus antigen and the anti-ovine rabbit anti-ovine IgG on a chromatography microporous membrane in sequence, spraying a detection line on the chromatography microporous membrane by using a film spraying machine, wherein the concentration of the 2019 novel coronavirus antigen coated in the detection line is 0.8-1.2 mg/m L, the line width of the detection line is 0.8mm, controlling the distance between the detection line and a quality control line to be 0.5cm, spraying the quality control line, wherein the concentration of the anti-ovine species antibody coated in the quality control line is 1.0-1.5 mg/m L, the line width of the quality control line is 0.8mm, drying for 2 hours at 37 ℃ after spraying is finished, sealing the detection line for 30 minutes at 37 ℃ in a PBS solution with the concentration of 10 mmol/L pH7.0 and containing 5% BSA, rinsing the detection line by using PBS solution with the concentration of 10 mmol/L.0, and drying the detection line at 37 ℃ in sequence to obtain a chromatography microporous membrane with the quality control line and drying the detection line, and drying the PBS solution at 37 ℃ in sequence, thus obtaining the microporous membrane.
(4) The sample pad provided in example 4(1), the binding pad to which the colloidal gold particles adsorbing goat anti-human IgA complex provided in example 4(2), the chromatography microporous membrane with the sequentially arranged detection line and quality control line provided in example 4(3), and the water absorption pad provided in example 4(1) were sequentially arranged on either side of the substrate provided in example 4(1), and the test strip for 2019 novel coronavirus was obtained.
The test paper for the 2019 novel coronavirus consists of a sample pad, a combination pad, a water absorption pad, a back lining and a nitrocellulose membrane 5, wherein the sample pad is made of common filter paper, the combination pad is made of a glass fiber membrane, a colloidal gold-labeled goat anti-human IgA is sprayed on the combination pad, the nitrocellulose membrane is an NC membrane, a quality control line on the nitrocellulose membrane is rabbit anti-goat IgG, and a detection line 2019-nCoV virus antigen is detected.
Example 5
Preparation detection device for 2019 novel coronavirus
As shown in fig. 5, the test paper for 2019 coronavirus prepared in example 4 and a plastic card are provided, wherein the plastic card comprises a base and a surface cover, one side of the base is provided with a fixing groove for placing the test paper and a latch engaged with the surface cover; the surface cover is provided with a sample adding hole (S), an observation window and a latch embedded with the base; letters T and C are printed beside the observation window respectively, wherein T corresponds to a detection line of the detection test paper and represents the detection line; c, corresponding to the quality control line of the test paper, representing a comparison line; t is the end near the loading hole.
And placing the test paper for the 2019 novel coronavirus in a fixing groove of the plastic card base, and embedding the surface cover with the latch of the base through a card pool to obtain a closed detection device.
The appearance of the detection device is rectangular, the outer layer is an environment-friendly plastic shell, and the middle part is a test paper core. The shell surface is covered with a sample adding hole and an observation window, letters T and C are respectively printed at two ends of the observation window, T is arranged at one side close to the sample adding hole, and C is arranged at one side far from the sample adding hole. The test paper core has uniform width, standard material pasting, firm adhesion and no damage to the NC film.
Performance analysis test:
(I) sensitivity of detecting immunoglobulin target to 2019-nCoV virus antigen
The five immunoglobulin targets provided in example 2 were subjected to an enzyme linked immunosorbent assay (E L SA) to detect the immunoglobulin targets, comparing the sensitivity of the five immunoglobulins to the 2019-nCoV virus antigen.
The 2019-nCoV virus antigen coated plate prepared in example 1 is used, blank control and negative control are set in each experiment, 50 mu L serum diluted samples containing 2019-nCoV virus antibodies (stock solution; 1: 2; 1: 5; 1: 10; 1: 20; 1: 40; 1: 50; 1: 100; 1:1501: 200; 1: 250; 1:500) are added into each hole except blank control holes, 5 detections are repeated at each dilution, 50 mu L of the five (1) - (5) immunoglobulin solutions (10 mu g/m L) are added, a sealing film is attached, the plate is incubated at 37 ℃ for 30 minutes, samples in each hole are removed, the plate is dried, washed by washing liquor for 6 times, the blank holes are added into each hole, 50 mu L of a blank enzyme-labeled rabbit anti-sheep working solution is added, the sealing film is attached, the plate is placed at 37 ℃ for 30 minutes, after color development is finished, 50 mu L of each hole is added, the plate is incubated with light beating is carried out, the plate is mixed evenly, and the hole is measured by an enzyme-labeled rabbit anti-sheep working solution (OD) at zero wavelength of 630 nm.
