Method for detecting virus and method for preparing colloidal gold immunochromatographic test strip

Abstract

The present invention relates to the technical field of immunoassay analysis, and particularly relates to a method for detecting a virus and a method for preparing a colloidal gold immunochromatographic test strip. Detecting a test sample using a colloidal gold immunochromatographic test strip includes the following steps: adding the test sample to the sample pad of the colloidal gold immunochromatographic test strip, and performing an immunochromatographic reaction for 10 min - 15 min; observing the colors on the test line and the quality control line of the colloidal gold immunochromatographic test strip to obtain a test result; wherein, the colloidal gold immunochromatographic test strip includes a bottom plate, and a sample pad, a conjugate pad, a chromatographic membrane, and a water absorption pad sequentially arranged on the bottom plate, the chromatographic membrane is provided with a test line and a quality control line; the conjugate pad is coated with a rotavirus antibody labeled with colloidal gold, the particle size of the colloidal gold is 50 nm - 70 nm, the antibody at the test line includes a first antibody for recognizing a rotavirus antigen, and the antibody at the quality control line includes a second antibody for recognizing a rotavirus antibody.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay analysis, and particularly relates to a method for detecting a virus and a method for preparing a colloidal gold immunochromatographic test strip. Background Art

[0002] Colloidal gold immunochromatography technology is a technology that combines antigen-antibody immune reaction with colloidal gold labeling tracer technology for qualitative and quantitative detection of antigen and antibody content. The colloidal gold labeling of antibodies is mainly achieved by the strong binding between colloidal gold and proteins through electrostatic forces. Due to its advantages such as rapidity, simplicity, low cost, and good stability, it has especially opened up a new door for on-site rapid detection.

[0003] Rotavirus is one of the main pathogens causing infantile diarrhea. It mainly infects small intestinal epithelial cells, resulting in cell damage and diarrhea. Rotavirus infection ranges from asymptomatic, mild illness to severe illness, and in severe cases, it can cause fatal gastroenteritis, dehydration, and electrolyte imbalance. Summary of the Invention

[0004] The main object of the present invention is to provide a method for detecting a virus to improve the detection sensitivity of the virus.

[0005] To achieve the above object, a method for detecting a virus proposed by the present invention uses a colloidal gold immunochromatographic test strip to detect a sample to be tested, including the following steps:

[0006] Adding the sample to be tested to the sample pad of the colloidal gold immunochromatographic test strip and performing an immunochromatographic reaction for 10 min - 15 min;

[0007] Observing the colors on the test line and the quality control line of the colloidal gold immunochromatographic test strip to obtain the test result;

[0008] Wherein, the colloidal gold immunochromatographic test strip includes a bottom plate, and a sample pad, a conjugate pad, a chromatographic membrane, and an absorbent pad sequentially arranged on the bottom plate. The chromatographic membrane is provided with a test line and a quality control line;

[0009] The conjugate pad is coated with a colloidal gold-labeled rotavirus antibody. The particle size of the colloidal gold is 50 nm - 70 nm. The antibody at the test line includes a first antibody for recognizing rotavirus antigen, and the antibody at the quality control line includes a second antibody for recognizing the rotavirus antibody.

[0010] Optionally, the rotavirus antibody includes an antibody with a clone number of 11H4, and the first antibody includes an antibody with a clone number of 7F12.

[0011] Optionally, the minimum detection limit of the colloidal gold immunochromatographic test strip is 4×10 4, unit: number of virus particles / ml.

[0012] Optionally, the particle size of the colloidal gold is 60 nm;

[0013] And / or, the second antibody includes goat anti-mouse IgG;

[0014] And / or, the first antibody is a monoclonal antibody or a polyclonal antibody.

[0015] Optionally, the scribing concentration of the first antibody at the test line is 1 μL / cm;

[0016] And / or, the coating concentration of the colloidal gold-labeled rotavirus antibody on the conjugate pad is 10 μL / cm.

[0017] Optionally, the material of the bottom plate includes at least one of polyvinyl chloride, polyethylene and glass;

[0018] And / or, the material of the sample pad includes a glass fiber membrane, and the glass fiber membrane includes ahlstrom 8964;

[0019] And / or, the material of the conjugate pad includes a glass fiber membrane and / or a polyester fiber membrane, and the glass fiber membrane includes ahlstrom 8980;

[0020] And / or, the material of the chromatography membrane includes a nitrocellulose membrane, and the nitrocellulose membrane includes sartorius140;

[0021] And / or, the sample pad, the conjugate pad, the chromatography membrane and the absorbent pad are sequentially and mutually overlapped and attached to the bottom plate.

[0022] The present application also provides a preparation method of a colloidal gold immunochromatographic test strip, including the following steps:

[0023] Prepare a colloidal gold solution composed of colloidal gold particles with a mass-volume concentration of four ten-thousandths and a particle size of 50 nm - 70 nm, adjust the pH of the colloidal gold solution to 7.0 - 9.0, then add a rotavirus antibody with a concentration of 10 μg / mL - 30 μg / mL, react for a preset time, then add a blocking agent, block for a preset time, obtain the reacted solution and concentrate it to obtain an immune label, resuspend the immune label with a reconstitution solution to obtain an immune label reconstitution solution, coat the immune label reconstitution solution on the conjugate pad, and dry it for standby;

[0024] Set a test line and a quality control line on the chromatography membrane, fix the first antibody for identifying the rotavirus antigen on the test line, and fix the second antibody for identifying the rotavirus antibody on the quality control line for standby;

[0025] The sample pad, the conjugate pad fixed with the immunolabel, the chromatographic membrane fixed with the first antibody and the second antibody, and the absorbent pad are sequentially arranged on the bottom plate to form a colloidal gold immunochromatographic test strip for detecting rotavirus antigen.

[0026] Optionally, in the step of binding the rotavirus antibody to colloidal gold particles with a particle size of 50 nm - 70 nm to form an immunolabel, and fixing the immunolabel on the conjugate pad for standby, the following steps are included:

[0027] Prepare a colloidal gold solution composed of colloidal gold particles with a mass - volume concentration of four ten - thousandths and a particle size of 50 nm - 70 nm, adjust the pH of the colloidal gold solution to 7.5, then add rotavirus antibody with a concentration of 20 μg / mL, oscillate and react at room temperature for 4 h - 5 h, then add a BSA solution with a mass - volume concentration of 0.5% - 1.0% to cover the bare gold surface sites not coated with the antibody, incubate for 30 min - 60 min, centrifuge at a centrifugal speed of 6000 r / min - 8000 r / min for 10 min - 15 min, remove the supernatant to obtain an immunolabel concentrated 10 times, resuspend the immunolabel with a reconstitution solution, the reconstitution solution includes 50 mM of NaCl, a BSA with a mass - volume concentration of 0.1%, a sucrose with a mass - volume concentration of 1.5%, and a sodium caseinate with a mass - volume concentration of 1.5%; obtain an immunolabel reconstitution solution diluted 5 - 10 times, coat the immunolabel reconstitution solution on the conjugate pad, and dry it at 45 ± 2 °C for 16 h - 18 h for standby.

[0028] Optionally, before the step of binding the rotavirus antibody to colloidal gold particles with a particle size of 50 nm - 70 nm to form an immunolabel, and fixing the immunolabel on the conjugate pad for standby, the following steps are further included:

[0029] Prepare a conjugate pad pretreatment solution, soak the conjugate pad in the conjugate pad pretreatment solution, after soaking for a preset time, take out the conjugate pad and dry it for standby, wherein the components of the conjugate pad pretreatment solution include 10 mM - 20 mM PB, 100 mM - 150 mM NaCl, a casein with a mass - volume concentration of 1% - 1.5%, a sucrose with a mass - volume concentration of 3% - 5%, a Tween - 20 with a volume concentration of 0.5% - 1.0%, a PEG 20000 with a mass - volume concentration of 0.1% - 0.2%, and a proclin 300 with a volume concentration of 0.1% - 0.2%.

[0030] Optionally, before the step of sequentially arranging the sample pad, the conjugate pad fixed with the immunolabel, the chromatographic membrane fixed with the first antibody and the second antibody, and the absorbent pad on the bottom plate to form a colloidal gold immunochromatographic test strip for detecting rotavirus antigen, the following steps are further included:

[0031] Prepare a sample pad pretreatment solution. Immerse the sample pad in the sample pad pretreatment solution. After soaking for a preset time, take out the sample pad and dry it for standby. Among them, the components of the sample pad pretreatment solution include 10 mM - 20 mM PBS, 1% - 1.5% (v / v) Tween-20, 0.2% - 0.5% (w / v) S9, 4% - 6% (w / v) sucrose, 0.5% - 0.8% (w / v) PEG6000, 1.5% - 2% (w / v) BSA, and 0.1% - 0.2% (v / v) proclin 300.

