A method for rapid and simultaneous detection of MS-222 and malachite green and a dual-color detection kit

Abstract

The present invention relates to a method and a dual-color detection kit for the rapid and simultaneous detection of MS-222 and malachite green, and a technology for the rapid detection of veterinary drug residues in aquatic products. The invention utilizes red and blue colloidal gold with different particle sizes, combined with specific antibodies to construct different red and blue probes, respectively. The invention utilizes the principle that MG-BSA complete antigen and 3-aminobenzoic acid-BSA antigen coated on the T1 line and the T2 line, respectively, can compete with MG and MS-222 in the sample for binding to the blue probe and the red probe, thereby constructing a dual-color rapid detection test strip for MS-222 and malachite green that is clearly distinguishable to the naked eye and simple to operate.

Description

Technical Field

[0001] The present invention belongs to the technical field of rapid detection of veterinary drug residues in aquatic products, and specifically relates to a synchronous multivariate detection technology for veterinary drug residues in aquatic products, and especially relates to the preparation and application of a dual-color colloidal gold synchronous detection test strip for fishery anesthetics and malachite green. Background Art

[0002] Aquatic product safety has always been a key concern for the industry. Currently, 90% of substandard aquatic products are primarily due to veterinary drug residues and excessive levels of heavy metals. Veterinary drug residues primarily include malachite green and nitrofurans. Malachite green, known for its triple-hazardous effects, is listed in my country's "List of Veterinary Drugs and Their Compounds Prohibited for Use in Food Animals" (Ministry of Agriculture Announcement No. 193). However, due to its significant efficacy and low price, there are no effective alternatives.

[0003] In recent years, emerging veterinary anesthetics for fisheries have been increasingly used in the transport of live fish due to their low price, ease of use, and ability to effectively reduce stress during aquatic product transportation. This, however, also poses a potential hazard. MS-222 (ethyl 3-aminobenzoate methylsulfonate), also known as tricaine and fish diazepam, is a common anesthetic used in fisheries. The United States, Canada, the European Union, and other countries and regions allow the use of MS-222 in fresh aquatic products, with limited usage requirements. Therefore, the development of rapid detection technologies for anesthetics is urgently needed.

[0004] There are many rapid detection methods for malachite green MG, but each has different limitations and problems. CN211426360U discloses an electrochemical sensor for detecting malachite green. This method improves the secondary antibody loading capacity by improving gold nanorods to achieve high-sensitivity detection of malachite green, but it requires the configuration of a corresponding chemical workstation to realize signal acquisition and analysis, which requires high professional expertise of personnel. CN110006880A introduces a composite material and application for direct reading colorimetric rapid detection of malachite green. It uses potassium iodate / Ag@nano-titanium dioxide composite as a colorimetric agent, provides a good colorimetric substrate, and ensures colorimetric stability. However, its preparation process is relatively cumbersome and requires several chemical reactions to realize the synthesis of the composite material.

[0005] There are few detection methods for fishing anesthetics, which mainly focus on traditional instrument detection and ELISA method, and no rapid detection method has been reported. CN108760947A proposed a GC-MS / MS method for detecting seven anesthetics in aquatic products, but the sample pretreatment is cumbersome, the instrument detection time for a single sample is nearly 1 hour, and GC-MS / MS is relatively expensive. CN113092604A discloses a rapid detection method for three commonly used anesthetics in aquatic products, which uses a single quadrupole gas chromatography-mass spectrometer (GC-MS) in positive ion mode to detect eugenol, isoeugenol and MS-222. This method reduces equipment requirements and the instrument can complete the detection within 0.5 hours, but the pretreatment requires 4 centrifugations and 1 nitrogen blow, and cannot meet the needs of on-site detection. CN110938011A introduces a preparation and application method for benzocaine hapten, complete antigen, and antibody, and uses ELISA method to achieve accurate detection, but still cannot get rid of instruments and equipment such as enzyme labeling instruments, making it difficult to achieve rapid detection on site.

