Kit for detecting veterinary drug residues in meat food

Abstract

The invention discloses a kit for detecting veterinary drug residues in meat food, the kit comprises a test strip and a detection agent, the detection agent comprises a buffer solution and a signal probe, and the signal probe is a colloidal gold labeled monoclonal antibody of an anti-target antigen; the colloidal gold is modified by facultative ion polypeptide; the facultative ionic polypeptide is formed by combining acidic amino acid and basic amino acid and has good anti-pollution performance, so that the signal probe has strong hydrophilic property, a firm hydration layer can be formed on the surface, the effect of inhibiting adhesion and interference of matrix components is achieved, the stability of the detection process can be improved, and the detection sensitivity is improved. The non-specific adhesion of a complex matrix on the surface of the colloidal gold can be eliminated, so that the interference of the complex matrix on the detection process is inhibited; the detection line of the test strip is coated with a target antigen-BSA, the quality control line is coated with a cationic polymer, the kit is used for detecting veterinary drug residues in meat food, the cost is low, operation is easy and convenient, detection is efficient, and the kit can be used for on-site detection.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety detection, and in particular relates to a kit for detecting veterinary drug residues in meat food. Background Art

[0002] Excessive residues of veterinary drugs (such as florfenicol) in food can cause multiple hazards to human health, such as drug resistance, allergic reactions, teratogenicity and carcinogenicity, etc. Strengthening the detection of veterinary drug residues in animal-derived foods has become an urgent task in current food safety supervision.

[0003] In food safety testing, high performance liquid chromatography, mass spectrometry, liquid chromatography-mass spectrometry, and lateral flow immunochromatography are mainly used. Among them, high performance liquid chromatography, mass spectrometry, and liquid chromatography-mass spectrometry all rely on high-precision instruments, which are costly, time-consuming and labor-intensive, and cannot meet the needs of rapid on-site screening. Lateral flow immunochromatography (LFIA) detects target substances in samples based on the specific binding of antigens and antibodies under capillary chromatography, and has the advantages of low cost, simple operation, and efficient and rapid detection.

[0004] The signal probe used in LFIA is one of the factors that affect the detection sensitivity. The commonly used signal probe is colloidal gold, but the complex matrix interferes with the colloidal gold in LFIA, limiting the improvement of its detection performance and practical application. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a kit for detecting veterinary drug residues in meat food, which can suppress the interference of complex matrix on the detection process.

[0006] The technical solution of the present invention to solve the above problems is as follows:

[0007] A kit for detecting veterinary drug residues in meat food comprises a test strip and a detection agent, wherein the test strip comprises a bottom plate, a sample pad arranged above the bottom plate and arranged in sequence along the length direction of the bottom plate, an NC membrane, and a water-absorbing pad, wherein a detection line and a quality control line are arranged on the NC membrane, wherein the detection line is coated with a target antigen-BSA, and the quality control line is coated with a cationic polymer; the detection agent comprises a buffer solution and a signal probe, wherein the signal probe is a monoclonal antibody against the target antigen labeled with colloidal gold; the colloidal gold is modified with a zwitterionic polypeptide; and the zwitterionic polypeptide is formed by combining an acidic amino acid and a basic amino acid.

[0008] The target antigen-BSA refers to coupling the target antigen with BSA (bovine serum albumin).

[0009] Zwitterionic peptides formed by the combination of acidic amino acids and basic amino acids have good anti-fouling properties. Colloidal gold is modified with zwitterionic peptides, and then the monoclonal antibody against the target antigen labeled with colloidal gold is used as a signal probe, so that the signal probe has strong hydrophilic properties and can form a strong hydration layer on the surface, achieving the effect of inhibiting the adhesion and interference of matrix components, which can not only improve the stability of the detection process, but also inhibit the interference of complex matrices on the detection process. In addition, after the introduction of zwitterionic peptides, the monoclonal antibody against the target antigen labeled with colloidal gold no longer needs to be blocked with blocking agents such as BSA.

[0010] Preferably, the zwitterionic polypeptide is formed by alternating combinations of glutamic acid (E) and lysine (K).

[0011] Preferably, the zwitterionic polypeptide is EK 10 : CPPPP-EKEKEKEKEKEKEKEKEK.

