Immunocolloidal gold chromatography method for rapidly detecting norovirus in shellfish

Abstract

The application relates to the technical field of biological detection, and discloses an immunocolloidal gold chromatographic method for rapidly detecting norovirus in shellfish, which comprises the following steps: balancing a test card packaged with a chromatographic test strip and a sample extraction tube containing a sample extraction solution to a set temperature; obtaining a shellfish sample to be detected and transferring the sample to the sample extraction tube, performing a physical oscillation operation to prepare a sample extract; adding the sample extract into a sample adding hole of the test card, enabling capillary chromatography of the sample extract along the chromatographic test strip, triggering specific immunological combination reaction based on a double-antibody sandwich method, and reading the color development state of a quality control line and a detection line through an observation window within 10 to 15 minutes after adding the sample, and determining the positive and negative states of norovirus GI and GII in the sample. The application is free from the dependence on special laboratory instruments, the single detection period is short, and the application is suitable for rapid primary screening of shellfish samples on the spot.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to an immunochromatographic method for rapid detection of norovirus in shellfish using colloidal gold chromatography. Background Technology

[0002] With the widespread consumption of aquatic products, filter-feeding aquatic animals such as shellfish are highly susceptible to accumulating norovirus in the aquatic environment, thus posing corresponding food safety risks. To monitor this type of contamination, current testing for norovirus in shellfish primarily relies on nucleic acid molecular biology detection technologies such as real-time fluorescence PCR.

[0003] While these nucleic acid detection technologies possess high sensitivity and specificity, they also have significant limitations in practical grassroots engineering applications. The nucleic acid detection process typically involves complex sample pretreatment, virus enrichment, and nucleic acid extraction steps, making the overall operation cumbersome and highly dependent on large, sophisticated instruments such as specialized nucleic acid extractors and gene amplification instruments. Furthermore, the entire PCR amplification and detection cycle is relatively long, often requiring several hours to produce final diagnostic results. In addition, this method places strict requirements on the professional skills of operators and the cleanliness level of the experimental environment, directly resulting in a high overall cost per test.

[0004] The aforementioned objective limitations in equipment, time, and environment make existing nucleic acid testing technologies ill-suited for the low-cost, rapid preliminary screening of large quantities of shellfish samples in on-site environments such as grassroots aquaculture farms and seafood wholesale markets. Therefore, the industry needs a simple-to-operate testing method that is not constrained by large laboratory equipment and can achieve rapid on-site colorimetric interpretation to fill the technological gap in rapid on-site screening. Summary of the Invention

[0005] The technical problem solved by this invention is that the detection of norovirus in shellfish in the prior art mainly relies on nucleic acid detection technologies such as real-time fluorescence PCR. Such methods have high requirements for the detection environment, special instruments and equipment, and the professional skills of the operators. Moreover, the detection cycle is long, which makes it difficult to meet the practical application needs of low-cost and rapid preliminary screening of large batches of shellfish samples in grassroots aquaculture farms, seafood markets and other on-site environments.

[0006] To address the above problems, the present invention provides the following technical solution: This invention provides a rapid immunogold chromatography method for detecting norovirus in shellfish, applied to an immunogold chromatography system. The immunogold chromatography system includes a test card and a sample extraction component. The sample extraction component includes a sampling tool and a sample extraction tube loaded with sample extraction solution. The test card has a sample application hole and an observation window on its surface, and an immunogold chromatography test strip is encapsulated inside the test card. The method includes: Place the test card and the sample extraction tube containing the sample extraction solution in the operating environment to equilibrate the temperature; Based on the physical morphology of the sample to be tested, the corresponding sampling tool is selected to obtain the shellfish sample. The shellfish sample is transferred to the sample extraction tube, sealed, and a physical shaking operation is performed to mix the shellfish sample with the sample extract to prepare the sample extract. The sample extract is added dropwise into the sample well of the test card. The sample extract comes into contact with the immunochromatographic gold test strip and undergoes capillary chromatography along the immunochromatographic gold test strip, triggering a double antibody sandwich capture reaction. The color development of the immunochromatographic gold test strip is observed through the observation window of the test card to determine the positive or negative status of norovirus GI and GII types in the shellfish sample.

[0007] In one embodiment of the present invention, the internal structure of the immunochromatographic gold test strip includes, in sequence, a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorbent pad; the sample pad is located directly below the sample well of the test card and is used to initially filter large particulate impurities in the sample extract and buffer the initial flow rate of the buffer phase; the absorbent pad is located at the physical end of the nitrocellulose membrane and is used to provide continuous capillary driving force and absorb excess waste liquid after the chromatography reaction is completed.

[0008] In one embodiment of the present invention, the conjugate pad is mixed and uniformly coated with colloidal gold-labeled mouse anti-norovirus GI monoclonal antibody and colloidal gold-labeled mouse anti-norovirus GII monoclonal antibody; the surface of the nitrocellulose membrane is sequentially provided with a first detection line, a second detection line and a control line along the liquid phase flow direction; the first detection line is solid-phase coated with mouse anti-norovirus GI monoclonal antibody, the second detection line is solid-phase coated with mouse anti-norovirus GII monoclonal antibody, and the control line is solid-phase coated with goat anti-mouse IgG polyclonal antibody.

[0009] As one embodiment of the present invention, the preparation process of the immunochromatographic gold test strip includes: preparing colloidal gold particles using the trisodium citrate reduction method; electrostatically adsorbing and coupling the mouse anti-norovirus GI monoclonal antibody and the mouse anti-norovirus GII monoclonal antibody to the colloidal gold particles under suitable pH conditions; after the coupled gold-labeled antibody is blocked by bovine serum albumin, it is mixed in a set ratio and sprayed onto a glass fiber conjugate pad, and then freeze-dried for later use; using a membrane scribing instrument, mouse anti-norovirus GI monoclonal antibody, mouse anti-norovirus GII monoclonal antibody, and goat anti-mouse IgG polyclonal antibody with a concentration of 1.0-2.0 mg / mL are quantitatively scribed on the first detection line, the second detection line, and the control line area on the membrane surface, respectively, and then placed in an oven for drying and fixation.

[0010] In one embodiment of the present invention, the sample extract is used to dissociate the tissue mucus of shellfish samples and release virus particles. The formulation of the sample extract includes: the base solution of the sample extract is a phosphate buffer solution with a pH of 7.2-7.4, and the base solution contains 0.1%-1.0% by volume of a nonionic surfactant, 0.5%-2% of bovine serum albumin, and 0.8%-1.0% by mass of sodium chloride.

[0011] In one embodiment of the present invention, in the step of placing the test card and the sample extraction tube containing the sample extraction solution in the operating environment to balance the temperature: the temperature parameters of the operating environment are controlled so that the temperature of the test card and the sample extraction solution is within the range of 15 to 30°C; after the detection reagent has completed the temperature equilibration process, the external packaging structure of the test card is removed and the test card is placed flat on the worktable.