[0032] The virus detection method of the present application uses a colloidal gold immunochromatographic test strip to detect a sample to be tested, including the following steps: Add the sample to be tested to the sample pad of the colloidal gold immunochromatographic test strip, and perform an immunochromatographic reaction for 10 min - 15 min; Observe the colors on the test line and the control line of the colloidal gold immunochromatographic test strip to obtain the test result; Among them, the colloidal gold immunochromatographic test strip includes a bottom plate, and a sample pad, a conjugate pad, a chromatographic membrane, and an absorbent pad sequentially arranged on the bottom plate. The chromatographic membrane is provided with a test line and a control line; The conjugate pad is coated with a rotavirus antibody labeled with colloidal gold, and the particle size of the colloidal gold is 50 nm - 70 nm. The antibody at the test line includes a first antibody for recognizing rotavirus antigen, and the antibody at the control line includes a second antibody for recognizing rotavirus antibody. Using colloidal gold with a particle size of 50 nm - 70 nm can increase the effective collision between antibody molecules and colloidal gold, increase the binding efficiency of the antibody and colloidal gold, improve the effective labeling quantity of nanogold, coat the highly efficiently labeled nanogold onto the conjugate pad, and increase the quantity of effectively labeled nanogold on the conjugate pad, thereby being beneficial to improving the detection sensitivity of rotavirus; Moreover, the colloidal gold with a particle size of 50 nm - 70 nm has a small volume specific surface area, requires less antibody usage, reduces the antibody usage amount, that is, the present application not only improves the labeling efficiency of colloidal gold but also saves the cost of antibodies. Description of the Drawings

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on the structures shown in these drawings.

[0034] Figure 1 It is a schematic structural diagram of a colloidal gold immunochromatographic test strip according to an embodiment of the present application;

[0035] Figure 2 It is a schematic flowchart of the virus detection method of the present application.

[0036] Description of the attached drawing reference numerals:

[0037]

[0038] The realization, functional features and advantages of the purpose of the present invention will be further described with reference to the accompanying drawings in conjunction with embodiments. Specific embodiments

[0039] Hereinafter, embodiments of the colloidal gold immunochromatographic test strip for detecting rotavirus antigen of the present application will be specifically disclosed with appropriate reference to the accompanying drawings. However, there may be cases where unnecessary details are omitted. For example, there are cases where detailed descriptions of well-known matters and repeated descriptions of actually identical structures are omitted. This is to prevent the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the accompanying drawings and the following description are provided for those skilled in the art to fully understand the present application and are not intended to limit the subject matter recited in the claims.

[0040] The "range" disclosed in the present application is defined in the form of a lower limit and an upper limit. A given range is defined by

[0041] selecting a lower limit and an upper limit. The selected lower limit and upper limit define the boundaries of a particular range. The ranges defined in this way can include or exclude the end values and can be combined arbitrarily, that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a particular parameter, ranges of 60-110 and 80-120 are also contemplated. In addition, if the minimum range values 1 and 2 are listed, and if the maximum range values 3, 4, and 5 are listed, then the following ranges are all contemplated: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In the present application, unless otherwise stated, the numerical range "a-b" represents an abbreviated representation of any real number combination between a and b, where a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" have been fully listed herein, and "0-5" is only an abbreviated representation of these numerical combinations. In addition, when it is stated that a certain parameter is an integer ≥2, it is equivalent to disclosing that the parameter is, for example, the integers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0042] If there is no special instruction, all embodiments and optional embodiments of the present application can be combined with each other to form new technical solutions.

[0043] If there is no special instruction, all technical features and optional technical features of the present application can be combined with each other to form new technical solutions.

[0044] Unless otherwise specified, all steps of this application can be carried out sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) carried out sequentially, or may also include steps (b) and (a) carried out sequentially. For example, when it is mentioned that the method may further include step (c), it means that step (c) can be added to the method in any order. For example, the method may include steps (a), (b), and (c), or may include steps (a), (c), and (b), or may also include steps (c), (a), and (b), etc.

[0045] Unless otherwise specified, the terms "comprising" and "including" mentioned in this application mean open-ended, and can also be closed-ended. For example, the "comprising" and "including" can mean that other components not listed may also be included or comprised, or it may only include or comprise the listed components.

[0046] Unless otherwise specified, in this application, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) while B is true (or exists); or both A and B are true (or exist).

[0047] Immunogold technology is a new type of immunolabeling technology that uses colloidal gold as a tracer marker for antigen-antibody reactions. Colloidal gold is formed by the polymerization of chloroauric acid (HAuCl4) under the action of a reducing agent into gold particles of a specific size, and due to electrostatic interaction, it becomes a stable colloidal state, known as colloidal gold. Colloidal gold carries a negative charge in a weak alkaline environment and can form a firm bond with the positive charge groups of protein molecules. Since this bond is an electrostatic bond, it does not affect the biological properties of the protein. Based on some physical properties of colloidal gold, such as high electron density, particle size, shape, and color reaction, combined with the immunological and biological properties of the conjugate, colloidal gold is thus widely used in the fields of immunology, histology, pathology, and cell biology.

[0048] Rotavirus is one of the main pathogens causing diarrhea in infants and young children. It mainly infects intestinal epithelial cells, thereby causing cell damage and diarrhea. Rotavirus infections range from asymptomatic, mild illness to severe illness, and in severe cases, it can cause fatal gastroenteritis, dehydration, and electrolyte imbalance.

[0049] Improving the sensitivity of detecting rotavirus can timely and effectively detect rotavirus infections, enable early treatment, and effectively ensure human health and safety.

[0050] Improving the labeling efficiency is beneficial to improving the detection sensitivity. To this end, the present application provides a method for detecting a virus. During the detection process, the conjugate pad used is coated with a rotavirus antibody labeled with colloidal gold, and the rotavirus antibody labeled with colloidal gold has a high labeling efficiency to improve the detection sensitivity.

[0051] As Figure 2 shown, the method for detecting a virus of the present application uses a colloidal gold immunochromatographic test strip to detect a sample to be tested, including the following steps: adding the sample to be tested to the sample pad of the colloidal gold immunochromatographic test strip, and performing an immunochromatographic reaction for 10 min - 15 min; observing the colors on the test line and the control line of the colloidal gold immunochromatographic test strip to obtain a detection result; wherein, the colloidal gold immunochromatographic test strip includes a bottom plate, and a sample pad, a conjugate pad, a chromatographic membrane, and an absorbent pad sequentially arranged on the bottom plate. The chromatographic membrane is provided with a test line and a control line; the conjugate pad is coated with a rotavirus antibody labeled with colloidal gold, the particle size of the colloidal gold is 50 nm - 70 nm, the antibody at the test line includes a first antibody for recognizing the rotavirus antigen, and the antibody at the control line includes a second antibody for recognizing the rotavirus antibody. Using colloidal gold with a particle size of 50 nm - 70 nm can increase the effective collision between the antibody molecules and the colloidal gold, increase the binding efficiency of the antibody and the colloidal gold, improve the effective labeling quantity of the nanogold, coat the nanogold with high labeling efficiency onto the conjugate pad, and increase the quantity of the effectively labeled nanogold on the conjugate pad, thereby being beneficial to improving the detection sensitivity of rotavirus; moreover, the volume specific surface area of the colloidal gold with a particle size of 50 nm - 70 nm is small, the amount of antibody used is less, reducing the usage amount of the antibody, that is, the present application not only improves the labeling efficiency of the colloidal gold, but also saves the cost of the antibody.

[0052] The colloidal gold immunochromatographic test strip for detecting rotavirus antigen includes a bottom plate, and a sample pad, a conjugate pad, a chromatographic membrane, and an absorbent pad sequentially arranged on the bottom plate. The chromatographic membrane is provided with a test line and a control line; the conjugate pad is coated with a rotavirus antibody labeled with colloidal gold, the particle size of the colloidal gold is 50 nm - 70 nm, the antibody at the test line includes a first antibody for recognizing the rotavirus antigen, and the antibody at the control line includes a second antibody for recognizing the rotavirus antibody.

[0053] As Figure 1 shown, the colloidal gold immunochromatographic test strip 100 for detecting rotavirus antigen generally consists of a bottom plate, a sample pad 20, a conjugate pad 30, a chromatographic membrane 40, an absorbent pad 50, etc. The chromatographic membrane is marked with a test line 41 and a control line 43. After the solid-phase carrier labeled with the antigen or antibody fixed on the conjugate pad undergoes a specific immune reaction with the analyte in the sample, the formed complex undergoes a chromatographic effect on the chromatographic membrane, and a specific immune reaction occurs again at the test line. The solid-phase carrier serves as a signal amplification marker, and the detection result is obtained by visual observation or detection with a corresponding device.

