Goose astrovirus detection card and application thereof

Abstract

The invention relates to the field of animal medicine detection, in particular to a goose astrovirus detection card and application thereof. The invention discloses a goose astrovirus detection card which comprises a bottom plate and immunochromatography test paper located on the bottom plate, and the immunochromatography test paper comprises a sample pad, a combination pad, a detection pad and a water absorption pad in the chromatography direction of a sample to be detected; the combination pad contains a goose astrovirus monoclonal antibody, and the goose astrovirus monoclonal antibody is labeled with a marker. The goose astrovirus detection card has the advantages of being rapid, convenient and accurate in detection and free of operation of professional experimenters.

Description

Technical Field

[0001] The present specification relates to the field of animal medical testing, and in particular to a goose astrovirus detection card and its application. Background Art

[0002] Since 2017, goose farms in many provinces in China have discovered and reported a waterfowl infectious disease with fatal visceral and joint gout as the main clinical features. The disease is common in goslings and is highly lethal, with a mortality rate of 30% to 50%. The dead geese showed white and swollen kidneys, and a large amount of urate deposits in the thoracoperitoneum, heart surface, and ureters, causing serious economic losses to the goose industry. After pathogen detection, researchers have successively isolated and identified a new type of astrovirus from clinical samples in various regions, which is genetically different from the reported avian astrovirus and named it Goose-origin astrovirus (GoAstV).

[0003] Goose astrovirus (GoAstV) is a non-enveloped, single-stranded positive-strand RNA virus. GoAstV particles have an icosahedral symmetric structure, with a diameter between 28nm-30nm and a genome length between 7148-7299bp. GoAstV is one of the main pathogens causing fatal gout in geese. The main clinical manifestations are depression, lying on the ground, and reduced feeding. Autopsy shows severe urate deposition in internal organs and joint cavities, which brings huge economic losses to the breeding industry. It is currently one of the important epidemics that endanger the world's goose breeding industry. Because avian astrovirus can have multiple hosts and can spread horizontally, vertically, and across species; at the same time, the genetic recombination of avian astrovirus, the ability to adapt to new hosts, and the continuous discovery of new avian astrovirus strains make it extremely difficult to purify flocks infected with avian astrovirus. In addition, avian astrovirus has a strong resistance to physical and chemical factors, and it is very difficult to disinfect the environment contaminated by avian astrovirus. The virus is widely present in my country's breeding goose flocks, seriously affecting the economic benefits of the goose breeding industry.

[0004] At present, the detection methods for GoAstV and GoCV include electron microscopy observation, metagenomic sequencing and RT-PCR, but electron microscopy operation and metagenomic sequencing require certain skills, and it is not easy to centrally process a large number of samples. At the same time, the instrument costs are high, so electron microscopy detection and metagenomic sequencing have great limitations in GoAstV and GoCV monitoring. Conventional PCR and fluorescent quantitative PCR methods are commonly used to detect the virus. They show good sensitivity and specificity both qualitatively and quantitatively, but the detection conditions are relatively complex and require the use of expensive instruments and equipment. They are currently limited to laboratory diagnosis and the steps are cumbersome and time-consuming, and cannot be used for on-site real-time detection. Summary of the invention

[0005] In order to solve the above technical problems, the present application provides a detection card that can be used for rapid detection of goose astrovirus.

[0006] The present application provides a goose astrovirus detection card, comprising a bottom plate and an immunochromatographic test paper located on the bottom plate, wherein the immunochromatographic test paper comprises a sample pad, a conjugation pad, a detection pad and a water-absorbing pad along the chromatographic direction of the sample to be tested; the conjugation pad contains a goose astrovirus monoclonal antibody, and the goose astrovirus monoclonal antibody is marked with a marker.

[0007] The present application also provides a goose astrovirus detection kit, which comprises the above-mentioned goose astrovirus detection card and a diluent.

[0008] The present application also provides the use of the above-mentioned goose astrovirus detection card or goose astrovirus detection kit in the early screening of gout disease in geese.