[0012] In one embodiment of the present invention, in the step of preparing the sample extract: when the sample to be tested is a solid shellfish tissue, a sampling rod as the sampling tool is used to pick up a solid sample with a mass of 100 mg; when the sample to be tested is a liquid shellfish sample, a pipette as the sampling tool is used to draw up a liquid sample with a volume of 50 μL; the physical shaking operation is to shake vigorously to mix, and the shaking time is required to be maintained at 1 minute.

[0013] In one embodiment of the present invention, the top of the sample extraction tube is provided with a dropper in an openable and closable state; the dropper is opened and aligned with the sample well on the surface of the test card, and 4 drops of sample extract are added into the sample well while maintaining a vertical position, the total volume of the 4 drops of sample extract being 120 μL; within a time range of 10 to 15 minutes after the sample addition is completed, the colorimetric result of the immunochromatographic gold test strip is read through the observation window; if the reaction exposure time exceeds 15 minutes, any chromatographic colorimetric pattern presented in the observation window is considered an invalid result.

[0014] As one embodiment of the present invention, before determining the positive or negative status of norovirus GI and GII in the shellfish sample, the physical and biochemical validity of a single chromatography reaction process must be verified: when the control line shows a red reaction line, the chromatography test process is confirmed to be valid; if the control line area does not show a red reaction line through the observation window, the invalid result determination mechanism is triggered, and the system will determine that the test result is invalid.

[0015] As one embodiment of the present invention, after confirming that the control line shows a normal color development state, the infection status of the sample is classified and judged based on the color combination characteristics of the first detection line and the second detection line: if only one red reaction line is observed on the control line through the observation window, and no color band appears in the areas where the first detection line and the second detection line are located, it is determined that the shellfish sample does not contain norovirus GI and GII antigens; if two red bands are shown in the observation window, and these two reaction lines are respectively located in the physical arrangement areas of the control line and the first detection line, the system determines that the test result is positive for norovirus GI; if the two red bands are respectively corresponding to the control line and the second detection line, the test result is determined to be positive for norovirus GII; if three red bands are shown simultaneously in the observation window, that is, the control line, the first detection line and the second detection line all show color development reactions, the system will determine that the shellfish sample is jointly positive for norovirus GI and GII.

[0016] This invention provides a rapid immunochromatographic method for detecting norovirus in shellfish. It offers the following advantages: 1. This invention is based on the double-antibody sandwich method and the principle of immunochromatography. By mixing and coating two types of colloidal gold-labeled monoclonal antibodies on the conjugate pad, and setting corresponding first detection line, second detection line and quality control line on the nitrocellulose membrane, the sample reaction and result presentation are integrated into a single test card. This technical feature makes the detection process of shellfish samples free from the dependence on special laboratory equipment such as nucleic acid extractors, and shortens the single detection cycle to within 15 minutes, meeting the engineering needs of rapid preliminary screening at the grassroots level such as seafood markets and farms.

[0017] 2. This invention controls the stability of biochemical reactions by defining specific operating parameters and interpretation logic. Setting a temperature range of 15 to 30°C maintains the immunobinding activity of antibodies inside the reagent. Limiting the effective interpretation window to 10 to 15 minutes after sample addition avoids non-specific precipitation caused by moisture evaporation from the nitrocellulose membrane, reducing the risk of false positives. At the same time, combined with the pre-color development judgment mechanism of the quality control line, it effectively eliminates the problem of chromatography failure caused by insufficient sample addition or antibody inactivation, ensuring the objectivity of the test results.

[0018] 3. This invention constructs a methodological verification model based on parallel comparison. By introducing the chi-square test, the qualitative visual interpretation results are transformed into quantitative statistical evaluation indicators. By setting specific error observation variables and combining them with the corresponding theoretical chi-square critical values ​​for calculation and comparison, the statistical results confirm that the positive confirmation rate of this rapid detection method is not significantly different from that of the real-time fluorescence PCR technology specified in the national standard. From the data dimension, the accuracy and feasibility of the method of this invention in actual screening applications are confirmed. Attached Figure Description

[0019] Figure 1 This is a structural block diagram of an immunochromatographic colloidal gold chromatography system for rapid detection of norovirus in shellfish according to an embodiment of the present invention; Figure 2 A flowchart of an immunochromatographic method for rapid detection of norovirus in shellfish according to an embodiment of the present invention; Figure 3 This is a real-time fluorescence PCR amplification image of a GI-positive sample tested using the reference method in the method validation of this invention; Figure 4 This is a real-time fluorescence PCR amplification image of GII-positive samples tested using the reference method in the method validation of this invention; Figure 5 This is a real-time fluorescence PCR amplification image of the VIC channel of a negative sample tested by the reference method in the method validation of this invention; Figure 6 This is a real-time fluorescent PCR amplification image of the FAM channel of a negative sample tested by the reference method in the method validation of this invention. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] See attached document Figure 1 The present invention provides an immunochromatographic system for rapid detection of norovirus in shellfish, the system comprising: a test card and a sample extraction component.

[0022] The test card serves as the physical carrier for the immunochromatographic reaction. The outer surface of the test card has sample application wells and an observation window. The test card internally encapsulates an immunochromatographic colloidal gold test strip.

[0023] The sample extraction assembly is used to process shellfish samples before testing. The assembly includes a sample extraction tube, a sample extraction solution, and sampling tools. The sample extraction solution is loaded inside the sample extraction tube. A dropper is located at the top of the sample extraction tube and is in an openable / closable position. The sampling tools include a sampling rod and a pipette. The sampling rod is used to obtain shellfish samples in solid form. The pipette is used to obtain shellfish samples in liquid form.

[0024] See attached document Figure 2 Based on the aforementioned rapid immunogold chromatography system for detecting norovirus in shellfish, this invention provides a rapid immunogold chromatography method for detecting norovirus in shellfish, which may include the following steps: S100. Place the test card and the sample extraction tube containing the sample extraction solution in the operating environment. Control the temperature parameters of the operating environment to maintain the temperature of the test card and sample extraction solution within the range of 15 to 30°C. Remove the outer packaging of the test card and place the test card flat on a dry workbench.

[0025] S200. Select the appropriate sampling tool based on the physical morphology of the shellfish sample to obtain the sample. For solid shellfish samples, use a sampling rod to pick up a set mass of sample. For liquid shellfish samples, use a pipette to draw up a set volume of sample. Place the obtained shellfish sample into a sample extraction tube. Seal the sample extraction tube and perform a shaking operation to mix the shellfish sample with the sample extract to prepare the sample extract.