[0054] In the present application, the conjugate pad is coated with rotavirus antibodies labeled with colloidal gold. The antibodies at the test line include the first antibody for recognizing rotavirus antigen, and the antibodies at the control line include the second antibody for recognizing rotavirus antibodies. When a test sample is dropped onto the sample pad, the rotavirus antigen in the sample flows on the sample pad. After reaching the conjugate pad, the rotavirus antigen specifically binds to the rotavirus antibodies labeled with colloidal gold on the conjugate pad, forming an "antigen-antibody-gold conjugate". At the same time, the "antigen-antibody-gold conjugate" is released from the conjugate pad and flows towards the chromatographic membrane by capillary action. When the "antigen-antibody-gold conjugate" reaches the test line, the "antigen-antibody-gold conjugate" binds to the first antibody at the test line, forming an "antibody-antigen-antibody-gold conjugate" at the test line, thereby causing the aggregation and color development of the nano-gold at the test line. At the same time, the rotavirus antibodies labeled with colloidal gold that have not specifically bound to the rotavirus antibodies continue to flow towards the control line and bind to the second antibody after reaching the control line, forming a "secondary antibody-antibody-gold conjugate", and the nano-gold aggregates and develops color at the control line. Among them, the color development at the control line indicates that the test strip is effective.

[0055] The present application uses colloidal gold with a particle size of 50nm - 70nm, which can increase the effective collision between antibody molecules and colloidal gold when preparing rotavirus antibodies labeled with colloidal gold, increase the binding efficiency between the antibody and colloidal gold, improve the effective labeling quantity of nano-gold, coat the highly efficiently labeled nano-gold onto the conjugate pad, and increase the quantity of effectively labeled nano-gold on the conjugate pad, thereby facilitating the improvement of the detection sensitivity of rotavirus; moreover, the colloidal gold with a particle size of 50nm - 70nm has a small volume specific surface area and requires less antibody usage, reducing the amount of antibody used. That is, the present application not only improves the labeling efficiency of colloidal gold but also saves the cost of antibodies.

[0056] Among the above 50nm - 70nm, the values include the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 50nm, 55nm, 58nm, 59nm, 60nm, 61nm, 62nm, 65nm, 68nm, 70nm, etc.

[0057] Furthermore, the particle size of the colloidal gold is 60nm; and / or, the rotavirus antibody includes the antibody with the clone number 11H4, the first antibody includes the antibody with the clone number 7F12; and / or, the second antibody includes goat anti-mouse IgG; and / or, the minimum detection limit of the colloidal gold immunochromatographic test strip for detecting rotavirus antigen is 4×10 4, unit: number of virus particles / ml; and / or, the first antibody is a monoclonal antibody or a polyclonal antibody; and / or, the scribing concentration of the first antibody at the test line is 1 μL / cm; and / or, the coating concentration of the colloidal gold-labeled rotavirus antibody on the conjugate pad is 10 μL / cm; and / or, the material of the bottom plate includes at least one of polyvinyl chloride, polyethylene and glass; and / or, the material of the sample pad includes a glass fiber membrane, and the glass fiber membrane includes ahlstrom 8964; and / or, the material of the conjugate pad includes a glass fiber membrane and / or a polyester fiber membrane, and the glass fiber membrane includes ahlstrom 8980; and / or, the material of the chromatographic membrane includes a nitrocellulose membrane, and the nitrocellulose membrane includes sartorius 140; and / or, the sample pad, the conjugate pad, the chromatographic membrane and the absorbent pad are sequentially and mutually overlapped and attached to the bottom plate.

[0058] In this application, the particle size of the colloidal gold used is 60 nm. The volume specific surface area of the 60-nm colloidal gold is small, and the amount of antibody used is less.

[0059] In this application, the rotavirus antibody includes the antibody with the clone number 11H4 from Hangzhou Xianzhi Biotechnology Co., Ltd., and the first antibody includes the antibody with the clone number 7F12 from Hangzhou Xianzhi Biotechnology Co., Ltd. Using the above-mentioned pair of rotavirus antibodies, the detection sensitivity is high.

[0060] The second antibody includes goat anti-mouse IgG. Goat anti-mouse antibodies from different domestic and foreign suppliers were screened, including Shanghai Modis Medical Technology Co., Ltd., Zhuhai Bomei Biotechnology Co., Ltd., Hangzhou Xianzhi Biotechnology Co., Ltd., Hangzhou Longji Biotechnology Co., Ltd., and Ningbo Maiyue Biotechnology Co., Ltd. The selected goat anti-mouse antibody is used as the quality control line antibody from Ningbo Maiyue Biotechnology Co., Ltd., and the batch number of the goat anti-mouse antibody is EC00102.

[0061] The lowest detection limit of the colloidal gold immunochromatographic test strip for detecting rotavirus antigen is 4×10 4 , unit: number of virus particles / mL; the conjugate pad is coated with a colloidal gold-labeled rotavirus antibody, and the particle size of the colloidal gold is 50 nm - 70 nm, which can effectively improve the detection sensitivity of the test strip. The specific steps for the detection limit test are as follows: Prepare the colloidal gold immunochromatographic test strip for detecting rotavirus antigen in this application, and the detection antigen is a rotavirus culture. Use a concentration of 1×10 9 Inactivated rotavirus culture, dilute the culture with a sample diluent to different concentrations for testing. The lowest detection limit obtained by testing with the colloidal gold immunochromatographic test strip for detecting rotavirus antigen in this application is 4×10 4 , unit: number of virus particles / mL.

[0062] The first antibody is a monoclonal antibody or a polyclonal antibody. In this application, the first antibody is an antibody fixed at the test line for recognizing rotavirus antigen, which can be a monoclonal antibody or a polyclonal antibody.

[0063] The scribing concentration of the first antibody at the test line is 1 μL / cm. The steps for fixing the first antibody at the test line are as follows: Prepare a first antibody solution with a concentration of 0.5 mg / mL - 2 mg / mL for recognizing rotavirus antigen, use a three-dimensional membrane scribing instrument to scribe the first antibody solution on the chromatography membrane to form a test line, and after drying, reserve it. At this time, the scribing concentration of the first antibody at the test line is 1 μL / cm.

[0064] The coating concentration of the colloidal gold-labeled rotavirus antibody on the conjugate pad is 10 μL / cm. During the process of coating the colloidal gold-labeled rotavirus antibody on the conjugate pad, the colloidal gold-labeled rotavirus antibody is coated on the conjugate pad by means of spraying dots, and the concentration of the colloidal gold-labeled rotavirus antibody coated on the conjugate pad is 10 μL / cm.

[0065] The material of the bottom plate includes at least one of polyvinyl chloride, polyethylene, and glass, and is not specifically limited. The material of the sample pad includes a glass fiber membrane, and the glass fiber membrane includes ahlstrom 8964. The material of the conjugate pad includes a glass fiber membrane and / or a polyester fiber membrane, and the glass fiber membrane includes ahlstrom 8980. The material of the chromatography membrane includes a nitrocellulose membrane, and the nitrocellulose membrane includes sartorius 140. The sample pad, conjugate pad, chromatography membrane, and absorbent pad are sequentially and mutually overlapped and attached to the bottom plate.

[0066] Furthermore, this application provides a preparation method for a colloidal gold immunochromatographic test strip, including the following steps: Combine a rotavirus antibody with colloidal gold particles with a particle size of 50 nm - 70 nm to form an immunolabel, and fix the immunolabel on the conjugate pad for reserve; Set a test line and a quality control line on the chromatography membrane, fix the first antibody for recognizing rotavirus antigen on the test line, and fix the second antibody for recognizing rotavirus antibody on the quality control line for reserve; Arrange the sample pad, the conjugate pad fixed with the immunolabel, the chromatography membrane fixed with the first antibody and the second antibody, and the absorbent pad on the bottom plate in sequence to form a colloidal gold immunochromatographic test strip for detecting rotavirus antigen.

[0067] The rotavirus antibody is combined with colloidal gold particles with a particle size of 50 nm - 70 nm to form an immunolabel, and the immunolabel is fixed on the conjugate pad for standby; a test line and a control line are set on the chromatographic membrane, the first antibody for identifying rotavirus antigen is fixed on the test line, and the second antibody for identifying rotavirus antibody is fixed on the control line for standby; a sample pad, a conjugate pad fixed with the immunolabel, a chromatographic membrane fixed with the first antibody and the second antibody, and an absorbent pad are sequentially arranged on the bottom plate to form a colloidal gold immunochromatographic test strip for detecting rotavirus antigen.