[0012] Preferably, the target antigen-BSA concentration is 1-10 mg / mL, and the cationic polymer concentration is 0.020-0.040 mg / mL.

[0013] Preferably, the width of the test strip is 3.4-3.8 mm.

[0014] Preferably, the detection line is spaced 2 to 6 mm apart from the quality control line.

[0015] Preferably, the base plate is made of PVC.

[0016] The present invention also provides a method for preparing the above-mentioned kit for detecting veterinary drug residues in meat food, comprising the following steps:

[0017] S1. preparing colloidal gold-labeled monoclonal antibodies against target antigens;

[0018] S2, preparing an NC membrane containing the detection line and the quality control line;

[0019] S3, overlapping the sample pad, NC membrane and water absorbent pad in sequence along the length direction of the bottom plate, so that a first overlapping area is generated between the sample pad and the NC membrane, and a second overlapping area is generated between the NC membrane and the water absorbent pad.

[0020] Preferably, in step S1, preparing a colloidal gold-labeled monoclonal antibody against a target antigen comprises the following steps:

[0021] (1) Continuously heating and stirring the chloroauric acid solution until it boils, then adding the sodium citrate solution, heating until the solution turns wine red and the color remains unchanged, then continuing to heat for 5 to 15 minutes, removing the heat source and stirring, and cooling to room temperature to obtain colloidal gold AuNPs;

[0022] (2) Adding zwitterionic peptides to colloidal gold AuNPs, incubating overnight at room temperature with shaking, centrifuging, and resuspending to obtain zwitterionic peptide-modified colloidal gold AuNPs@(EK) n ;

[0023] (3) Add the EDC and NHS solutions sequentially to the zwitterionic peptide-modified colloidal gold Au NPs@(EK) n Solution, incubate for 20-40 min, centrifuge, discard the supernatant, and resuspend;

[0024] (4) Adjusting the pH with hydrochloric acid, adding monoclonal antibodies against the target antigen, incubating at room temperature for 1.5 to 2 hours, and then centrifuging to obtain colloidal gold-labeled monoclonal antibodies against the target antigen Au NPs@(EK) n @Ab.

[0025] Further preferably, in step S1, preparing the colloidal gold-labeled monoclonal antibody against the target antigen comprises the following steps:

[0026] (1) Continuously heating and stirring the chloroauric acid solution until it boils, then adding the sodium citrate solution, heating until the solution turns wine red and the color remains unchanged, then continuing to heat for 5 to 15 minutes, removing the heat source and stirring, and cooling to room temperature to obtain colloidal gold AuNPs;

[0027] (2) The zwitterionic peptide EK 10 Add to colloidal gold AuNPs, incubate at room temperature overnight, centrifuge, and resuspend to obtain zwitterionic peptide-modified colloidal gold AuNPs@EK 10 ;

[0028] (3) Add the EDC and NHS solutions sequentially to the zwitterionic peptide-modified colloidal gold Au NPs@EK 10 Solution, incubate for 20-40 min, centrifuge, discard the supernatant, and resuspend;

[0029] (4) Adjusting the pH with hydrochloric acid, adding monoclonal antibodies against the target antigen, incubating at room temperature for 1.5 to 2 hours, and then centrifuging to obtain colloidal gold-labeled monoclonal antibodies against the target antigen Au NPs@EK 10 @Ab.

[0030] Preferably, in step (2), the mass concentration of the zwitterionic polypeptide is 1 to 5 mg / mL.

[0031] Preferably, in step (3), the molar ratio of EDC:NHS is (2-4):1.

[0032] The EDC is 1-ethyl-(3-dimethylaminopropyl)carbodiimide; and the NHS is N-hydroxysuccinimide.

[0033] Preferably, in step (4), the pH range is 5 to 8.

[0034] Preferably, in step (4), the mass concentration of the monoclonal antibody against the target antigen is 0.1-1.1 mg / mL.

[0035] Preferably, in steps (2), (3) and (4), the centrifugation conditions are: 9000-11000 rpm, 12-18 min, 3-5°C.