[0026] S300. Open the dropper at the top of the sample extraction tube. Vertically add the set volume of sample extract into the sample well of the test card through the dropper. The sample extract contacts the immunochromatographic gold chromatography strip through the sample well and undergoes capillary chromatography along the immunochromatographic gold chromatography strip.

[0027] S400. Within 10 to 15 minutes after adding the sample extract, observe the color development of the immunogold chromatography strip through the observation window of the test card. Based on the color development results of the corresponding area of ​​the immunogold chromatography strip, determine the positive or negative status of norovirus GI and GII types in the shellfish sample. Observations exceeding 15 minutes are considered invalid.

[0028] S500. Real-time fluorescence PCR technology is used as a reference method to simultaneously detect shellfish samples. The detection results of the method of this invention and the detection results of the reference method are obtained. A chi-square test is introduced to statistically analyze the obtained detection results, and the chi-square value is calculated to verify the difference in the positive confirmation rate between the two detection methods.

[0029] To further elaborate on the detection mechanism within the test card, in this embodiment, the immunochromatographic gold chromatography method of the present invention is mainly based on the principle of the double-antibody sandwich method to achieve qualitative detection of the target antigen. The basic principle of the double-antibody sandwich method is to use two different specific antibodies to recognize different epitopes on the antigen to be tested, thereby forming an antibody-antigen-antibody complex in space, similar to a sandwich, and achieving macroscopic signal amplification and color development through the aggregation of the labeled material.

[0030] Specifically, the immunochromatographic strip inside the test card includes a conjugation pad and a nitrocellulose membrane arranged sequentially along the liquid phase migration direction. The conjugation pad is coated with colloidal gold-labeled mouse anti-norovirus type GI monoclonal antibody and colloidal gold-labeled mouse anti-norovirus type GII monoclonal antibody. To achieve spatial separation and detection of different viral antigen types, the nitrocellulose membrane has a first detection line, a second detection line, and a control line. The first detection line is solid-phase coated with mouse anti-norovirus type GI monoclonal antibody, while the second detection line is solid-phase coated with mouse anti-norovirus type GII monoclonal antibody. The control line is solid-phase coated with goat anti-mouse IgG polyclonal antibody.

[0031] After the sample extract is added, it permeates and contacts the conjugate pad. At this point, the system will exhibit different reaction pathways depending on whether the sample solution contains the test antigen. As a typical positive reaction pathway, when the sample extract contains norovirus GI type test antigen, the free GI type test antigen specifically binds to the pre-coated colloidal gold-labeled mouse anti-norovirus GI type monoclonal antibody on the conjugate pad, forming a primary antigen-antibody complex. Similarly, when the sample extract contains GII type test antigen, the test antigen binds to the corresponding colloidal gold-labeled mouse anti-norovirus GII type monoclonal antibody, forming the corresponding primary antigen-antibody complex. Conversely, if the sample extract does not contain the test antigen, the corresponding colloidal gold-labeled antibody remains in a free state. As a preferred method, the antibody conjugation technology on the conjugate pad and the preparation process of the colloidal gold particles can be achieved by those skilled in the art using conventional electrostatic adsorption or chemical cross-linking processes. The specific preparation and coating methods are well-known techniques in the field and will not be elaborated here.

[0032] Along with the aforementioned binding reaction, the reaction solution, driven by capillary forces, migrates chromatographically along the nitrocellulose membrane in a direction away from the sample well. The reaction solution carries the formed primary antigen-antibody complex and unbound free colloidal gold-labeled antibody.

[0033] When the reaction solution migrates to the coated regions of the first and second detection lines, a double-antibody sandwich capture reaction is triggered. Taking the capture process of GI antigen as an example, the monoclonal antibody solid-coated on the first detection line specifically captures the primary antigen-antibody complex migrating in the liquid phase, thus forming a double-antibody sandwich complex. As the liquid phase continues to chromatographically, a large number of double-antibody sandwich complexes aggregate in the solid phase in the first detection line region. The aggregated colloidal gold particles increase the local concentration in this region, macroscopically causing the first detection line to show a red band. The GII antigen follows the same capture mechanism in the second detection line region, forming the corresponding complex and showing a corresponding red band. For the negative reaction pathway, since the sample does not contain the analyte antigen, a complex acting as a bridge cannot be formed in the liquid phase. The free colloidal gold-labeled antibody cannot be recognized and captured by the monoclonal antibody solid-coated on the first and second detection lines, thus directly bypassing the detection region, macroscopically manifested as a colorless detection line.

[0034] It should be noted that regardless of whether the sample contains the analyte antigen, after the reaction solution crosses the first and second detection lines, it will continue to migrate along the nitrocellulose membrane to the control line region. The remaining free colloidal gold-labeled antibody in the reaction solution undergoes non-competitive specific binding with the goat anti-mouse IgG polyclonal antibody on the control line, forming a conjugate product of the polyclonal antibody and the free gold-labeled antibody. A large amount of colloidal gold-labeled antibody is retained by the solid phase of the control line, resulting in a stable red band on the control line. This red band serves as an indicator, typically used to show that the reaction solution volume is sufficient and the chromatography process has completely covered the entire detection area, thus confirming the physical validity of a single chromatographic test.

[0035] In this embodiment, based on the aforementioned system architecture, the immunochromatographic gold test strip serves as a biochemical reaction carrier. The layout of its internal structure and the reagent coating method help improve the chromatographic stability and the reliability of the detection results.

[0036] The immunochromatographic gold test strip is entirely attached to a support base plate for structural support. Along the direction of capillary migration of the sample extract, the internal structure of the immunochromatographic gold test strip sequentially includes a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorbent pad. The sample pad is positioned directly below the sample well of the test card and is used for preliminary filtration of large particulate impurities in the sample extract and to buffer the initial flow rate of the buffer phase. The absorbent pad is located at the physical end of the nitrocellulose membrane and is used to provide continuous capillary driving force and absorb excess waste liquid after the chromatography reaction. The specific material selection and assembly connection method of the support base plate, sample pad, and absorbent pad can be implemented by those skilled in the art according to conventional chromatography test strip manufacturing processes; its basic assembly structure is well-known in the field and will not be described in detail here.

[0037] As the pre-reaction region in the double-antibody sandwich method, the conjugate pad is typically made of glass fiber, whose three-dimensional mesh structure facilitates the attachment and release of labeled antibodies. The conjugate pad is uniformly coated with colloidal gold-labeled mouse anti-norovirus type GI monoclonal antibody and colloidal gold-labeled mouse anti-norovirus type GII monoclonal antibody. Preferably, colloidal gold particles, as chromogenic markers, have high electron density and exhibit a visible red color when aggregated in large quantities. Since the colloidal gold-labeled antibodies exist primarily on the conjugate pad in a physically adsorbed and dried state, the labeled antibodies can be rapidly resoluble and released with the liquid phase when the liquid sample passes through the conjugate pad, thus ensuring thorough mixing and contact with the sample extract.