[0009] The beneficial effects brought about by the present application include but are not limited to: (1) during the use stage, the present application can achieve the effect of convenient and on-site operation, while the existing technologies require professional laboratory personnel to operate in the laboratory; (2) and the reaction time of the detection card of the present application when in use is shorter than that of the existing technology; (3) the detection accuracy of the detection card of the present application is consistent with that of the fluorescent quantitative PCR product; therefore, the detection card of the present application can achieve fast, convenient and accurate results. DETAILED DESCRIPTION

[0010] As shown in this specification and claims, unless the context clearly indicates an exception, the words "a", "an", "an" and / or "the" do not refer to the singular and may also include the plural. Generally speaking, the terms "comprise" and "include" only indicate the inclusion of the steps and elements that have been clearly identified, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements.

[0011] The present application provides a goose astrovirus detection card, comprising a bottom plate and an immunochromatographic test paper located on the bottom plate, wherein the immunochromatographic test paper comprises a sample pad, a conjugation pad, a detection pad and a water-absorbing pad along the chromatographic direction of the sample to be tested; the conjugation pad contains a goose astrovirus monoclonal antibody, and the goose astrovirus monoclonal antibody is marked with a marker.

[0012] In some embodiments, the immunochromatographic test strip can be fixed to the base plate by an adhesive.

[0013] In some embodiments, the sample pad may correspond to a sample loading hole of a detection card.

[0014] In some embodiments, the marker may include any one or more of latex particles, fluorescent molecular markers, quantum dots, fluorescent microspheres, colored microspheres, colloidal gold, colloidal selenium, colloidal silver, or colloidal carbon. In some embodiments, preferably, the marker may include fluorescent microspheres.

[0015] Fluorescent microspheres refer to fluorescent substances, such as fluorescein, that are marked on the surface of particles or embedded inside microspheres through physical and chemical methods. They are generally spherical in shape, with diameters ranging from nanometers to micrometers, and can emit fluorescence visible to the naked eye when excited by excitation light.

[0016] The shape and size of fluorescent microspheres are relatively stable, with uniform particle size, good monodispersity, high luminous efficiency, good repeatability, good various reactions after interaction with organisms, strong adsorption, large specific surface area, strong surface reaction ability and large agglutination effect. Basically unaffected by the external environment, the optical stability of fluorescent substances is good, and the fluorescence intensity needs to be read by an immunofluorescence analyzer. It is to use the functional groups (such as carboxyl groups) modified on the surface of fluorescent microspheres to covalently bind with antigens or antibodies, and to perform quantitative detection through the fluorescent signals emitted by fluorescent substances.

[0017] In some embodiments, the fluorescent microspheres may be carboxylated modified microspheres containing high-brightness rare earth fluorescent pigments.

[0018] In some embodiments, the detection pad may be provided with a detection line and a quality control line. In some embodiments, preferably, the detection line may be coated with an antibody against goose astrovirus. In some embodiments, preferably, the quality control line may be coated with a goat anti-mouse monoclonal antibody.

[0019] In some embodiments, the material of the sample pad may include any one or more of glass fiber membrane, polyester fiber membrane or non-woven fabric.

[0020] In some embodiments, the detection pad can be a nitrocellulose membrane.

[0021] In some embodiments, the conjugate pad can be a glass cellulose membrane.

[0022] In some embodiments, the base plate is made of PVC.

[0023] In some embodiments, the absorbent pad is made of absorbent filter paper.

[0024] In some embodiments, the goose Astrovirus is goose Astrovirus type 4.

[0025] The present application also provides a goose astrovirus detection kit, which comprises the above-mentioned goose astrovirus detection card and a diluent.

[0026] In some embodiments, the diluent can be phosphate buffered saline.

[0027] The present application also provides the use of the above-mentioned goose astrovirus detection card or goose astrovirus detection kit in the early screening of gout disease in geese.

[0028] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0029] Example 1 - Preparation of test strips

[0030] 1. Preparation of the test pad:

[0031] Prepare a goat anti-mouse monoclonal antibody coating solution with a concentration of 10uL / mL, which is used for the quality control line, and a G4 monoclonal antibody coating solution with a concentration of 200uL / mL, which is used for the detection line. Pipette the two coating solutions into the film marking machine pipeline separately, and stick the nitrocellulose membrane to the middle position of the PVC plate, parallel to the lower edge of the absorbent paper.