[0006] The multivariate synchronous detection technology of test strips is in its infancy, and it mainly realizes synchronous detection by combining a device with several test strips and setting multiple detection lines. CN 213689646U proposes a six-in-one detection card for detecting chemical residues in aquatic products. The implementation method is to fix several test strips on a device, but it is necessary to drip samples on 4 test strips for testing, which increases the workload and detection cost. CN108931644A discloses a foot-and-mouth disease virus colloidal gold double test strip, which realizes the simultaneous determination of whether there is foot-and-mouth disease virus infection and whether there is foot-and-mouth disease vaccine immunity by setting dual detection lines on the test strip and combining colloidal gold color development. However, the color development of the dual detection lines and the quality control line is consistent, and qualitative detection based on naked eye observation is prone to misjudgment results. CN112198310A provides a test strip, a kit, and a method for preparing the test strip for the combined detection of C-reactive protein and serum amyloid protein A. CRP antibody and CRP broadening rabbit anti-two antibodies are coated on the dual detection lines respectively, and jointly labeled fluorescent microspheres are used to achieve the purpose of dual-element synchronous detection. However, the detection cost is high and fluorescence signal analysis equipment is required.

[0007] In summary, existing veterinary drug residue detection technologies have the following problems: 1) The detection of fishery anesthetics is mainly based on instrumental analysis methods, and there is a lack of rapid detection technologies suitable for on-site detection; 2) The rapid detection technology of malachite green has high technical requirements for operators, is difficult to prepare materials, and lacks synchronous multiplex rapid detection methods; 3) Most of the currently reported multiplex detection test strips require specialized instruments and equipment or multiple test strips, resulting in high detection costs. Summary of the Invention

[0008] The present invention aims to provide a MS-222 and MG dual-element dual-color colloidal gold immunoassay strip and a kit that are clearly distinguishable by the naked eye, simple to operate, and suitable for on-site rapid simultaneous detection.

[0009] The present invention utilizes red and blue colloidal gold with different particle sizes, combined with specific antibodies to construct different red and blue probes, respectively. Utilizing the principle that the MG-BSA complete antigen and 3-aminobenzoic acid-BSA antigen coated on the T1 line and T2 line, respectively, can compete with MG and MS-222 in the sample for binding to the blue and red probes, a competitive dual-color rapid test strip and kit for simultaneous detection are constructed, thereby realizing rapid and simultaneous detection of MS-222 and malachite green.

[0010] The technical solution is as follows: A method for rapid and simultaneous detection of MS-222 and malachite green, comprising the following steps:

[0011] (1) Mix the red probe and the blue probe with the sample to be tested and the chromatography solution and then drop them onto the sample pad of the immunochromatographic test paper;

[0012] The red probe and the blue probe are prepared by linking one of the MS-222 antibody and the MG antibody to the red nano-colloidal gold particles and the blue nano-colloidal gold particles, respectively;

[0013] The immunochromatographic test paper is provided with a test line 1, a test line 2 and a quality control line; the test line 1 and the test line 2 are coated with carboxylated malachite green-BSA complete antigen and 3-aminobenzoic acid-BSA complete antigen respectively; the quality control line is coated with goat anti-mouse IgG;

[0014] (2) Observe the color of the test strip.

[0015] The red nano colloidal gold particles have a particle size of 10-40 nm, and the preparation method comprises the following steps: mixing a boiling chloroauric acid solution with a sodium citrate solution, heating and stirring for 5-20 minutes; the chloroauric acid concentration in the reaction system is 0.5-5 mmol / L, and the sodium citrate concentration is 3-40 mmol / L;

[0016] The blue colloidal gold nanoparticles have a particle size of 75-100 nm, and the preparation method comprises the following steps: mixing red colloidal gold particles with a particle size of 10-40 nm with a chloroauric acid solution, a sodium citrate solution, and a p-diphenol solution, and heating the mixture; in the reaction system, the molar ratio of the red colloidal gold to the chloroauric acid is 1:40-150;

[0017] Preferably, the molar ratio of red colloidal gold to chloroauric acid is 1:50-100, more preferably 1:50-80.

[0018] Preferably, the particle size of the red nano-colloidal gold particles is 15-25 nm, and the particle size of the blue nano-colloidal gold particles is 80-90 nm.

[0019] Preferably, the red probe is connected to the MS-222 antibody, and the blue probe is connected to the malachite green antibody.