[0038] The nitrocellulose membrane, receiving the reconstituted reaction solution, is the region that specifically captures the antigen and presents the final colorimetric result. The nitrocellulose membrane has a uniform three-dimensional microporous structure, providing a physical channel for the smooth chromatographic migration of the antigen-antibody complex. On the surface of the nitrocellulose membrane, along the liquid flow direction, are sequentially arranged a first detection line, a second detection line, and a control line. Based on the logic of determining the validity of the chromatographic detection, the control line must be located downstream of the first and second detection lines. This structural design ensures that the reaction solution can only reach the control line and trigger color development after completely passing through all detection areas, thus accurately indicating whether the overall chromatographic process has been effectively completed. These three coating lines maintain a predetermined spatial spacing to prevent interference and are all directly opposite the viewing window on the test card casing.

[0039] The first detection line is coated with a mouse anti-norovirus GI monoclonal antibody using a streak-spray method. This antibody specifically targets and captures the specific antigenic epitopes of norovirus GI. The second detection line is coated with a mouse anti-norovirus GII monoclonal antibody, used to independently recognize and capture norovirus GII antigens. Based on system quality control requirements, the control line is coated with a goat anti-mouse IgG polyclonal antibody. From an immunological binding perspective, this polyclonal antibody does not react with norovirus antigens but specifically recognizes and binds to mouse-derived monoclonal antibodies from the binding pad using species specificity. Through flow rate adjustment and drying control of the spraying equipment, these specific antibodies form well-defined band structures on the nitrocellulose membrane and are firmly adsorbed onto the inner surface of the micropores. This solid-phase coating method reduces the risk of antibody detachment or physical migration during liquid-phase washing, helping to maintain the morphological integrity of the chromatographic bands.

[0040] To achieve high-efficiency detection using the aforementioned immunochromatographic system, this embodiment further discloses the preparation process of the immunochromatographic colloidal gold test strip and the formulation of the sample extraction solution.

[0041] In the preparation of the immunochromatographic gold test strip, the gold-labeled antibody was prepared by the trisodium citrate reduction method to prepare colloidal gold particles. Subsequently, mouse anti-norovirus GI monoclonal antibody and mouse anti-norovirus GII monoclonal antibody were electrostatically adsorbed and coupled to the colloidal gold particles under suitable pH conditions. The coupled gold-labeled antibody was blocked with bovine serum albumin (BSA), mixed in a predetermined ratio, and sprayed onto a glass fiber conjugation pad 131. After freeze-drying, it was ready for use. For the coating of the nitrocellulose membrane 132, mouse anti-norovirus GI monoclonal antibody, mouse anti-norovirus GII monoclonal antibody, and goat anti-mouse IgG polyclonal antibody at concentrations of 1.0–2.0 mg / mL were quantitatively streaked onto the first detection line 1321, the second detection line 1322, and the control line 1323 on the membrane surface using a streaking device. The membrane was then placed in an oven for drying and fixation.

[0042] The sample extraction solution 22 not only serves as a physical suspension but also needs to have lysis and environmental buffering functions. As a preferred formulation system, the base solution of the sample extraction solution 22 is phosphate buffered saline (PBS, pH 7.2-7.4), which contains 0.1%-1.0% by volume of a nonionic surfactant (such as Tween-20 or Triton X-100) to dissociate the tissue mucus of the shellfish sample and release virus particles; it also contains 0.5%-2% bovine serum albumin (BSA) and 0.8%-1.0% by mass of sodium chloride (NaCl) to maintain the conformational stability of the released antigen and reduce non-specific background binding during chromatography.

[0043] Regarding the environmental and reagent preparation in step S100, in this embodiment, in order to reduce the interference of ambient temperature and physical placement on the colloidal gold chromatography reaction, the present invention standardizes the pre-preparation process for detection reagents and environment.

[0044] In practice, the refrigerated or cryogenically stored test card and the sample extraction tube containing the sample extraction solution are transferred to the operating environment. The test card and sample extraction solution are allowed to stand to allow their internal physical temperature to match the operating environment temperature. During the pre-preparation stage, the temperature of the test card and sample extraction solution needs to be restored to room temperature (15°C to 30°C) through natural heat exchange.

[0045] In one embodiment of the present invention, the determination of the above-mentioned temperature threshold range is based on the fundamental thermodynamic laws of immunological reactions. 15°C is typically set as the lower limit temperature for maintaining the kinetic activity of the antigen-antibody binding reaction; temperatures below this level may lead to a slowdown in molecular thermal motion, causing a decrease in the binding reaction rate and an increase in the viscosity of the chromatographic liquid phase. 30°C serves as the upper limit temperature to prevent structural thermal denaturation of the internally coated protein antibodies, and is also used to control excessive evaporation of the reaction solution during chromatography. Restoring the temperature of the detection reagent to the aforementioned defined room temperature range helps maintain the thermodynamic binding activity of the antibodies coated inside the test card, reduces the risk of slow capillary flow rate due to low temperatures, and also helps control protein denaturation caused by high temperatures.

[0046] Once the test reagent has completed its temperature equilibration process, the external packaging of the test card can be removed. The test card is typically sealed inside an aluminum foil bag with moisture-proof properties. Based on the principle of physical condensation, premature opening before temperature equilibration may cause gaseous moisture in the operating environment to condense and adhere to the surface of the nitrocellulose membrane inside the test card due to temperature differences. Condensed moisture may affect the three-dimensional microporous structure of the nitrocellulose membrane, thereby increasing the risk of uneven distribution of the chromatographic liquid phase. Regarding the specific material selection and packaging process of the aforementioned moisture-proof packaging bag, those skilled in the art can implement it according to conventional in vitro diagnostic reagent packaging specifications. The packaging materials and sealing technology are well-known in the field and will not be elaborated upon here.

[0047] After unpacking the reagents, place the test card flat on the worktable. This flat placement requires the physical support surface of the test card to be level. Maintaining a level test card reduces the influence of gravity on the flow rate distribution in liquid chromatography capillary chromatography, facilitating the uniform propagation of subsequently added sample extract within the nitrocellulose membrane. As a preferred method, keeping the worktable surface dry and clean reduces the risk of free water droplets or microparticles from the external environment seeping in through the sample application port or observation window of the test card, thereby contaminating the internal immunochromatographic microenvironment.

[0048] Regarding the sample extraction and pretreatment in step S200, in this embodiment, the pretreatment quality of shellfish samples is closely related to the release efficiency of the target viral antigen. Specifically, the cap of the sample extraction tube needs to be opened to allow the sample to be transferred in. The system provides differentiated sampling methods for sample matrices with different physical forms.