[0032] Turn on the three-dimensional gold spraying film scribing instrument and follow the "Standard Operating Procedures for Three-Dimensional Gold Spraying Film Scratching Instrument". Set the scribing volume to 1ul / cm, start the program, evenly draw the quality control line (C line) and the test line (T line) on the nitrocellulose membrane, record the time, and use a black pen to mark the beginning, end and uneven parts of the membrane.

[0033] Lay the scribed nitrocellulose membranes flat one by one in the oven, avoid overlapping, control the temperature at 37±2℃, and dry for 2-4 hours.

[0034] 2. Preparation of conjugate pad:

[0035] Take 10ul of fluorescent microspheres with a solid content of 1% (the fluorescent microspheres used are carboxyl-modified microspheres containing high-brightness rare-earth fluorescent pigments).

[0036] Add 1 ml of MES buffer, centrifuge at 11500 r / min for 15 min, discard the supernatant, and resuspend the washed fluorescent microspheres in 1 ml of MES buffer to obtain a fluorescent microsphere dispersion.

[0037] An equal volume of EDC solution was added to the fluorescent microsphere dispersion at a volume of 0.48% of the volume, and the mixture was shaken and mixed. The mixture was reacted at room temperature (15-25° C.) in the dark for 15 min.

[0038] After the reaction was completed, 6 μl of goose astrovirus type 4 monoclonal antibody was added to the activated fluorescent microspheres, mixed thoroughly, and reacted at room temperature (15-25° C.) in the dark for 40 minutes.

[0039] After the reaction, add 100ul of Gly solution, mix well, and react at room temperature (15-25°C) in the dark for 15 minutes. Then add 100ul of 10% BSA solution, mix well, and react at room temperature (15-25°C) in the dark for 15 minutes.

[0040] After the reaction is completed, centrifuge at 11500r / min for 15min, discard the supernatant, resuspend the precipitate with 100ul of labeling solution, add 2-3ul of pigment, and mix well.

[0041] Turn on the three-dimensional gold spraying film scratching instrument and follow the "Standard Operating Procedures for Three-Dimensional Gold Spraying Film Scratching Instrument". Set the spraying volume to 3ul / cm, spray on the glass cellulose membrane, and mark the uneven spraying parts.

[0042] Place the labeled fluorescent microsphere pad in an oven at 37±2℃ and dry for 2-4 hours.

[0043] 3. Assembly of the test strip card

[0044] Using a non-absorbent PVC adhesive board as the base, overlap and paste the absorbent paper, test pad, binding pad, and sample pad on the base from top to bottom, with each component overlapping 1-2mm at adjacent locations. Use a strip cutter to cut the pasted PVC board into 3mm wide test strips, which are combined with the card case through the card slot in the card holder. The T line on the test pad is close to the sample pad side, and the corresponding card cover has a "T" mark; the quality control line C line is close to the absorbent pad side, and the corresponding card cover has a "C" mark.

[0045] The card cover has a specification of 70mm*20mm*5mm, and there is a 16mm*3mm viewing window in the center of the card cover. There is a sample addition hole marked as "S" in the area corresponding to the card cover and the sample pad, and there is an arrow indicating the detection direction below the sample addition hole.

[0046] Example 2 - Detection of goose astrovirus type 4 in goose anal swabs

[0047] 1. Use a cotton swab to collect freshly excreted feces, or take samples directly from the rectum; insert the cotton swab into the collection tube containing the sample diluent, and repeatedly rotate it on the tube wall for at least 10 times, and mix the solution to dissolve the specimen in the solution as much as possible. Squeeze the cotton swab on the wall of the centrifuge tube to squeeze out as much liquid as possible, and discard the cotton swab.

[0048] 2. Take out the test strip from the aluminum foil bag, add 80 μL of sample treatment solution into the well, and let it react for 15 minutes.

[0049] 3. Insert the test strip with the sample well facing inwards into the portable fluorescent immunoassay analyzer after 15 minutes of reaction, and click "Quick Test" to read the result. After the timer ends, the result after 25 minutes will be invalid.

[0050] 4. Qualitative judgment criteria: T / C value <0.1 is negative; T / C value ≥0.1 is positive.