[0068] Colloidal gold with a particle size of 50 nm - 70 nm is used, so that when preparing the colloidal gold-labeled rotavirus antibody, the effective collision between the antibody molecules and the colloidal gold can be increased, the binding efficiency of the antibody and the colloidal gold can be increased, the effective labeling quantity of the nano gold can be improved, the highly efficiently labeled nano gold is coated on the conjugate pad, and the quantity of the effectively labeled nano gold on the conjugate pad is increased, thereby being beneficial to improving the detection sensitivity of rotavirus; moreover, the volume specific surface area of the colloidal gold with a particle size of 50 nm - 70 nm is small, the amount of the antibody used is less, the usage amount of the antibody is reduced, that is, the present application not only improves the labeling efficiency of the colloidal gold, but also saves the cost of the antibody.

[0069] Further, in the step of combining the rotavirus antibody with colloidal gold particles with a particle size of 50 nm - 70 nm to form an immunolabel, and fixing the immunolabel on the conjugate pad for standby, the following steps are included: preparing a colloidal gold solution composed of colloidal gold particles with a mass-volume concentration of four ten-thousandths and a particle size of 50 nm - 70 nm, adjusting the pH of the colloidal gold solution, then adding a rotavirus antibody with a concentration of 10 μg / mL - 30 μg / mL, reacting for a preset time, adding a blocking agent, blocking for a preset time, obtaining the reacted solution and concentrating it to obtain the immunolabel, resuspending the immunolabel with a reconstitution solution to obtain an immunolabel reconstitution solution, coating the immunolabel reconstitution solution on the conjugate pad, and drying it for standby.

[0070] The mass-volume concentration refers to the mass of the solute divided by the volume of the solvent to obtain the mass-volume concentration. For example, the mass of the nano gold divided by the volume of the solvent obtains the concentration of the nano gold solution.

[0071] A colloidal gold solution with a concentration of four ten-thousandths is used, and the particle size of the nano gold is 50 nm - 70 nm. Compared with the conventional colloidal gold solution with a concentration of one ten-thousandth, the present application uses a high-concentration colloidal gold solution, which can increase the effective collision between the antibody molecules and the colloidal gold, increase the binding efficiency of the antibody and the colloidal gold, and compared with the colloidal gold with a particle size of 40 nm, the colloidal gold with a particle size of 50 nm - 70 nm has a small volume specific surface area and uses less antibody. Therefore, by using the high-concentration large-particle colloidal gold to prepare the immunolabel, the usage amount of the antibody is reduced, the labeling efficiency of the colloidal gold is improved, and the cost of the antibody is saved.

[0072] In the process of preparing the immunolabel, adjust the pH of the colloidal gold solution, and then add rotavirus antibody with a concentration of 10 μg / mL - 30 μg / mL, preferably with a rotavirus antibody concentration of 20 μg / mL - 25 μg / mL, so that the antibody is positively charged in the colloidal gold solution and adsorbed on the negatively charged surface of the colloidal gold. React for a preset time, then add a blocking agent and block for a preset time to cover the bare gold surface sites not coated with the antibody, obtaining a reacted solution. Concentrate the reacted solution, for example, by centrifugation, remove the supernatant to obtain the immunolabel, resuspend the immunolabel with a reconstitution solution to obtain an immunolabel reconstitution solution, coat the immunolabel reconstitution solution on the conjugate pad, and dry it for standby.

[0073] Among the above-mentioned 10 μg / mL - 30 μg / mL rotavirus antibodies, the values include the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 10 μg / mL, 15 μg / mL, 20 μg / mL, 21 μg / mL, 22 μg / mL, 23 μg / mL, 24 μg / mL, 25 μg / mL, 30 μg / mL, etc.

[0074] Further, in the step of binding the rotavirus antibody to colloidal gold particles with a particle size of 50 nm - 70 nm to form an immunolabel and fixing the immunolabel on the conjugate pad for standby, the following steps are included: Prepare a colloidal gold solution composed of colloidal gold particles with a mass-volume concentration of four ten-thousandths and a particle size of 50 nm - 70 nm, and adjust the pH of the colloidal gold solution. Then add rotavirus antibody with a concentration of 10 μg / mL - 30 μg / mL, react for a preset time, then add a blocking agent and block for a preset time to obtain a reacted solution and concentrate it to obtain an immunolabel concentrated 10 times. Resuspend the immunolabel with a reconstitution solution to obtain an immunolabel reconstitution solution diluted 5 - 10 times, coat the immunolabel reconstitution solution on the conjugate pad, and dry it for standby.

[0075] In the process of preparing the immunolabel, concentrate the obtained reacted solution to obtain an immunolabel concentrated 10 times. Resuspend the immunolabel with a reconstitution solution to obtain an immunolabel reconstitution solution diluted 5 - 10 times, coat the immunolabel reconstitution solution on the conjugate pad, and dry it for standby.

[0076] Among the above-mentioned 5 - 10 times, the values include the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, etc.

[0077] Further, in the step of binding rotavirus antibody to colloidal gold particles with a particle size of 50 nm - 70 nm to form an immunolabel, and fixing the immunolabel on the conjugate pad for standby, the following steps are included: Prepare a colloidal gold solution composed of colloidal gold particles with a mass-volume concentration of four ten-thousandths and a particle size of 50 nm - 70 nm, and adjust the pH of the colloidal gold solution to 7.0 - 9.0. Then add rotavirus antibody with a concentration of 10 μg / mL - 30 μg / mL, and react with shaking at room temperature for 4 h - 5 h. Then add a BSA solution with a mass-volume concentration of 0.5% - 1.0% to cover the bare gold surface sites not coated with antibody, and incubate for 30 min - 60 min. Centrifuge at a centrifugal speed of 6000 r / min - 8000 r / min for 10 min - 15 min, remove the supernatant, and obtain an immunolabel concentrated 10 times. Resuspend the immunolabel with a reconstitution solution to obtain an immunolabel reconstitution solution diluted 5 - 10 times. Coat the immunolabel reconstitution solution on the conjugate pad, and dry it at 45 ± 2 °C for 16 h - 18 h for standby.

[0078] When the pH value of the colloidal gold solution is 7.0 - 9.0, the colloidal gold is negatively charged, which is beneficial to the binding of the antibody to the colloidal gold. React with shaking at room temperature for 4 h - 5 h to make the reaction complete. Then add a BSA solution with a mass-volume concentration of 0.5% - 1.0% to cover the bare gold surface sites not coated with antibody, and incubate for 30 min - 60 min. Centrifuge at a centrifugal speed of 6000 r / min - 8000 r / min for 10 min - 15 min, remove the supernatant, and obtain an immunolabel concentrated 10 times. Resuspend the immunolabel with a reconstitution solution to obtain an immunolabel reconstitution solution diluted 5 - 10 times. Coat the immunolabel reconstitution solution on the conjugate pad, and dry it at 45 ± 2 °C for 16 h - 18 h for standby.

[0079] In the above pH value of 7.0 - 9.0, the values include the minimum and maximum values of this range, as well as each value between such minimum and maximum values. Specific examples include but are not limited to the point values in the examples and 7.0, 7.5, 8.0, 8.5, 9.0, etc.

[0080] In the above reaction with shaking at room temperature for 4 h - 5 h, the values include the minimum and maximum values of this range, as well as each value between such minimum and maximum values. Specific examples include but are not limited to the point values in the examples and 4 h, 4.5 h, 5 h, etc.

[0081] In the above 0.5% - 1.0% BSA solution, the values include the minimum and maximum values of this range, as well as each value between such minimum and maximum values. Specific examples include but are not limited to the point values in the examples and 0.5%, 0.7%, 0.9%, 1.0%, etc.

[0082] Furthermore, the pH of the colloidal gold solution is 7.5; and / or, the concentration of the rotavirus antibody is 20 μg / mL; and / or, the reconstitution solution includes 50 mM of NaCl, 0.1% (m / v) of BSA, 1.5% (m / v) of sucrose, and 1.5% (m / v) of sodium caseinate; and / or, the rotavirus antibody includes the 11H4 clone antibody of Hangzhou Xianzhi Biotech Co., Ltd.; and / or, the first antibody includes the 7F12 clone antibody of Hangzhou Xianzhi Biotech Co., Ltd.; and / or, the second antibody includes goat anti-mouse IgG; and / or, the scribing concentration of the first antibody at the test line is 1 μL / cm; and / or, the coating concentration of the immunolabel on the conjugate pad is 10 μL / cm; and / or, the concentration of the first antibody is 0.5 mg / mL - 2 mg / mL; and / or, the material of the bottom plate includes at least one of polyvinyl chloride, polyethylene, and glass; and / or, the material of the sample pad includes a glass fiber membrane, and the glass fiber membrane includes ahlstrom 8964; and / or, the material of the conjugate pad includes a glass fiber membrane, and the glass fiber membrane includes ahlstrom 8980; and / or, the material of the chromatographic membrane includes a nitrocellulose membrane, and the nitrocellulose membrane includes sartorius140.