[0036] Preferably, in step S2, the method for preparing the NC membrane containing the detection line and the quality control line comprises the following steps:

[0037] The target antigen-BSA is diluted to 1-10 mg / mL using a film-scratching diluent, and the diluted target antigen-BSA is streaked on the detection line of the NC membrane at 0.6-1.0 μL / cm using a film-scratching instrument; 0.020-0.040 mg / mL of cationic polymer is streaked on the quality control line of the NC membrane at 0.6-1.0 μL / cm; and the membrane is dried at 37-40°C for 2-4 hours to form a detection NC membrane containing a detection line and a quality control line.

[0038] Preferably, the membrane diluent used to dilute the target antigen-BSA is selected from at least one of 0.01M PB (pH=7.4, 1wt% sucrose) and 0.01M PBS (pH=7.4, 1wt% sucrose).

[0039] More preferably, the streaking dilution for diluting the target antigen-BSA is 0.01 M PB (pH=7.4, 1 wt% sucrose).

[0040] Preferably, the target antigen-BSA and the monoclonal antibody against the target antigen are both known commercially available products.

[0041] Preferably, in step S3, the lengths of the first overlapping region and the second overlapping region are both 1-2 mm.

[0042] The present invention also provides a method for detecting veterinary drug residues in meat food, comprising the following steps:

[0043] S1, preparing standard solutions containing the target antigen in different concentration gradients, and mixing the standard solutions of each concentration gradient with the detection agent in the kit respectively to obtain the color development depth of the test strip in the kit under each concentration gradient;

[0044] S2. Establish the corresponding relationship between the concentration gradient of the standard solution and the color development depth;

[0045] S3, homogenize the sample to be tested, take the supernatant, mix it with the detection agent in the kit, incubate it, and obtain the test solution; drop the test solution onto the sample pad, and observe the result after 5 to 8 minutes;

[0046] S4. Result determination: If both the test line and the quality control line are clearly colored, the test sample does not contain the test target; if the quality control line is clearly colored and the test line is lightly colored or not colored, the test sample contains the test target;

[0047] The kit is the kit described above.

[0048] If the test target exists in the sample to be tested, the antigen in the sample to be tested and the target antigen coated on the test line will compete for binding with the monoclonal antibody against the target antigen labeled with colloidal gold. The target antigen coated on the test line is suppressed and competed away. The monoclonal antibody and the antigen in the sample to be tested continue to chromatograph until they are combined with the quality control line. At this time, the test line does not show color or shows a lighter color, and the color depth of the test line is inversely proportional to the content of the test target. The quality control line shows obvious color, and the result is judged as positive.

[0049] If there is no detection target in the sample to be tested, during the chromatography process, the monoclonal antibody against the target antigen labeled with colloidal gold will first bind to the target antigen coated on the test line, and the remaining monoclonal antibody against the target antigen labeled with colloidal gold will continue to chromatograph until it binds to the quality control line. At this time, both the test line and the quality control line are obviously colored, and the result is judged to be negative;

[0050] If the quality control line in the result does not show color, the test strip is scrapped and cannot be used.

[0051] The present invention has the following beneficial effects:

[0052] The invention provides a kit for detecting veterinary drug residues in meat food, comprising a test strip and a detection agent, wherein the detection agent comprises a buffer solution and a signal probe, wherein the signal probe is a monoclonal antibody against a target antigen labeled with colloidal gold; the colloidal gold is modified by a zwitterionic polypeptide; the zwitterionic polypeptide is formed by combining an acidic amino acid and a basic amino acid, and has good anti-fouling performance, so that the signal probe has a strong hydrophilic property, and can form a firm hydration layer on the surface, so as to achieve the effect of inhibiting the adhesion and interference of matrix components, and can not only improve the stability of the detection process, but also eliminate the nonspecific adhesion of complex matrix on the surface of colloidal gold, thereby inhibiting the interference of complex matrix on the detection process; the detection line of the test strip is coated with the target antigen-BSA, and the quality control line is coated with a cationic polymer. The kit is used to detect veterinary drug residues in meat food, with low cost, simple operation, and high detection efficiency, and can be used for on-site detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is a technical principle diagram of a kit for detecting veterinary drug residues in meat food according to the present invention;

[0054] Figure 2 It is a graph showing the test results of the florfenicol standard substance in Examples 1 to 4 and Comparative Example 1;

[0055] Figure 3 This is a picture of the centrifuge tube described in Example 5;

[0056] Figure 4 This is a graph showing the stability test results of AuNPs in Example 5;

[0057] Figure 5 For the AuNPs@EK in Example 5 10 Stability test result graph;

[0058] In the figure, 1-bottom plate, 2-sample pad, 3-NC membrane, 31-detection line, 32-quality control line, 4-water absorbent pad. DETAILED DESCRIPTION

[0059] The present invention is further described in detail below based on the accompanying drawings and implementation examples.