And analyzing the sensitivity and stability of the five different types of immunoglobulin targets on the detection of the 2019-nCoV viral antigen, and further judging the immunoglobulin target for the detection of the 2019-nCoV viral antigen.
(II) detection of specificity of the test paper prepared in example 4
The test strips prepared in example 4 were used to detect 6 coronavirus antigens, i.e., 2019-nCoV, SARS-CoVer, MERS-CoV, HCoV-229E, HCoV-OC43, and HCoV-N L63, respectively, while PBS buffer was used as a negative control to observe the reaction of the test strips to detect the specificity of the test strips prepared in example 4.
(III) sensitivity of the test paper prepared in example 4
The 2019-nCoV cytotoxic prepared in the embodiment 1 and the 2019-nCoV antigenic protein prepared by adopting eukaryotic expression are used for detecting the test strip, and the lower detection limit of the test strip is observed.
(IV) sample detection is carried out by using the test paper for the 2019 novel coronavirus prepared in example 4
(1) Sample preparation
① treatment of Positive quality control reagent
The 2019-nCoV virus antigen expressed by pronucleus is taken, after affinity purification, each milliliter contains 0.5mg as a contrast, and the antigen is subpackaged in a finger-shaped tube with the diameter of 1.5m L for later use at the temperature of minus 20 ℃.
② handling of negative quality control reagents
The negative sample was PBS buffer and was dispensed into 1.5m L finger tubes.
③ treatment of the liquid to be tested
And (3) puncturing the finger tip of the human finger to be detected by using a blood sugar blood sampling finger tip needle, and extruding a small amount of blood to obtain the sample to be detected.
Collecting venous blood of an upper arm of a human to be detected by using a venous blood collection needle, adding an EDTA (ethylene diamine tetraacetic acid) anticoagulation tube, and uniformly mixing the materials upside down to obtain a sample to be detected.
(2) Sucking a sample to be detected with the volume of 50 mu L and directly dripping the sample to be detected on a sample pad of the test strip, observing the result 3-10 min after sample application, and determining the negative and positive results of the visual inspection result, wherein the detection result is discarded after 30 min.
Analysis of results
The detection results are analyzed one by one aiming at the performance analysis test, and the result analysis is as follows:
(I) sensitivity of detecting immunoglobulin target to 2019-nCoV virus antigen
The sensitivity and stability of five different types of immunoglobulin targets on the detection of the 2019-nCoV virus antigen are analyzed by adopting an E L SA experiment, the results are shown in the following table 1, when the test strip is used for detecting the optimal target IgA of the 2019-nCoV virus antibody compared with other four subtypes of immunoglobulin (IgG, IgM, IgE and IgD), the result can still be detected under the condition that the dilution degree is 1:200 when the IgA is used as the immunoglobulin target for detection, and the results can be detected under the conditions that the dilution degrees are 1:10, 1:150, 1:50 and 1:200 when the IgD, IgE, IgM and IgG are used as the immunoglobulin target for detection.
TABLE 1
(II) detection of specificity of the test paper prepared in example 4
The test paper prepared in example 4 was used to detect 6 coronavirus antigens, 2019-nCoV, SARS-CoVer, MERS-CoV, HCoV-229E, HCoV-OC43 and HCoV-N L63, and PBS buffer was used as a negative control, and the results are shown in FIG. 3. when the test paper reacts only with 2019-nCoV antigen, the test paper showed 2 lines, and other virus antigens reacted only with the test paper showed one line, which demonstrated that the test paper reacted only with 2019-nCoV antigen, and the test paper showed good specificity.
(III) sensitivity of the test paper prepared in example 4
The test result shows that the lower limit of the detection of the cytotoxicity is 23 mu L when the 2019-nCoV cytotoxic prepared in example 1 is used for detection, and the sensitivity of the 2019-nCoV antigenic protein detection is 50ng/m L-0.5 mg/m L when the 2019-nCoV antigenic protein prepared in eukaryotic expression is used for detection.
(IV) sample detection is carried out by using the test paper for the 2019 novel coronavirus prepared in example 4
Sucking a sample to be detected with the volume of 50 mu L and directly dripping the sample to be detected on a sample pad of the test strip, observing the result 3-10 min after sample application, and analyzing the result as follows:
as shown in FIG. 4, when C, T lines appear simultaneously, it indicates that the sample contains the antigen to be detected, and the sample is judged to be positive; if the C line appears and the T line does not appear, the sample does not contain the antigen to be detected, and the sample is judged to be negative; if neither line C, T nor line C appears on line T, the test is invalid, which indicates that the test strip has quality problem or improper operation, and the test strip should be retested or replaced.