[0083] The pH of the colloidal gold solution is preferably 7.1 - 8.5. Since the isoelectric points of different antibodies vary, it is necessary to explore the labeling efficiency of antibodies with colloidal gold at different pH values. The pH values explored were 7.0, 7.1, 7.3, 7.5, 8.0, 8.1, 8.5, and 9.0. When the pH value was 7.5, the antibody carried a positive charge in the colloidal gold solution and adsorbed on the negatively charged surface of the colloidal gold. At this time, the labeling efficiency was the highest. Therefore, the preferred pH value is 7.5.

[0084] The concentration of the rotavirus antibody is 20 μg / ml to avoid incomplete labeling caused by too low a concentration of the rotavirus antibody and to avoid wasting the antibody due to too high a concentration.

[0085] The reconstitution solution includes 50 mM of NaCl, 0.1% (m / v) of BSA, 1.5% (m / v) of sucrose, and 1.5% (m / v) of sodium caseinate.

[0086] Rotavirus antibodies include the antibody with clone number 11H4 from Hangzhou Xianzhi Biotechnology Co., Ltd.; the first antibody includes the antibody with clone number 7F12 from Hangzhou Xianzhi Biotechnology Co., Ltd.; the second antibody includes goat anti-mouse IgG; the coating concentration of the immunolabel on the conjugate pad is 10 μL / cm. Using the above colloidal gold immunochromatographic test strip, the obtained immunolabel has a high labeling efficiency, and the concentration of the colloidal gold-labeled rotavirus antibody coated on the conjugate pad is 10 μL / cm. By coating with a high concentration of the colloidal gold-labeled rotavirus antibody, it helps to effectively detect low-concentration rotavirus; the material of the bottom plate includes at least one of polyvinyl chloride, polyethylene, and glass; the material of the sample pad includes a glass fiber membrane, and the glass fiber membrane includes ahlstrom8964; the material of the conjugate pad includes a glass fiber membrane, and the glass fiber membrane includes ahlstrom 8980; the material of the chromatographic membrane includes a nitrocellulose membrane, and the nitrocellulose membrane includes sartorius 140.

[0087] The step of immobilizing the first antibody at the test line is as follows: Prepare a first antibody solution with a concentration of 0.5 mg / mL - 2 mg / mL for recognizing rotavirus antigen, and use a three-dimensional membrane scribing instrument to scribe the first antibody solution on the chromatographic membrane to form a test line. After drying, it is reserved. At this time, the scribing concentration of the first antibody at the test line is 1 μL / cm. The first antibody solution with a concentration of 0.5 mg / mL - 2 mg / mL avoids too low a concentration where the immobilization amount of the first antibody is insufficient and the color development at the test line is not obvious. At the same time, it also avoids too high a concentration, resulting in waste of the antibody. Among the above 0.5 mg / mL - 2 mg / mL, the values include the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.5 mg / mL, 1.8 mg / mL, 2 mg / mL, etc.

[0088] Furthermore, before the step of combining the rotavirus antibody with colloidal gold particles with a particle size of 50 nm - 70 nm to form an immunolabel and immobilizing the immunolabel on the conjugate pad for standby, the following steps are also included: Prepare a conjugate pad pretreatment solution, soak the conjugate pad in the conjugate pad pretreatment solution, take out the conjugate pad after soaking for a preset time, and dry it for standby. Among them, the components of the conjugate pad pretreatment solution include 10 mM - 20 mM PB, 100 mM - 150 mM NaCl, casein with a mass-volume concentration of 1% - 1.5%, sucrose with a mass-volume concentration of 3% - 5%, Tween-20 with a volume concentration of 0.1% - 5%, PEG 20000 with a mass-volume concentration of 0.1% - 0.2%, and proclin 300 with a volume concentration of 0.1% - 0.2%.

[0089] The PB buffer system stabilizes the pH of the solution. The salt ion substance is sodium chloride, which can remove the viscous substances in the sample, causing the viscous substances to settle without affecting the flow and penetration of the sample on the test strip. Tween-20 is a surfactant that functions to moisten the conjugate pad faster, facilitating the attachment and reconstitution of the immunolabels and helping the analyte to bind better with the immunolabels. The role of sucrose is to protect the antibody protein labeled on the colloidal gold, making the antibody more stable. Casein serves to protect the antibody protein labeled on the colloidal gold, making the protein more stable. Additionally, it has an inhibitory effect on non-specific reactions, helping to prevent non-specific binding and enabling complete release. The macromolecular substance PEG 20000 acts as a framework for the uniform dispersion of the colloidal gold after drying, facilitating the rapid dissolution and release of the gold-labeled substance. Proclin 300 acts as a preservative, which can stabilize the conjugate pad treatment solution and extend its shelf life. The above components work together synergistically, making the buffer system of this conjugate pad treatment solution conducive to the binding and release of immunolabels on the conjugate pad.

[0090] Among them, the value concentration range of 10 mM - 20 mM PB includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM. The value concentration range of 100 mM - 150 mM NaCl includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 100 mM, 120 mM, 140 mM, 150 mM. The value concentration range of casein with a mass - volume concentration of 1% - 1.5% includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 1%, 1.2%, 1.4%, 1.5%. The value concentration range of sucrose with a mass - volume concentration of 3% - 5% includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 3%, 3.5%, 4%, 5%. The value concentration range of Tween - 20 with a volume concentration of 0.1% - 5% includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 0.1%, 0.5%, 0.7%, 0.9%, 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%. The value concentration range of PEG 20000 with a mass - volume concentration of 0.1% - 0.2% includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 0.1%, 0.15%, 0.2%. The value concentration range of proclin 300 with a volume concentration of 0.1% - 0.2% includes the minimum value and the maximum value of this range, as well as each value between this minimum value and the maximum value. Specific examples include but are not limited to the point values in the examples and 0.1%, 0.15%, 0.2%.

[0091] Among them, the soaking time is 20 min - 30 min, the drying temperature is 45°C - 50°C, and the drying time is 16 h - 18 h.

[0092] The steps for treating the conjugate pad are as follows: Prepare a conjugate pad pretreatment solution with the composition of 10 - 20 mM PB, 100 - 150 mM NaCl, 1 - 1.5% casein, 3 - 5% sucrose, 0.1% - 5% Tween - 20, 0.1 - 0.2% PEG 20000, and 0.1 - 0.2% proclin 300. Pour the prepared liquid into a container made of acrylic or stainless steel, with the size of the container being approximately 30 cm × 30 cm × 10 cm. The conjugate pad material used is a glass fiber membrane, model ahlstrom 8980. Place it into the liquid, ensuring that the liquid submerges the glass fiber membrane, and the soaking time is about 20 min - 30 min. After the soaking, take out the glass fiber membrane, lay it flat on a sieve, and put it into a forced air drying oven set at a temperature of 45°C - 50°C. The drying time is about 16 h - 18 h. After drying, it serves as a pretreated conjugate pad and is stored sealed at room temperature for later use.

[0093] Further, before the steps of sequentially arranging the sample pad, the conjugate pad fixed with immune labels, the chromatographic membrane fixed with the first antibody and the second antibody, and the absorbent pad on the bottom plate to form a colloidal gold immunochromatographic test strip for detecting rotavirus antigen, the following steps are also included: Prepare a sample pad pretreatment solution, soak the sample pad in the sample pad pretreatment solution, and after soaking for a preset time, take out the sample pad and dry it for later use. The components of the sample pad pretreatment solution include 10 mM - 20 mM PBS, 1% - 1.5% Tween - 20 by volume concentration, 0.2% - 0.5% S9 by mass - volume concentration, 4% - 6% sucrose by mass - volume concentration, 0.5% - 0.8% PEG6000 by mass - volume concentration, 1.5% - 2% BSA by mass - volume concentration, and 0.1% - 0.2% proclin 300 by volume concentration.

[0094] PBS serves as a buffer system to stabilize the pH of the system. Through the blocking agent BSA, the hydrophilic macromolecule PEG6000, the surfactants Tween - 20 and S9, it effectively prevents sample substances from non - specifically adsorbing onto the immunochromatographic test strip. Using the above - mentioned treatment solution to treat the sample pad can increase the hydrophilicity of the sample pad, contribute to the rapid wetting of the sample pad, and promote the occurrence of the chromatographic effect. At the same time, due to the suspension and dispersion effect and the stable pH environment, the sample pad can also protect the antibodies in the sample and reduce non - specific binding, which helps to effectively detect low - concentration rotavirus and avoid false - positive problems.