[0060] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0061] The antigen florfenicol-BSA and florfenicol monoclonal antibody used in the following examples and comparative examples are both products of Shenzhen Kejie Industrial Co., Ltd., model KJ01FFAb.

[0062] Example 1

[0063] A kit for detecting veterinary drug residues in meat food, comprising a test strip and a detection agent, wherein the width of the test strip is 3.4 to 3.8 mm. Figure 1 As shown, it includes a PVC base plate 1 and a sample pad 2 , an NC membrane 3 , and a water absorbent pad 4 on the base plate 1 .

[0064] The preparation method of the above test strip is as follows:

[0065] The method for preparing the sample pad 2 is as follows: a glass cellulose membrane is soaked in a phosphate buffer solution and then dried to prepare the sample pad 2 .

[0066] The preparation method of NC membrane 3 is as follows:

[0067] The stripping diluent for the antigen florfenicol-BSA is 0.01M PB (pH=7.4, 1wt% sucrose). The antigen florfenicol-BSA diluted to 5 mg / mL is stripped on the detection line 31 (T line) of the NC membrane 3 at 0.8 μL / cm using a stripper, and 0.035 mg / mL of polydiallyldimethylammonium chloride (PDDA) is stripped on the quality control line 32 (C line) of the NC membrane 3. The membrane is dried at 37-40°C for 2-4 hours to form a detection NC membrane 3 containing a detection line 31 and a quality control line 32.

[0068] The test strip is assembled as follows: along the flow direction of the sample to be tested, the sample pad 2, the NC membrane 3, and the absorbent pad 4 are overlapped in sequence to form a first overlapping area between the sample pad and the NC membrane, and a second overlapping area between the NC membrane and the absorbent pad. The lengths of the first overlapping area and the second overlapping area are both 1 to 2 mm.

[0069] The detection agent includes a buffer solution and a signal probe, wherein the preparation method of the signal probe includes the following steps:

[0070] S1. Add 100 mL of 0.01 wt% chloroauric acid solution to a 250 mL conical flask, continue heating and stirring until boiling; quickly add 4 mL of 1 wt% sodium citrate solution, heat until the solution turns wine red and the color remains unchanged, continue heating for 15 min, remove the heat source and stir, cool to room temperature, and obtain AuNPs;

[0071] S2. EK 10 The solution was added to 1 mL of AuNPs, EK 10The final concentration was 1 mg / mL. After incubation at room temperature overnight, the mixture was centrifuged at 10,000 rpm for 15 min and resuspended in PBS to obtain Au NPs@EK 10 ;

[0072] S3, EDC / NHS were added to the Au NPs@EK prepared above in a molar ratio of 2:1. 10 In the solution, after activating the carboxyl groups for 30 min, the mixture was centrifuged at 10,000 rpm for 15 min, the supernatant was discarded, and the mixture was resuspended in PBS;

[0073] S4. Adjust the pH of the solution to about 5 with hydrochloric acid, and then take 3.8 μL of the diluted florfenicol monoclonal antibody (1.1 mg / mL) and add it to the activated Au NPs@EK 10 The solution was shaken at room temperature for 1.5 to 2 h, centrifuged at 10,000 rpm for 15 min, and resuspended in PBS to obtain Au NPs@EK 10 @Ab.

[0074] The method for detecting veterinary drug residues (florfenicol) by the above kit is as follows:

[0075] Florfenicol standard solutions with different concentration gradients (final concentrations of 0, 1, 10, 50, 100 ng / mL) were added dropwise to the detection solution with the signal probe for incubation to obtain the test solution, which was then added dropwise to the sample pad 2 of the test strip. With the chromatography, the solution passed through the T line and the C line in sequence, and the results were observed after 5 to 8 minutes.