[0049] When the sample to be tested is solid shellfish tissue, use a sampling stick to pick up 100 mg of solid sample. As a preferred method, the macroscopic volume reference standard for this mass is roughly equivalent to the size of a standard match head. Setting this specific sampling amount aims to balance the amount of antigen acquired with the matrix effect of the sample extract. Excessive solid tissue may result in excessively high viscosity of the mixed liquid phase, increasing the risk of subsequent blockage of the micropores inside the immunochromatographic gold test strip; while insufficient sampling amount may result in the concentration of the target antigen falling below the reagent's limit of detection.

[0050] If the sample to be tested is a liquid shellfish sample, use a pipette to draw a volume of 50 μL of liquid sample. This quantitative volume parameter ensures a suitable liquid-phase dilution ratio with the sample extract pre-loaded in the sample extraction tube. A reasonable dilution ratio helps maintain the pH and ionic strength of the mixture system, placing it in a biochemical state conducive to subsequent specific immunobinding.

[0051] After sample collection, the obtained solid or liquid shellfish samples are transferred to the sample extraction solution in the sample extraction tube. The sample extraction tube is then tightly sealed and subjected to physical agitation. In the general pretreatment principle of immunoassay, the sample extraction solution typically contains appropriate amounts of surfactants and buffer salts. The surfactants are used to dissociate mucoproteins and cellular structures in the shellfish samples, while the buffer system is used to stabilize the molecular conformation of free antigens. Combined with external physical agitation, the above-mentioned biochemical lysis and elution processes can be significantly accelerated.

[0052] To ensure full antigen release, vigorous shaking and mixing should be maintained for one minute. Continuous physical shaking helps disrupt the local tissue aggregation morphology of the shellfish sample, promoting the release of norovirus particles attached to or encapsulated within the sample, and their uniform dispersion in the liquid carrier, thus preparing a homogeneous sample extract. This time threshold is set primarily based on a comprehensive consideration of on-site operational efficiency and extraction effectiveness. Too short a shaking time may lead to insufficient tissue dissociation, limiting antigen release; while prolonged shaking not only reduces the overall timeliness of rapid on-site testing but may also cause excessive foaming in the sample extract system, interfering with subsequent quantitative sample addition accuracy. Regarding the specific chemical composition of the sample extract, those skilled in the art can use conventional antigen elution formulations containing surfactants and buffer salts; the basic component ratios and preparation processes are well-known in the field and will not be elaborated upon here.

[0053] Regarding the sample addition and chromatographic reaction control in the aforementioned steps S300 and S400, in this embodiment, precise sample addition and strict reaction time control help ensure the objectivity and accuracy of the chromatographic results.

[0054] After obtaining a homogeneous sample extract, open the dropper at the top of the sample extraction tube. Align the dropper with the sample well on the test card surface and, while maintaining a vertical position, add 4 drops of sample extract into the well. The total volume of these 4 drops is 120 μL. As a preferred method, using a vertical dropping posture helps to form a continuous droplet of standard volume solely by gravity, preventing droplets from adhering to or flowing off the outer wall of the dropper, thereby improving the consistency of the single drop volume. The 120 μL sample volume parameter is designed to match the liquid phase capacity of the internal structure of the immunochromatographic gold test strip. Insufficient liquid input may lead to a decrease in the capillary driving force of the system, causing the reaction solution to stagnate and fail to completely penetrate all downstream specific detection and quality control areas. Conversely, if the sample volume is overloaded, excess liquid phase may easily escape from the preset microporous chromatography channel and overflow onto the surface of the nitrocellulose membrane, potentially causing nonspecific background color development.

[0055] After the addition operation is completed, the sample extract comes into contact with the structure below the well. Based on the conventional immunochromatographic kinetics, the sample extract, as the mobile phase, is propelled by capillary forces within the micropores of the nitrocellulose membrane, migrating chromatographically along the stationary phase of the immunogold immunochromatographic test strip towards the observation window. During this fluid propulsion process, the norovirus target antigen carried in the mobile phase comes into full contact with the pre-coated specific antibody molecules within the test strip, thereby triggering a specific immune binding reaction during migration.

[0056] To obtain reliable test results, it is necessary to control the timing of colorimetric interpretation. Specifically, the colorimetric results of the immunochromatographic test strip should be read through the observation window within 10 to 15 minutes after sample addition. This observation window is primarily based on the capillary flow rate of the porous membrane and the antigen-antibody binding kinetics. In general chromatographic physics, 10 minutes is typically the lower limit of the time required for the liquid reaction stream to flow through all test and control lines and for the aggregated colloidal gold particles to reach a visually recognizable concentration. Before reaching this time, the nitrocellulose membrane background area of ​​the test strip often retains a large amount of unwashed free colloidal gold, resulting in a slightly reddish background that can easily interfere with the visual interpretation of the true bands.

[0057] Regarding the upper limit of time control, any chromatographic color pattern appearing in the observation window after the reaction exposure time exceeds 15 minutes is considered an invalid result. From the perspective of physicochemical changes in the chromatographic system, as the standing time continues to extend, moisture inside the micropores of the nitrocellulose membrane will continuously evaporate into the open environment. This one-way loss of moisture directly leads to a gradual increase in the concentration of buffer salt ions in the local area. This environmental change may promote the biological precipitation and non-specific aggregation of free non-specific proteins or unbound colloidal gold. This physical evaporation and precipitation phenomenon increases the possibility of inducing false red bands in the coating area where there is no corresponding target antigen, thereby increasing the risk of interpreting false positive results.

[0058] In this embodiment, to ensure the reliability of the detection conclusions, the system needs to verify the physical and biochemical validity of a single chromatographic reaction process before interpreting the positive or negative status of the target virus.

[0059] Based on the internal layout logic of immunochromatography, the control line is physically located at the end of the nitrocellulose membrane chromatography path. After flowing through the first and second detection lines, the liquid sample continues to migrate towards the control line. At this point, the liquid phase carries free colloidal gold-labeled mouse monoclonal antibody derived from the binding pad. The solid phase on the control line is coated with goat anti-mouse IgG polyclonal antibody. This polyclonal antibody utilizes a conventional species-specific recognition mechanism to non-competitively bind to the aforementioned mouse-derived monoclonal antibody, causing colloidal gold particles to aggregate in large quantities in the control line region, resulting in color development. For the species-specific binding mechanism of antigen and antibody, those skilled in the art can refer to basic immunological theory; the reaction principle is well-known in the field and will not be elaborated further here.