[0095] Among them, the value concentration range of 10 mM - 20 mM PBS includes the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM. The value concentration range of 1% - 1.5% Tween-20 includes the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 1%, 1.3%, 1.5%. The value concentration range of 0.2% - 0.5% S9 includes the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 0.2%, 0.3%, 0.4%, 0.5%. The value concentration range of 4% - 6% sucrose includes the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 4%, 5%, 6%. The value concentration range of 0.5% - 0.8% PEG6000 includes the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 0.5%, 0.6%, 0.7%, 0.8%. The value concentration range of 1.5% - 2% BSA sucrose includes the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 1.5%, 1.8%, 2%. The value concentration range of 0.1% - 0.2% proclin 300 includes the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 0.1%, 0.15%, 0.2%.

[0096] Among them, the soaking time is 20 min - 30 min, the drying temperature is 45°C - 50°C, and the drying time is 16 h - 18 h.

[0097] The processing steps of the sample pad are as follows: Prepare a sample pad treatment solution with a composition of 10 mM - 20 mM PBS, 1% - 1.5% Tween-20, 0.2% - 0.5% S9, 4% - 6% sucrose, 0.5% - 0.8% PEG6000, 1.5% - 2% BSA, and 0.1% - 0.2% proclin 300. Pour the prepared liquid into a container made of acrylic or stainless steel. The sample pad material used is a glass fiber membrane with the model ahlstrom 8964. Place it in the liquid, ensuring that the liquid submerges the glass fiber membrane. The soaking time is about 20 min - 30 min. After soaking, take out the glass fiber membrane, lay it flat on a sieve, and put it into a forced air drying oven set at a temperature of 45°C - 50°C. The drying time is about 16 h - 18 h. After drying, it serves as a pretreated sample pad and is stored sealed at room temperature for later use.

[0098] In the above method, both the conjugate pad and the sample pad are treated with corresponding pretreatment solutions, which helps to reduce non-specific binding during the detection process and reduce false positives. Based on the improvement of the above-mentioned various performances, the test strip prepared by the preparation method of the colloidal gold immunochromatographic test strip has high sensitivity and low false positives.

[0099] This application also provides a colloidal gold immunochromatographic test strip kit for detecting rotavirus antigen, including a test strip, a diluent for the sample to be tested, and the above-mentioned colloidal gold immunochromatographic test strip for detecting rotavirus antigen, or a colloidal gold immunochromatographic test strip for detecting rotavirus antigen prepared by the preparation method of the colloidal gold immunochromatographic test strip for detecting rotavirus antigen as described above.

[0100] Furthermore, the colloidal gold immunochromatographic test strip kit for detecting rotavirus antigen includes a cassette and the above-mentioned colloidal gold immunochromatographic test strip for detecting rotavirus antigen. The cassette includes a cassette cover and a cassette bottom. The cassette cover and the cassette bottom enclose a space for accommodating the colloidal gold immunochromatographic test strip for detecting rotavirus antigen. The cassette is provided with a sample addition area and a color development area; and / or, the diluent for the sample to be tested includes: 10 mM - 20 mM PBS, 0.05% - 0.1% Triton X100, 0.05% - 0.1% Tween 20, and 0.1% - 0.2% fish gelatin.

[0101] The cassette includes a cassette cover and a cassette bottom. The cassette cover and the cassette bottom enclose a space for accommodating the colloidal gold immunochromatographic test strip for detecting rotavirus antigen. The cassette is provided with a sample addition area and a color development area. When testing a sample, the diluted sample is dropped into the sample addition area, and after the reaction time, the color is observed in the color development area to judge the test result.

[0102] The PBS buffer solution serves as a buffering matrix to keep the basic physical and chemical properties of the sample stable. The Triton X-100 solution and Tween-20 mixture are added to the diluent as surfactants, which can effectively remove dirt and expose the viral antigen. Moreover, the surfactants selected in the present invention are all mild types, which is beneficial to the stability of the protein. Fish gelatin is a common biopolymer, extracted from fish skin and fish bones and then processed into a high-molecular polypeptide polymer. There are generally more than 20 kinds of amino acids that make up fish gelatin, mainly glycine, proline, hydroxyproline and alanine. On the one hand, fish gelatin has poor antigenicity, and on the other hand, it has a far genetic relationship with mammals, which determines the superiority of fish gelatin replacing BSA in ELISA kits. The inclusion of fish gelatin in the diluent can avoid false positives and shield interfering substances in the sample. Fish gelatin has good emulsifying properties, as well as foaming and film-forming properties manifested by reducing the surface tension of the gas-liquid phase by increasing the phase viscosity of the solution, which can protect the structure of the protein from being damaged and enhance the stability of the protein.

[0103] The sample diluent is an important component of the colloidal gold immunochromatographic test strip for detecting rotavirus antigen in rotavirus. Its composition can be 10 mM PBS, 0.05% Triton X100, 0.05% Tween 20, 0.2% fish gelatin. The sample diluent is dispensed into an extraction tube. The sample collected with a sampling swab is added to the extraction tube. The dropper of the extraction tube is tilted to the sample addition hole, and 5-6 drops of the diluent are added. After 15 minutes, the test result is observed. This sample diluent is used in conjunction with the colloidal gold immunochromatographic test strip for detecting rotavirus antigen, which can effectively lyse rotavirus sufficiently and improve the sensitivity of the viral antigen.

[0104] The value concentration range of 10 mM - 20 mM PBS includes the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM. The value concentration range of 0.05% - 0.1% Triton X100 includes the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 0.05%, 0.07%, 0.09%, 0.1%. The value concentration range of 0.05% - 0.1% Tween 20 includes the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 0.05%, 0.07%, 0.09%, 0.1%. The value concentration range of 0.1% - 0.2% fish gelatin includes the minimum and maximum values of this range, as well as each value between this minimum and maximum value. Specific examples include but are not limited to the point values in the examples and 0.1%, 0.15%, 0.2%.

[0105] The present application also provides a detection method, which uses the above-mentioned colloidal gold immunochromatographic test strip for detecting rotavirus antigen to detect a sample to be tested, including the following steps: adding the sample to be tested to the sample pad of the colloidal gold immunochromatographic test strip for detecting rotavirus antigen, and performing an immunochromatographic reaction for 10 min - 15 min; observing the colors on the test line and the quality control line of the colloidal gold immunochromatographic test strip for detecting rotavirus antigen to obtain the detection result.

[0106] Using the above detection method, the colloidal gold test strip measures the rotavirus antigen according to the specific reaction of the antigen and the antibody. The rotavirus antigen can be detected through a one-time operation, and the result can be read quickly and intuitively, and the detection result can be obtained within 15 minutes. Moreover, the method for detecting rotavirus antigen of the present invention does not require special instrument equipment, nor does it require the operation of professional personnel, getting rid of the dependence on professional instruments. Compared with the traditional double antibody sandwich method, the detection method of the present invention improves the labeling efficiency of colloidal gold by improving the gold concentration (from one ten-thousandth to four ten-thousandths) and the particle size (from 40 nm to 60 m), and improves the detection sensitivity. The overall coincidence rate of the detection results of the present invention is relatively high, with a specificity of 97% and a sensitivity of 96%, which is suitable for on-site detection.

[0107] Examples

[0108] 1) Preparation and characterization of colloidal gold

[0109] First, take 200 mL of ultrapure water in a 500 mL round-bottom spherical distillation flask, add 2.0 mL of 1% HAuCl4 solution, place it in a digital display constant temperature magnetic heating mantle for heating. After boiling, add 1.5 mL of 1% trisodium citrate solution while stirring. After the color changes, continue heating for 15 min until the color remains unchanged. Take out the distillation flask, wait for the gold solution to cool to room temperature, centrifuge at 6000 r, remove the supernatant, add ultrapure water to make up the volume to 50 mL, and then place it at 4 °C for standby.

[0110] Take out 2 mL of the standby colloidal gold solution, test it with a UV-visible spectrophotometer, scan in the wavelength range of 400 nm to 700 nm, the scanning interval is 1 nm, and the highest absorption peak at 541 nm ± 2 nm is qualified.

[0111] Operate according to the above method for preparing colloidal gold. The results show that the colloidal gold solution is wine red. Test the colloidal gold sample with a UV-visible spectrogram: it shows that the peak of the prepared colloidal gold is located at 539 nm, and the peak shape is narrow, indicating that the prepared gold particles have uniform particle size, the gold particle size is 60 nm, and the dispersibility is good.