[0076] The results are as follows Figure 2 As shown in A (in each test strip, the two strips from top to bottom are line C and line T), the T line of the florfenicol standard of different concentrations has good color development, and as the concentration increases, the color depth of the T line gradually becomes lighter, which is in line with the expected results.

[0077] Example 2

[0078] Compared with Example 1, the main difference is that the concentration of the antigen florfenicol-BSA on the detection line 31 in the NC membrane 3 is 1 mg / mL.

[0079] The test results of the kit for detecting veterinary drug residues (florfenicol) are as follows Figure 2 As shown in B, although florfenicol can be detected, the color on the T line is light and difficult to observe with the naked eye.

[0080] Example 3

[0081] Compared with Example 1, the main difference is that in step S4 of preparing the signal probe, the pH of the solution is about 7.5-8.

[0082] The test results of the kit for detecting veterinary drug residues (florfenicol) are as follows Figure 2 As shown in C, although florfenicol can be detected, there is almost no color on the T line, and the color on the C line is diffuse, so the detection effect is not good.

[0083] Example 4

[0084] The preparation method of NC membrane 3 is as follows: the antigen stripping diluents are 0.01M PB (pH=7.4, 1wt% sucrose), 0.01M PB (pH=7.4, 1wt% methanol), and 0.01M PBS (pH=7.4, 1wt% sucrose), respectively. The antigen florfenicol-BSA is diluted to 5 mg / mL using the above stripping diluents, and is striped on the detection line 31 of the NC membrane 3 at 0.8 μL / cm using a stripper, and 0.035 mg / mL PDDA is striped on the quality control line 32 of the NC membrane 3, and dried at 37-40°C for 2-4 hours to form a detection NC membrane 3 containing a detection line 31 and a quality control line 32.

[0085] The method for detecting veterinary drug residues (florfenicol) by the above kit is as follows:

[0086] Add 0 and 1 ng / mL florfenicol standard solutions to the detection agent with the signal probe and incubate to obtain the test solution, then add the test solution to the sample pad 2 of the test strip. With the chromatography, it flows through the T line and the C line in turn. Observe the results after 5 to 8 minutes. Figure 2 As shown in Figure E (0 ng / mL on the left and 1 ng / mL on the right), the scratching diluents are 0.01M PB (pH=7.4, 1wt% sucrose), 0.01M PB (pH=7.4, 1wt% methanol), and 0.01M PBS (pH=7.4, 1wt% sucrose) from left to right. When the scratching diluent is 0.01M PB (pH=7.4, 1wt% sucrose), the T line is well colored; when the scratching diluent is 0.01M PB (pH=7.4, 1wt% methanol), the T line is almost not colored; when the scratching diluent is 0.01MPB (pH=7.4, 1wt% sucrose), the T line is colored, but the C line shows color diffusion.

[0087] Example 5

[0088] Take 4 1.5 ml centrifuge tubes, such as Figure 3 As shown, they are numbered 1, 2, 3, and 4. Tubes 1 and 2 are AuNPs and AuNPs@EK diluted with ultrapure water, respectively. 10 Tubes 3 and 4 are AuNPs and AuNPs@EK diluted with PBS buffer, respectively. 10; After mixing, take 100 μL of the diluted solution for UV-visible spectroscopy analysis to observe the gold nanoparticles modified EK 10 The changes in the UV absorption peaks before and after the reaction were used to determine the stability of the particles. Figure 4 and 5 As shown, modified EK 10 The UV absorption peak of the colloidal gold after modification has no obvious change, while the color of the unmodified colloidal gold solution has changed and the expected UV absorption peak is not observed. 10 The modification improves the stability of gold nanoparticles.

[0089] Comparative Example 1

[0090] Compared with Example 1, the main difference is that in step S3 of preparing the signal probe, the molar ratio of EDC to NHS is 1:1. The test results of the kit for detecting veterinary drug residues (florfenicol) are as follows Figure 2 As shown in D, the color rendering on the T line is poor, making it difficult to observe and the effect is not good.