Respectively detecting the 2019-nCoV positive quality control substance liquid and the 2019-nCoV negative quality control substance liquid by using test paper, wherein a detection line (T line) and a control line (C line) of the test paper of the 2019-nCoV positive quality control substance liquid simultaneously appear, and judging the test paper to be positive; and the T line of the 2019-nCoV negative quality control product liquid test paper has no strip, and the test paper is judged to be negative; if the C, T line does not appear or the T line does not appear, the detection is invalid, which indicates that the quality of the test strip is poor or the test strip is not operated properly, and the test strip should be re-detected or replaced.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. The test paper for the 2019 novel coronavirus comprises a sample pad, a combination pad, a chromatography microporous membrane and a water absorption pad which are sequentially overlapped on any one side of a substrate, and is characterized in that the combination pad is combined with a compound with colloidal gold particles for adsorbing goat anti-human IgA antibodies; the chromatography microporous membrane is sequentially provided with a detection line and a quality control line, the detection line is arranged at the position of the chromatography microporous membrane close to the binding pad, and the quality control line is arranged at the position of the chromatography microporous membrane far away from the binding pad; the detection line is a 2019 novel coronavirus antigen, and the quality control line is an antibody for resisting sheep species.
2. The test paper for the 2019 novel coronavirus according to claim 1, wherein the line width of the detection line is 0.7-0.8 cm; and/or the presence of a gas in the gas,
the line width of the quality control line is 0.7-0.8 cm.
3. The test strip for the 2019 novel coronavirus according to claim 1, wherein the distance between the detection line and the quality control line is 0.5-0.6 cm.
4. The test strip for detecting the 2019 novel coronavirus as claimed in any one of claims 1 to 3, wherein the colloidal gold particles have a particle size of 15 to 25 nm.
5. The test paper for the 2019 novel coronavirus, according to claim 1, which comprises a sample pad, a combination pad, a chromatography microporous membrane and a water absorption pad, wherein the sample pad, the combination pad, the chromatography microporous membrane and the water absorption pad are sequentially overlapped on any one side of a substrate, colloidal gold particles are combined with the combination pad to adsorb a sheep anti-human IgA antibody compound, the chromatography microporous membrane is sequentially provided with a detection line and a quality control line, the detection line is 2019 novel coronavirus antigen, the quality control line is rabbit anti-sheep IgG antibody, the concentration of the 2019 novel coronavirus antigen is 0.8-1.2 mg/m L, and the concentration of the rabbit anti-sheep IgG antibody is 1.0-1.5 mg/m L.
6. A detection device for 2019 novel coronavirus, which is characterized by comprising a card sleeve and the detection test paper for 2019 novel coronavirus according to any one of claims 1-5.
7. A preparation method of test paper for 2019 novel coronavirus is characterized by comprising the following steps:
providing a compound with colloidal gold particles adsorbing goat anti-human IgA, and depositing the compound with colloidal gold particles adsorbing goat anti-human IgA to a combination pad to obtain the combination pad with the compound with colloidal gold particles adsorbing goat anti-human IgA;
providing a 2019 novel coronavirus antigen and an anti-sheep species antibody, and spraying the 2019 novel coronavirus antigen and the anti-sheep species antibody on the chromatography microporous membrane in sequence to obtain a chromatography microporous membrane with a detection line and a quality control line in sequence;
and sequentially arranging a sample pad, the combination pad combined with the compound for adsorbing the goat anti-human IgA by the colloidal gold particles, the chromatography microporous membrane and the water absorption pad which are sequentially provided with the detection line and the quality control line on any one side of the substrate to obtain the test paper for the 2019 novel coronavirus.
8. The test strip for the 2019 novel coronavirus according to claim 7, wherein the concentration of the 2019 novel coronavirus antigen is 0.8-1.2 mg/m L.
9. The test strip for 2019 novel coronavirus according to claim 7, wherein the concentration of the anti-ovine species antibody is 1.0-1.5 mg/m L.
10. The method for preparing a test strip for a 2019 novel coronavirus according to claim 7, wherein the method for preparing the goat anti-human IgA adsorbed complex by the colloidal gold particles comprises the following steps:
mixing the colloidal gold solution and the goat anti-human IgA, adding bovine serum albumin, mixing, and standing at 4-6 ℃ to obtain a goat anti-human IgA mixture;
subjecting the sheep anti-human IgA mixture to first centrifugation, collecting sheep anti-human IgA mixture supernatant, subjecting the sheep anti-human IgA mixture supernatant to second centrifugation, and collecting sheep anti-human IgA mixture precipitate;
providing a first buffer solution, and adding the first buffer solution into the sheep anti-human IgA mixture precipitate for mixing to obtain a compound with the sheep anti-human IgA adsorbed by the colloidal gold particles.
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