[0112] 2) Pretreatment of conjugate pad

[0113] Prepare the conjugate pad pretreatment solution, the composition is 15 mM PB, 125 mM NaCl, 1.25% casein, 4% sucrose, 0.7% Tween-20, 0.15% PEG 20000, 0.15% proclin 300. Pour the prepared liquid into a container made of acrylic or stainless steel, and the size of the container is about 30 cm × 30 cm × 10 cm. The conjugate pad material used is a glass fiber membrane, model ahlstrom 8980. Put it into the liquid, and make sure the liquid submerges the glass fiber membrane. The soaking time is about 20 min. After the soaking is over, take out the glass fiber membrane, lay it flat on the screen, put it into a blast drying oven with a set temperature of 45 °C - 50 °C, and the drying time is about 17 h. After drying, it is used as a pretreated conjugate pad and stored at room temperature in a sealed manner for standby.

[0114] 3) Sample pad treatment

[0115] Prepare the sample pad treatment solution, which consists of 15 mM PBS, 1.25% Tween-20, 0.3% S9, 5% sucrose, 0.7% PEG6000, 1.5% BSA, and 0.15% proclin 300. Pour the prepared liquid into a container made of acrylic or stainless steel. The sample pad is made of glass fiber membrane with the model number ahlstrom 8964. Put it into the liquid, and make sure the liquid submerges the glass fiber membrane. The soaking time is about 20 min - 30 min. After soaking, take out the glass fiber membrane, lay it flat on the sieve, and put it into a forced air drying oven set at a temperature of 45°C - 50°C. The drying time is about 17 h. After drying, it serves as a pretreated sample pad and is stored sealed at room temperature for later use.

[0116] 4) Preparation of the gold-labeled antibody conjugate pad

[0117] Prepare a colloidal gold solution composed of colloidal gold particles with a mass-volume concentration of four ten-thousandths and a particle size of 60 nm, and adjust the pH of the colloidal gold solution to 7.5. Then add rotavirus antibody (clone number 11H4 antibody from Hangzhou Xianzhi Biotechnology Co., Ltd.) with a concentration of 20 μg / mL, and react with shaking at room temperature for 4 h - 5 h. Then add a 1.0% BSA solution by mass-volume to cover the bare gold surface sites not coated with antibodies, and incubate for 30 min - 60 min. Centrifuge at a centrifugal speed of 7000 r / min for 10 min - 15 min, remove the supernatant, and obtain an immunolabel concentrated 10 times. Resuspend the immunolabel with a reconstitution solution to obtain an immunolabel reconstitution solution diluted 7 times. Coat the immunolabel reconstitution solution on the conjugate pad and dry it at 45 ± 2°C for 17 h for later use.

[0118] 5) Assembly of the colloidal gold immunochromatographic test strip for detecting rotavirus antigen

[0119] Use goat anti-mouse IgG (secondary antibody) with the batch number EC00102DE from Ningbo Maiyue Biotechnology Co., Ltd. and rotavirus antibody (primary antibody) (clone number 7F12 antibody from Hangzhou Xianzhi Biotechnology Co., Ltd.) as the quality control line and the test line respectively, and coat them on the nitrocellulose membrane (chromatographic membrane). Then stick the sample pad, the gold-labeled antibody conjugate pad, the nitrocellulose membrane, and the absorbent paper on the bottom plate in sequence as follows Figure 1 Just assemble it.

[0120] Use the prepared test strip to detect the analyte. When both the test line and the quality control line show red bands, it indicates that the analyte sample contains rotavirus antigen. If the tested sample does not contain rotavirus antigen, when the sample moves to the test line, no red band will be shown, and only a red band will be shown at the quality control line; as long as the quality control line does not show color, it proves that the test strip is invalid and the sample needs to be retested.

[0121] 6) Screening of rotavirus antibody pairs

[0122] Screening steps: Under the condition that other experimental conditions remain unchanged, different rotavirus antibody pairs (rotavirus antibody and primary antibody) are used to prepare different gold-labeled antibody conjugate pads and chromatographic membranes, and different test strips are assembled. The different test strips are used to test the analyte under the same conditions, and the sensitivities of the test strips are tested. The results are shown in the following table. The test strip prepared with the 7F12 clone antibody (primary antibody) of Hangzhou Xianzhi Biotechnology Co., Ltd. and the 11H4 clone antibody (rotavirus antibody) of Hangzhou Xianzhi Biotechnology Co., Ltd. has higher sensitivity. Therefore, this rotavirus antibody is selected.

[0123] Table 1 Screening list of different antibody pairs

[0124]

[0125] 7) Stability test of colloidal gold immunochromatographic test strip for detecting rotavirus antigen

[0126] Three batches of rotavirus antigen detection test strips were continuously produced and tested with inactivated rotavirus cultures at a concentration of 1×10 9 . They were diluted to 1×10 7 , 1×10 5 , 4×10 4 and subjected to 10 repeated tests. The test results showed no difference. The products were accelerated and damaged at 50 °C for 45 days. At 0 day, 7 days, 14 days, 21 days, 28 days, 35 days, 42 days, and 45 days, they were tested with rotavirus cultures at a concentration of 1×10 7 , 1×10 5 , 4×10 4 . The test results showed no difference. Using the Arrhenius equation to convert to long-term stability, it can be stably stored at 4 °C - 30 °C for 18 months, and the test card will not show abnormal results, indicating that the prepared rotavirus antigen detection test strip has good stability.

[0127] 8) Specificity test of colloidal gold immunochromatographic test strip for detecting rotavirus antigen

[0128] Use the prepared test strips to detect other pathogens (including influenza virus A, influenza virus B, novel coronavirus, Escherichia coli, Candida albicans, Gardnerella vaginalis, Legionella pneumophila, Mycobacterium tuberculosis, group A streptococcus, group B streptococcus, norovirus), and at least 5 different human-derived rotavirus positive reference products, and analyze the specificity of the test strips by observing the results. The experimental results show that the test results for other pathogens are negative, indicating that the test strips of this application have no cross-reaction with other pathogens.

[0129] Table 2 List of different pathogen detections

[0130]

[0131] 9) Detection limit comparison experiment

[0132] Use the rotavirus antigen detection test strip prepared in this application to compare with the kit of Guangzhou Wondfo Biotech Co., Ltd. The antigen to be detected is the rotavirus culture, and the inactivated rotavirus culture with a concentration of 1×10 9 is used for testing. It is diluted to 1×10 7 , 1×10 5 , 2×10 5 , 4×10 4 for testing. The results are shown in the following table. The lowest detection limit of the colloidal gold immunochromatographic test strip for detecting rotavirus antigen prepared in this application is 4×10 4 , while the lowest detection limit of the colloidal gold immunochromatographic test strip for detecting rotavirus antigen of Guangzhou Wondfo Biotech Co., Ltd. is 2×10 5 . The sensitivity of the colloidal gold immunochromatographic test strip for detecting rotavirus antigen prepared by this method is higher than that of the colloidal gold immunochromatographic test strip for detecting rotavirus antigen of Guangzhou Wondfo Biotech Co., Ltd.

[0133]

[0134] 10) Comparison experiment of different colloidal gold particle sizes

[0135] Under the same other experimental conditions, change the particle size of the colloidal gold to 40nm, 50nm, 58nm, 60nm, 62nm, 70nm, 80nm respectively to prepare colloidal gold immunochromatographic test strips for detecting rotavirus antigen, and use the prepared different test strips to detect rotavirus positive test substances with different concentrations. Each group of tests is parallel tested 3 times. The results are shown in the following table.

[0136]

[0137] 11) Under the condition that other experimental conditions are the same, change the concentration of colloidal gold, which are respectively one ten-thousandth, four ten-thousandths, and five ten-thousandths of the mass-volume concentration, to prepare colloidal gold immunochromatographic test strips for detecting rotavirus antigen, and use the prepared different test strips to detect rotavirus-positive test substances with different concentrations. Each group of tests is parallel tested 3 times, and the results are shown in the following table.

[0138]

[0139] 12) Under the condition that other experimental conditions remain unchanged, change the formulations of the sample pad pretreatment solution, the conjugate pad pretreatment solution, and the test sample dilution solution to prepare different colloidal gold immunochromatographic test strips for detecting rotavirus antigen, and use the prepared different test strips to detect 200 negative test substances with the same concentration to study the false positive rate of the test strip detection.

[0140]

[0141] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention. Any equivalent structural transformation made by using the content of the specification and drawings of the present invention under the inventive concept of the present invention, or direct / indirect application in other related technical fields is included in the patent protection scope of the present invention.