[0091] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A kit for detecting veterinary drug residues in meat food, comprising a test strip and a detection agent, wherein the test strip comprises a bottom plate (1), a sample pad (2) arranged above the bottom plate (1) and arranged in sequence along the length direction of the bottom plate (1), an NC membrane (3), and a water-absorbing pad (4), wherein a detection line (31) and a quality control line (32) are arranged on the NC membrane (3), and the test strip comprises: The detection line (31) is coated with the target antigen-BSA, and the quality control line (32) is coated with a cationic polymer; the detection agent includes a buffer solution and a signal probe, and the signal probe is a monoclonal antibody against the target antigen labeled with colloidal gold; the colloidal gold is modified with a zwitterionic polypeptide; and the zwitterionic polypeptide is formed by the combination of acidic amino acids and basic amino acids.

2. A kit for detecting veterinary drug residues in meat products according to claim 1, characterized in that: The zwitterionic polypeptide is formed by alternating combinations of glutamic acid and lysine.

3. A kit for detecting veterinary drug residues in meat products according to claim 1 or 2, characterized in that: The zwitterionic polypeptide is EK 10 : CPPPP-EKEKEKEKEKEKEKEKEK.

4. A kit for detecting veterinary drug residues in meat products according to claim 1, characterized in that: The target antigen-BSA concentration is 1-10 mg / mL, and the cationic polymer concentration is 0.020-0.040 mg / mL.

5. The method for preparing a kit for detecting veterinary drug residues in meat products according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. preparing colloidal gold-labeled monoclonal antibodies against target antigens; S2, preparing an NC membrane (3) containing the detection line (31) and the quality control line (32); S3, overlapping the sample pad (2), the NC membrane (3), and the water absorbent pad (4) in sequence along the length direction of the bottom plate (1), so that a first overlapping area is generated between the sample pad (2) and the NC membrane (3), and a second overlapping area is generated between the NC membrane (3) and the water absorbent pad (4).

6. The preparation method according to claim 5, characterized in that: In step S1, preparing a colloidal gold-labeled monoclonal antibody against a target antigen comprises the following steps: (1) Continuously heating and stirring the chloroauric acid solution until it boils, then adding the sodium citrate solution, heating until the solution turns wine red and the color remains unchanged, then continuing to heat for 5 to 15 minutes, removing the heat source and stirring, and cooling to room temperature to obtain colloidal gold AuNPs; (2) Adding zwitterionic peptides to colloidal gold AuNPs, incubating overnight at room temperature with shaking, centrifuging, and resuspending to obtain zwitterionic peptide-modified colloidal gold AuNPs@(EK) n ; (3) Add the EDC and NHS solutions sequentially to the zwitterionic peptide-modified colloidal gold Au NPs@(EK) n Solution, incubate for 20-40 min, centrifuge, discard the supernatant, and resuspend; (4) Adjusting the pH with hydrochloric acid, adding monoclonal antibodies against the target antigen, incubating at room temperature for 1.5 to 2 hours, and then centrifuging to obtain colloidal gold-labeled monoclonal antibodies against the target antigen Au NPs@(EK) n @Ab.

7. The preparation method according to claim 6, characterized in that: In the step (2), the mass concentration of the zwitterionic polypeptide is 1 to 5 mg / mL.

8. The preparation method according to claim 6, characterized in that: In the step (3), the molar ratio of EDC:NHS is (2-4):

1.

9. The preparation method according to claim 6, characterized in that: In the step (4), the pH range is 5-8.

10. A method for detecting veterinary drug residues in meat products, characterized in that: The following steps are involved: S1, preparing standard solutions containing the target antigen in different concentration gradients, and mixing the standard solutions of each concentration gradient with the detection agent in the kit respectively to obtain the color development depth of the test strip in the kit under each concentration gradient; S2. Establish the corresponding relationship between the concentration gradient of the standard solution and the color development depth; S3, homogenizing the sample to be tested, taking the supernatant, mixing it with the detection agent in the kit, incubating it, and obtaining a test solution; dropping the test solution onto the sample pad (2), and observing the result after 5 to 8 minutes; S4, result determination: if both the test line (31) and the quality control line (32) are clearly colored, the test sample does not contain the test target; if the quality control line (32) is clearly colored and the test line (31) is lightly colored or not colored, the test sample contains the test target; The kit is the kit according to any one of claims 1 to 4.

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