[0060] As a preferred method, observing the color development of the control line through the observation window of the test card constitutes the preliminary logical judgment benchmark for the entire system's result interpretation. When a red reaction line is observed on the control line, this color development indicates, at the physical level, that the amount of sample extract added to the sample well is sufficient, and that the chromatographic phase has completely penetrated the previous specific detection area. At the biochemical level, this phenomenon indicates that the colloidal gold-labeled antibody and the solid-phase polyclonal antibody inside the reagent maintain the expected immunogenicity. In actual visual interpretation, as long as a visually identifiable red band appears in the control line area, regardless of its intensity, it should be considered as meeting the color development standard. The satisfaction of the above conditions confirms the validity of this chromatographic test process, thus supporting the operator to further interpret the positive or negative result of the target antigen based on the color development of the test line.

[0061] Conversely, if the control line area is not shown in the observation window, the invalid result determination mechanism is triggered. The absence of color development in the control line may be due to insufficient sample volume leading to interruption of chromatography, physical damage to the microporous structure of the nitrocellulose membrane, or inactivation of internal antibody proteins caused by improper reagent storage. In this abnormal state, since it cannot be confirmed whether the liquid phase flows normally through the detection area or whether the reaction system is functioning properly, the system will determine the test result to be invalid. At this time, regardless of whether the first or second detection line shows a red band, this color feature loses its diagnostic significance. The standard procedure for handling this type of invalid test is to discard the current test card and extract a new shellfish sample to re-execute the complete testing procedure.

[0062] Furthermore, in this embodiment, once the control line is confirmed to show a normal color development state, the system will classify and judge the infection status of the sample based on the color combination characteristics of the first detection line and the second detection line.

[0063] For negative results, if only a single red reaction line is observed through the observation window of the test card at the control line, while no colored bands appear in the areas containing the first and second test lines, then the shellfish sample is determined to be free of norovirus GI and GII antigens. This visual presentation corresponds to the physical state where the sample extract lacks the corresponding target, causing the free colloidal gold-labeled antibody to pass directly through the detection area without being captured by the solid phase. In practice, to eliminate interference from non-specific binding, a negative result typically requires that the nitrocellulose membrane background within the observation window remain clear and its original color intact.

[0064] When a single type of viral antigen is present in the sample, the system exhibits a dual-line colorimetric characteristic. Specifically, if two red bands appear in the observation window, and these two reaction lines are located in the physical arrangement areas of the control line and the first test line, respectively, the system determines the test result to be positive for norovirus GI. This result indicates that the sample matrix contains GI antigen and has successfully mediated the formation of a local double-antibody sandwich complex. Similarly, if the two red bands appear to correspond to the control line and the second test line, respectively, the test result is determined to be positive for norovirus GII, indicating the independent presence of GII antigen in the sample.

[0065] Besides a single infection state, when two types of viral antigens are present in a sample simultaneously, the system will exhibit a triple-line simultaneous display characteristic. In this case, if three red bands appear simultaneously in the observation window—that is, the control line, the first detection line, and the second detection line all show colorimetric reactions—the system will determine that the shellfish sample is co-positive for norovirus types GI and GII. This triple-line simultaneous display confirms that the specific capture reactions of the two types are completed independently and effectively within their respective solid-phase regions.

[0066] As a preferred approach, the initial concentrations of viral antigens enriched in different shellfish samples vary physically, resulting in varying numbers of colloidal gold particles trapped in the first or second detection line region. This manifests as different shades of red bands on a macroscopic visual scale. In the actual visual assessment, as long as a visually identifiable red mark appears in the corresponding detection line region, regardless of its color intensity or line width, it should be considered as indicating specific immune binding and a positive result. Setting such a broad colorimetric acceptance standard helps reduce the risk of missed detections in low-concentration antigen samples in rapid testing scenarios at the grassroots level.

[0067] Regarding the methodological verification and reference consistency analysis in step S500 above, in order to verify the accuracy and reliability of the immunochromatographic colloidal gold chromatography method described in this invention in the detection of actual shellfish samples, this embodiment conducted a parallel comparison experiment in accordance with GB4789.42-2025 "National Food Safety Standard for Microbiological Examination of Food - Norovirus Examination".

[0068] Experimental design and reference methods: The norovirus (GI / GII) nucleic acid detection kit developed by Huiduda Technology Co., Ltd. was used, with real-time fluorescence PCR technology as the reference method. The judgment criteria were defined as follows: a positive result was defined as the presence of an amplification curve and a Ct value < 40; a negative result was defined as the absence of a Ct value or a Ct value ≥ 40. A total of 20 shellfish norovirus GI-positive samples, 20 GII-positive samples, and 20 shellfish norovirus-negative samples were obtained. These 60 samples were simultaneously tested using both the reference method and the immunochromatographic gold chromatography method of this invention to determine the compatibility of the two methods.

[0069] Experimental results data: Comparison results of GI positive samples: Reference Figure 3 , Figure 3 The real-time fluorescence PCR amplification curves for GI-positive samples tested by the reference method are shown, where the vertical axis represents fluorescence value (540-560 nm) and the horizontal axis represents cycle number. According to Table 1, of the 20 GI-positive samples (Ct values ​​ranging from 29.5 to 39.0) detected by the reference method, 18 were positive (+) and 2 were negative (-) when tested using the immunogold immunoassay of this invention.

[0070] Table 1. Comparison of results between the reference method and immunochromatographic assay for GI-positive samples. Comparison results of GII positive samples: Reference Figure 4, Figure 4 The real-time fluorescence PCR amplification curves for GII-positive samples tested using the reference method are shown, where the vertical axis represents fluorescence value (465-510 nm) and the horizontal axis represents cycle number. Combined with the data in Table 2, it can be seen that of the 20 GII-positive samples (Ct values ​​ranging from 27.1 to 39.8) detected by the reference method, 17 were positive (+) and 3 were negative (-) when tested using the immunogold immunoassay of this invention.

[0071] Table 2. Comparison of results between the reference method and immunochromatographic assay for GII-positive samples. Negative sample comparison results: Reference Figure 5 and Figure 6 , Figure 5 and Figure 6 The real-time fluorescence PCR amplification curves of negative samples tested using the reference method in the VIC and FAM channels are shown respectively. Figure 5 In the graph, the vertical axis represents fluorescence intensity (540-590 nm), and the horizontal axis represents the cycle number. Figure 6 In the figure, the vertical axis represents fluorescence value (465-510nm), and the horizontal axis represents cycle number. According to the data in Table 3, out of 20 negative samples, the immunogold assay of this invention detected 18 negative (-) samples and 2 false positive (+) samples.

[0072] Table 3. Comparison of results of negative samples determined by the reference method and immunochromatographic gold chromatography. (Note: "-" represents negative, "+" represents positive, and "ND" represents not detected) Methodological consistency analysis conclusions: The overall experimental results show that: 2 samples were confirmed as GI positive by the immunogold assay to be confirmed but negative by the reference method; 3 samples were confirmed as GII positive but negative by the reference method; and 2 samples were confirmed as negative but positive by the reference method. The chi-square test was used for consistency analysis, and the statistical significance of the differences was calculated. It is 0.57.