[0051] Example 3 - Precision and sensitivity experiments of goose astrovirus type 4 fluorescent microsphere test strips

[0052] Select 10 prepared intermediates, cut 1 strip from each plate to test the same positive sample for intra-plate repeatability; select 1 intermediate, cut 10 strips continuously to test the same positive sample for inter-plate repeatability. The test results are shown in Table 1:

[0053] Table 1

[0054]

[0055] The experimental results showed that the CV values ​​within and between plates were both below 15%, indicating that the goose star type 4 fluorescent microsphere test strips had good precision.

[0056] A positive standard was selected for 100-fold, 1000-fold, 5000-fold, and 10000-fold dilutions and then tested. The test results are shown in Table 2 below:

[0057] Table 2

[0058]

[0059] The experimental results showed that the goose star-shaped type 4 fluorescent microsphere test strip could still detect positive results when the positive standard was diluted 10,000 times, indicating that it had good sensitivity.

[0060] Example 4-Comparison experiment of goose astrovirus type 4 fluorescent microsphere test strip and fluorescent quantitative PCR product

[0061] Comparison was made with commercially available fluorescent quantitative PCR products, and 10 anal swabs from uninfected geese and 10 anal swabs from infected geese were tested. The test results are shown in Tables 3 and 4 below:

[0062] Table 3

[0063]

[0064] Table 4

[0065]

[0066] The above experimental results prove that the accuracy of the fluorescent microsphere test strips is consistent with that of the fluorescent quantitative PCR product.

[0067] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only for example and does not constitute a limitation of this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements and corrections to this specification. Such modifications, improvements and corrections are suggested in this specification, so such modifications, improvements and corrections still belong to the spirit and scope of the exemplary embodiments of this specification.

[0068] At the same time, this specification uses specific words to describe the embodiments of this specification. For example, "one embodiment", "an embodiment", and / or "some embodiments" refer to a certain feature, structure or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more in different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures or characteristics in one or more embodiments of this specification can be appropriately combined.

[0069] In some embodiments, numbers describing the number of components and attributes are used. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise specified, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may change according to the required features of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the setting of such numerical values ​​is as accurate as possible within the feasible range.

[0070] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, as an example and not a limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.

Claims

1. A goose astrovirus detection card, characterized in that: It comprises a bottom plate and an immunochromatographic test paper located on the bottom plate, wherein the immunochromatographic test paper comprises a sample pad, a binding pad, a detection pad and a water-absorbing pad along the chromatography direction of the sample to be tested; The conjugate pad contains goose astrovirus monoclonal antibody, and the goose astrovirus monoclonal antibody is labeled with a marker.

2. The goose astrovirus detection card according to claim 1, characterized in that: The marker includes any one or more of latex particles, fluorescent molecular markers, quantum dots, fluorescent microspheres, colored microspheres, colloidal gold, colloidal selenium, colloidal silver or colloidal carbon; preferably, the marker includes fluorescent microspheres.

3. The goose astrovirus detection card according to claim 2, characterized in that: The fluorescent microspheres are carboxyl modified microspheres containing high-brightness rare earth fluorescent pigments.

4. The goose astrovirus detection card according to claim 1, characterized in that: The detection pad is provided with a detection line and a quality control line. Preferably, the detection line is coated with anti-goose astrovirus antibodies, and the quality control line is coated with goat anti-mouse monoclonal antibodies.

5. The goose astrovirus detection card according to claim 1, characterized in that: The material of the sample pad includes any one or more of glass fiber membrane, polyester fiber membrane or non-woven fabric; And / or, the detection pad is a nitrocellulose membrane.

6. The goose astrovirus detection card according to claim 1, characterized in that: The conjugate pad is a glass cellulose membrane; And / or, the absorbent pad is made of absorbent filter paper.

7. The goose astrovirus detection card according to claim 1, characterized in that: The material of the bottom plate is PVC.

8. The goose astrovirus detection card according to claim 1, characterized in that: The goose astrovirus is goose astrovirus type 4.

9. A goose astrovirus detection kit, characterized in that: The kit comprises the goose astrovirus detection card according to any one of claims 1 to 8 and a diluent.

10. Use of the goose astrovirus detection card according to any one of claims 1 to 8 or the goose astrovirus detection kit according to claim 9 in early screening of gout in geese.

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