Claims

1. A colloidal gold immunochromatographic test strip cassette for detecting rotavirus antigen, characterized in that, The colloidal gold immunochromatographic test strip kit includes a test sample diluent and a colloidal gold immunochromatographic test strip. The colloidal gold immunochromatographic test strip includes a base plate, and a sample pad, a conjugate pad, a chromatographic membrane, and an absorbent pad sequentially arranged on the base plate. A test line and a control line are provided on the chromatographic membrane; The conjugate pad is coated with a rotavirus antibody labeled with colloidal gold. This rotavirus antibody is the antibody with the clone number 11H4 of Hangzhou Xianzhi Biotechnology Co., Ltd. The particle size of the colloidal gold is 50nm - 70nm. The antibody at the test line is: the first antibody for recognizing rotavirus antigen, and the antibody at the control line is the second antibody for recognizing the rotavirus antibody; The first antibody is the antibody with the clone number 7F12 of Hangzhou Xianzhi Biotechnology Co., Ltd.; The conjugate pad is treated with a conjugate pad pretreatment solution. The components of the conjugate pad pretreatment solution are: 10mM - 20mM PB, 100mM - 150mM NaCl, casein with a mass - volume concentration of 1% - 1.5%, sucrose with a mass - volume concentration of 3% - 5%, Tween - 20 with a volume concentration of 0.5% - 1.0%, PEG 20000 with a mass - volume concentration of 0.1% - 0.2%, and proclin 300 with a volume concentration of 0.1% - 0.2%; The sample pad is treated with a sample pad pretreatment solution. The components of the sample pad pretreatment solution are: 10mM - 20mM PBS, Tween - 20 with a volume concentration of 1% - 1.5%, S9 with a mass - volume concentration of 0.2% - 0.5%, sucrose with a mass - volume concentration of 4% - 6%, PEG6000 with a mass - volume concentration of 0.5% - 0.8%, BSA with a mass - volume concentration of 1.5% - 2%, and proclin 300 with a volume concentration of 0.1% - 0.2%; The components of the test sample diluent are: 10mM - 20mM PBS, 0.05% - 0.1% Triton X100, 0.05% - 0.1% Tween 20, and 0.1% - 0.2% fish gelatin; The preparation method of the conjugate pad coated with the rotavirus antibody labeled with colloidal gold includes: preparing a colloidal gold solution composed of colloidal gold particles with a mass - volume concentration of four ten - thousandths and a particle size of 50nm - 70nm, adjusting the pH of the colloidal gold solution to 7.0 - 9.0, then adding a rotavirus antibody with a concentration of 10μg / mL - 30μg / mL, reacting for a preset time, adding a blocking agent, blocking for a preset time, obtaining the reacted solution and concentrating it to obtain an immunolabel, resuspending the immunolabel with a re - solution to obtain an immunolabel re - solution, coating the immunolabel re - solution on the conjugate pad, and drying for standby.

2. The colloidal gold immunochromatographic test strip for detecting rotavirus antigen according to claim 1, wherein The lowest detection limit of the colloidal gold immunochromatographic test strip is 4×10 4 , with the unit: number of virus particles / mL.

3. The colloidal gold immunochromatographic test strip for detecting rotavirus antigen according to claim 1 or 2, characterized in that, The particle size of the colloidal gold is 60nm; And / or, the second antibody includes goat anti - mouse IgG.

4. The colloidal gold immunochromatographic test strip cassette for detecting rotavirus antigen according to claim 1, wherein The scribing concentration of the first antibody at the test line is 1μL / cm; And / or, the coating concentration of the conjugate pad coated with the rotavirus antibody labeled with colloidal gold is 10μL / cm.

5. The colloidal gold immunochromatographic test strip for detecting rotavirus antigen according to claim 1, wherein The material of the base plate includes at least one of polyvinyl chloride, polyethylene, and glass; And / or, the material of the sample pad includes a glass fiber membrane, and the glass fiber membrane includes Ahlstrom 8964; And / or, the material of the conjugate pad includes a glass fiber membrane or a polyester fiber membrane, and the glass fiber membrane includes Ahlstrom 8980; And / or, the material of the chromatographic membrane includes a nitrocellulose membrane, and the nitrocellulose membrane includes Sartorius 140; And / or, the sample pad, the conjugate pad, the chromatographic membrane, and the absorbent pad are sequentially and mutually overlapped and attached to the bottom plate.

6. A preparation method of a colloidal gold immunochromatographic test strip box for detecting rotavirus antigen as described in claim 1, characterized in that, Including the following steps: Prepare a colloidal gold solution composed of colloidal gold particles with a mass-volume concentration of four ten-thousandths and a particle size of 50 nm - 70 nm, adjust the pH of the colloidal gold solution to 7.0 - 9.0, then add a rotavirus antibody with a concentration of 10 μg / mL - 30 μg / mL, react for a preset time, then add a blocking agent and block for a preset time, obtain the reacted solution and concentrate it to obtain an immunolabel, resuspend the immunolabel with a reconstitution solution to obtain an immunolabel reconstitution solution, coat the immunolabel reconstitution solution on the conjugate pad, and dry it for later use; Set a test line and a control line on the chromatographic membrane, fix the first antibody for identifying rotavirus antigen on the test line, and fix the second antibody for identifying the rotavirus antibody on the control line for later use; Arrange the sample pad, the conjugate pad fixed with the immunolabel, the chromatographic membrane fixed with the first antibody and the second antibody, and the absorbent pad on the bottom plate in sequence to form a colloidal gold immunochromatographic test strip for detecting rotavirus antigen.

7. The preparation method of the colloidal gold immunochromatographic test strip for detecting rotavirus antigen according to claim 6, characterized in that, Combining the rotavirus antibody with colloidal gold particles with a particle size of 50 nm - 70 nm to form an immunolabel, and fixing the immunolabel on the conjugate pad, including the following steps: Prepare a colloidal gold solution composed of colloidal gold particles with a mass-volume concentration of four ten-thousandths and a particle size of 50 nm - 70 nm, adjust the pH of the colloidal gold solution to 7.5, then add a rotavirus antibody with a concentration of 20 μg / mL, oscillate and react at room temperature for 4 h - 5 h, then add a BSA solution with a mass-volume concentration of 0.5% - 1.0% to cover the exposed gold surface sites not coated with the antibody, block for 30 min - 60 min, centrifuge at a centrifugal speed of 6000 r / min - 8000 r / min for 10 min - 15 min, remove the supernatant to obtain an immunolabel concentrated 10 times, resuspend the immunolabel with a reconstitution solution, and the reconstitution solution includes 50 mM NaCl, a BSA with a mass-volume concentration of 0.1%, a sucrose with a mass-volume concentration of 1.5%, and a sodium caseinate with a mass-volume concentration of 1.5%; obtain an immunolabel reconstitution solution diluted 5 - 10 times, coat the immunolabel reconstitution solution on the conjugate pad, and dry it at 45 ± 2 °C for 16 h - 18 h for later use.

8. The preparation method of the colloidal gold immunochromatographic test strip for detecting rotavirus antigen according to claim 6 or 7, characterized in that, Combining the rotavirus antibody with colloidal gold particles with a particle size of 50 nm - 70 nm to form an immunolabel, and fixing the immunolabel on the conjugate pad, further includes the following steps: Prepare a binding pad pretreatment solution. Immerse the binding pad in the binding pad pretreatment solution and soak for a preset time. Then take out the binding pad and dry it for standby. The components of the binding pad pretreatment solution are as follows: 10 mM - 20 mM PB, 100 mM - 150 mM NaCl, casein with a mass - volume concentration of 1% - 1.5%, sucrose with a mass - volume concentration of 3% - 5%, Tween - 20 with a volume concentration of 0.5% - 1.0%, PEG 20000 with a mass - volume concentration of 0.1% - 0.2%, and proclin 300 with a volume concentration of 0.1% - 0.2%.

9. The preparation method of the colloidal gold immunochromatographic test strip for detecting rotavirus antigen according to claim 6, characterized in that, Before the step of sequentially arranging the sample pad, the binding pad fixed with the immunolabel, the chromatographic membrane fixed with the first antibody and the second antibody, and the absorbent pad on the bottom plate to form a colloidal gold immunochromatographic test strip for detecting rotavirus antigen, the following steps are further included: Prepare a sample pad pretreatment solution. Immerse the sample pad in the sample pad pretreatment solution and soak for a preset time. Then take out the sample pad and dry it for standby. The components of the sample pad pretreatment solution are as follows: 10 mM - 20 mM PBS, Tween - 20 with a volume concentration of 1% - 1.5%, S9 with a mass - volume concentration of 0.2% - 0.5%, sucrose with a mass - volume concentration of 4% - 6%, PEG6000 with a mass - volume concentration of 0.5% - 0.8%, BSA with a mass - volume concentration of 1.5% - 2%, and proclin 300 with a volume concentration of 0.1% - 0.2%.

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