[0073] Due to the calculation Strictly meet The statistical limits were reached. This data fully demonstrates that the positive confirmation rate of the norovirus immunochromatographic colloidal gold chromatography method provided by this invention is not significantly different from that of the real-time fluorescence PCR reference method specified in the national standard within the 95% confidence interval. It fully meets the requirements for on-site rapid testing, thereby greatly shortening the testing cycle and reducing equipment dependence while maintaining high accuracy.

[0074] Regarding the methodological verification and reference consistency analysis in step S500, in this embodiment, in order to verify the accuracy and reliability of the above-mentioned immunochromatographic colloidal gold chromatography method in the actual detection of shellfish samples, the system introduces a parallel comparison verification model.

[0075] The implementation of this validation model presupposes the construction of a reasonable test sample set. Specifically, a validation set of 60 shellfish samples was configured, consisting of: 20 positive samples known to be infected with norovirus GI, 20 positive samples known to be infected with norovirus GII, and 20 negative samples without norovirus. This reasonable configuration of positive and negative samples and genotyping helps to comprehensively evaluate the specificity and sensitivity of the detection method of this invention when facing different target states.

[0076] After establishing the sample set, the validation model adopted the technical requirements specified in the national standard GB 4789.42-2025 as the qualitative reference standard. In practice, a commercially available norovirus nucleic acid detection kit was selected, and the aforementioned 60 validation samples were simultaneously tested in parallel using real-time fluorescence PCR technology. For the nucleic acid extraction, primer and probe design, and temperature cycling procedures involved in real-time fluorescence PCR technology, those skilled in the art can perform routine operations according to the aforementioned national standard; the basic molecular biological amplification principles are well-known in the field and will not be elaborated upon here.

[0077] To establish a quantitative and objective comparison benchmark, the system set the positive / negative determination boundary for the real-time fluorescence PCR amplification cycle threshold based on the performance parameters of the reference kit. Specifically, the amplification cycle threshold for real-time fluorescence PCR was defined as follows: Based on the objective requirements of the disclosure and verification standards, the criteria for determining the reference method are defined as follows: when the amplification curve of the tested sample is normal and the corresponding cycle threshold is met... When the reference test result for the shellfish sample is positive, the result is determined to be negative; conversely, when the sample does not produce an effective amplification curve, or its cycle threshold is met, the result is determined to be negative. If the result is negative, the reference test result for the shellfish sample is determined to be negative.

[0078] The definition of the aforementioned cycle threshold constant, setting 40 amplification cycles as the critical point, is primarily based on the substrate consumption patterns and the risk of non-specific amplification in the PCR amplification system. In conventional nucleic acid exponential amplification processes, the fluorescence signal increase that appears after more than 40 cycles is usually due to primer dimer accumulation or non-specific extension of trace amounts of background nucleic acids, and does not possess true diagnostic significance for the target pathogen. By establishing this objective cycle threshold criterion, the validation model provides a reliable data reference benchmark for subsequent statistical analysis and evaluation of the positive confirmatory rate of the immunochromatographic method.

[0079] After completing the above parallel comparisons, in this embodiment, in order to quantitatively evaluate the detection consistency between the immunochromatographic colloidal gold chromatography method and the reference method, the system performed statistical tests on the aforementioned verification sample comparison data.

[0080] After completing the parallel comparison experiment, the detection results of the immunochromatographic colloidal gold chromatography method of this invention and the real-time fluorescence PCR reference method were obtained. The system recorded the number of samples where the two methods were inconsistent and defined the corresponding error observation variables. Specifically, the number of samples that were determined to be GI positive by the method of this invention but negative by the reference method was defined as the GI false positive number. The actual observations based on the validation set are The number of samples that are determined to be GII positive by the method of this invention but are negative by the reference method is defined as the GII false positive number. Its actual observed value is Similarly, the number of samples that are determined to be negative by the method of this invention but positive by the reference method is defined as the false negative number. Its actual observed value is .

[0081] To verify the difference in positive confirmation rates between the two methods, the chi-square test from consistency analysis is introduced as a statistical evaluation tool. In general statistical principles, the chi-square test is commonly used to compare the degree of deviation in the frequency distributions of two categorical variables, and is suitable for analyzing the consistency between two qualitative detection methods. The chi-square test statistic is defined as follows: The error observation variables recorded above (including the number of false positives (NFP) for GI, the number of false positives (NFP) for GI and GII, and the number of false negatives (NFN)) along with the corresponding true positive and true negative observation values ​​are used as input parameters for the statistical contingency table and substituted into the chi-square test calculation model. Based on the observed false positive and false negative data, the chi-square test statistic calculated statistically is: .

[0082] In hypothesis testing, it is necessary to establish statistical boundaries for determining whether a significant difference exists. As a preferred approach, the confidence interval for this consistency comparison is set at 95%, i.e., a significance level threshold of 0.05. Since this comparison involves cross-comparison of negative and positive states, forming a typical 2×2 contingency table structure in statistics, its statistical degrees of freedom are defined as 1. Combining the aforementioned significance level and degrees of freedom parameters, the theoretical chi-square critical value obtained from the table is 3.84.

[0083] According to the basic criteria of the chi-square test, the chi-square test statistic is satisfied when it is strictly less than the theoretical chi-square critical value. This indicates that the difference in results produced by the two detection methods is attributed to random error, and that they are statistically equivalent. Substituting the actual calculated data from the aforementioned experiment into this judgment logic, due to the chi-square test statistic... The system yielded a comparison result of 0.57 < 3.84.

[0084] The data comparison shows that the actual calculated chi-square test statistic is strictly less than the set theoretical chi-square critical value. Based on this statistical analysis logic, it can be deduced that the positive confirmation rate of the norovirus immunochromatographic rapid detection method provided by this invention and the reference method specified in the national standard are not significantly different within the 95% confidence interval. The above verification data and statistical conclusions objectively demonstrate that this system and method help meet the accuracy requirements for on-site screening of norovirus in shellfish samples and have engineering feasibility for replacing complex nucleic acid testing for preliminary rapid screening.

Claims

1. A rapid immunogold chromatography method for detecting norovirus in shellfish, applied to an immunogold chromatography system, the immunogold chromatography system comprising a test card and a sample extraction component, the sample extraction component comprising a sampling tool and a sample extraction tube loaded with sample extraction solution, the test card having a sample application hole and an observation window on its surface, and an immunogold chromatography test strip encapsulated inside the test card, characterized in that... Includes the following steps: Place the test card and the sample extraction tube containing the sample extraction solution in the operating environment to equilibrate the temperature; Based on the physical morphology of the sample to be tested, the corresponding sampling tool is selected to obtain the shellfish sample. The shellfish sample is transferred to the sample extraction tube, sealed, and a physical shaking operation is performed to mix the shellfish sample with the sample extract to prepare the sample extract. The sample extract is added dropwise into the sample well of the test card. The sample extract comes into contact with the immunochromatographic gold test strip and undergoes capillary chromatography along the immunochromatographic gold test strip, triggering a double antibody sandwich capture reaction. The color development of the immunochromatographic gold test strip is observed through the observation window of the test card to determine the positive or negative status of norovirus GI and GII types in the shellfish sample.

2. The rapid immunochromatographic method for detecting norovirus in shellfish according to claim 1, characterized in that, The internal structure of the immunochromatographic gold test strip includes, in sequence, a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorbent pad. The conjugate pad is mixed and uniformly coated with colloidal gold-labeled mouse anti-norovirus type GI monoclonal antibody and colloidal gold-labeled mouse anti-norovirus type GII monoclonal antibody. The surface of the nitrocellulose membrane is sequentially provided with a first detection line, a second detection line, and a quality control line along the liquid phase flow direction. The first detection line is coated with mouse anti-norovirus type GI monoclonal antibody, the second detection line is coated with mouse anti-norovirus type GII monoclonal antibody, and the quality control line is coated with goat anti-mouse IgG polyclonal antibody.

3. The rapid immunochromatographic method for detecting norovirus in shellfish according to claim 2, characterized in that, The preparation process of the immunochromatographic gold test strip includes: Colloidal gold particles were prepared using the trisodium citrate reduction method. The mouse anti-norovirus type GI monoclonal antibody and the mouse anti-norovirus type GII monoclonal antibody were electrostatically adsorbed and coupled to the colloidal gold particles under suitable pH conditions. After being conjugated, the gold-labeled antibodies were blocked with bovine serum albumin, mixed in a set ratio, and sprayed onto a glass fiber conjugate pad. After being freeze-dried, they were ready for use. A membrane scribing instrument was used to quantitatively scribble mouse anti-norovirus type GI monoclonal antibody, mouse anti-norovirus type GII monoclonal antibody, and goat anti-mouse IgG polyclonal antibody at concentrations of 1.0–2.0 mg / mL onto the first detection line, the second detection line, and the quality control line area on the membrane surface, respectively. The membrane was then placed in an oven to dry and fix.

4. The rapid immunochromatographic method for detecting norovirus in shellfish according to claim 1, characterized in that, The sample extract is used to dissociate the tissue mucus of shellfish samples and release virus particles. The formulation of the sample extract includes: The base solution of the sample extract is a phosphate buffer solution with a pH of 7.2-7.

4. The base solution contains 0.1%-1.0% by volume of nonionic surfactant, 0.5%-2% of bovine serum albumin, and 0.8%-1.0% by mass of sodium chloride.

5. The rapid immunochromatographic method for detecting norovirus in shellfish according to claim 1, characterized in that, In the steps of placing the test card and the sample extraction tube containing the sample extract in the operating environment to equilibrate the temperature, and preparing the sample extract: Control the temperature parameters of the operating environment to keep the temperature of the test card and the sample extract solution within the range of 15 to 30°C; After the test reagent has completed the temperature equilibrium process, remove the external packaging structure of the test card and place the test card flat on the worktable; When the sample to be tested is a solid shellfish tissue, a sampling rod with a mass of 100 mg is used as the sampling tool to pick up a solid sample. When the sample to be tested is a shellfish sample in liquid form, a pipette used as the sampling tool is used to draw a liquid sample with a volume of 50 μL. The physical oscillation operation involves vigorous oscillation to mix the materials, and the vigorous oscillation and mixing time must be maintained at 1 minute.

6. The rapid immunochromatographic method for detecting norovirus in shellfish according to claim 1, characterized in that, The top of the sample extraction tube is equipped with a dropper that can be opened and closed. Turn on the dropper, align the dropper with the sample well on the surface of the test card, and add 4 drops of sample extract into the sample well while keeping it vertical. The total volume of the 4 drops of sample extract is 120 μL. Within 10 to 15 minutes after the sample addition is completed, the colorimetric result of the immunochromatographic gold test strip is read through the observation window; Any chromatographic color pattern appearing in the observation window after the reaction exposure time exceeds 15 minutes is considered an invalid result.

7. The rapid immunochromatographic method for detecting norovirus in shellfish according to claim 2, characterized in that, Before determining the positive or negative status of norovirus GI and GII in the shellfish samples, the physical and biochemical validity of the single chromatographic reaction process must be verified: When the control line shows a red reaction line, the validity of this chromatography test is confirmed. If the observation window confirms that no red reaction line appears in the quality control line area, the invalid result determination mechanism is triggered, and the system will determine that the test result is invalid.

8. The rapid immunochromatographic method for detecting norovirus in shellfish according to claim 7, characterized in that, Once the control line is confirmed to show normal color development, the infection status of the sample is classified and judged based on the color combination characteristics of the first detection line and the second detection line: If only one red reaction line is observed through the observation window on the quality control line, and no colored bands appear in the areas where the first detection line and the second detection line are located, then it is determined that the shellfish sample does not contain norovirus type GI and type GII antigens. If two red bands appear in the observation window, and these two reaction lines are located in the physical arrangement area of ​​the quality control line and the first detection line, respectively, the system determines that the test result is positive for norovirus GI type. If the two red bands appear to correspond to the control line and the second test line respectively, the test result is determined to be positive for norovirus GII type. If three red bands appear simultaneously in the observation window, that is, the control line, the first detection line and the second detection line all show a color reaction, the system will determine that the shellfish sample is positive for both norovirus GI and GII types.

9. The rapid immunochromatographic method for detecting norovirus in shellfish according to claim 1, characterized in that, The method further includes methodological validation and reference consistency analysis steps, which include setting reference baseline conditions: Construct a validation set that includes positive samples known to be infected with norovirus GI, positive samples known to be infected with norovirus GII, and negative samples that do not contain norovirus; The validation set was simultaneously measured using real-time fluorescence PCR as a reference method, and the judgment criteria were defined as follows: A positive result is defined as the presence of an amplification curve and a Ct value < 40; a negative result is defined as the absence of a Ct value or a Ct value ≥ 40.

10. The rapid immunochromatographic method for detecting norovirus in shellfish according to claim 9, characterized in that, After determining the results based on the aforementioned reference standard method, a consistency statistical test was performed: Record the number of samples where the two methods are inconsistent, and define the corresponding error observation variables; The error observation variables recorded above, together with the corresponding true positive and true negative observation values, are used as input parameters for the statistical contingency table. They are then substituted into the chi-square test calculation model, and the chi-square test statistic is obtained through statistical calculation. When the calculated chi-square test statistic is strictly less than the theoretical chi-square critical value of 3.84, it indicates that the difference in results produced by the two detection methods is attributed to random error, and the two are statistically equivalent.

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