[0020] In step (2), the color development time is 5-20 min, preferably 10-15 min; the color can be qualitatively observed by the naked eye, or the gray value can be extracted and quantitatively determined after taking a picture.

[0021] The red probe or blue probe is prepared by adding a red colloidal gold solution or a blue colloidal gold solution to either a malachite green antibody or an MS-222 antibody, along with BSA and a carbonate solution, mixing thoroughly, centrifuging, and washing the precipitate; the carbonate is potassium carbonate or sodium carbonate, and the carbonate concentration in the reaction system is 0.001-0.002 mol / L. Preferably, the pH of the reaction system is 6-10.

[0022] Preferably, in step (1), the chromatography fluid is a phosphate buffer, borate buffer, or tris-aminomethane buffer containing 20-50 g / L Tween-20 and 0-300 g / L BSA, with a pH of 7.3-7.5. More preferably, the chromatography fluid is a phosphate buffer containing 30-50 g / L Tween-20 and 50-150 g / L BSA, with a pH of 7.4.

[0023] The sample to be tested is a water sample or an extract of aquatic products. The extract of aquatic products is prepared by the following method: the aquatic product is ultrasonically extracted with an organic solvent for 5-15 minutes, a purifier is added to the supernatant, and ultrasonically vibrates for 2-5 minutes. After centrifugation, the supernatant is dried and reconstituted with a buffer solution.

[0024] The organic solvent is acetonitrile; the purifier is one or a combination of N-propylethylenediamine, sodium chloride, magnesium sulfate, and graphite carbon; and the buffer is a phosphate buffer, a borate buffer, or a trisaminomethane buffer with a pH of 7.3-7.5.

[0025] A test paper for rapid detection of MS-222 and MG is an immunochromatographic test strip, comprising a test line 1, a test line 2 and a quality control line; the test line 1 and the test line 2 are respectively coated with carboxylated malachite green-BSA complete antigen and 3-aminobenzoic acid-BSA complete antigen; the quality control line is coated with goat anti-mouse IgG.

[0026] The preparation method comprises the following steps:

[0027] 1) Nitrocellulose Membrane Coating: Use a membrane streaker to spray 3-aminobenzoic acid-BSA complete antigen, carboxymalachite green-BSA complete antigen, and goat anti-mouse IgG onto the corresponding areas of the nitrocellulose membrane. The distance between the MG test line (T1 line), the MS-222 test line (T2 line), and the quality control line (C line) should be 3-8 mm. The membrane should then be dried in an oven at 37°C for 2-6 h.

[0028] 2) Test Strip Assembly: Assemble the sample pad, conjugate pad, nitrocellulose membrane, absorbent pad, and PVC base in sequence. To ensure smooth sample flow, overlap the test strip components by 0.5-2.5 mm. Cut the strips into 2-10 mm wide strips and seal them in a dry place for storage.

[0029] The use of the above test paper can quickly detect MS-222 and MG at the same time with high sensitivity and no misjudgment.

[0030] The present invention provides a method for preparing blue colloidal gold, which can be achieved by simply adding one extra step to the existing method for synthesizing colloidal gold. The synthesis conditions are mild and simple, which is conducive to the development of multi-color colloidal gold test strips. The invention also provides a design concept for a dual-element, dual-color synchronous detection test strip, which improves the naked eye's recognition and realizes the simultaneous visual detection of two substances. The detection limit is low, and the possibility of misjudgment is avoided, which can be extended to the design and development of other test strips in the future.

[0031] This rapid synchronous detection test strip for MS-222 and MG is easy to carry and operate, and it only takes 10 minutes to interpret the qualitative results. It is suitable for rapid on-site testing and initial screening of large quantities of samples. It can also be combined with mobile phone photography and ImageJ software to collect grayscale quantitative detection.

[0032] The beneficial effects of the present invention are:

[0033] 1) Utilizing the distinct color differences between the red and blue probes and the purple control line, this method addresses the issue with existing dual-element rapid test strips, where the two test lines and the control line display the same color, making them prone to misinterpretation by the naked eye. In one preferred embodiment of the present invention, higher MG content results in a lighter blue T1 line, while higher MS-222 content results in a lighter red T2 line. The control line appears purple because it captures both probes simultaneously.

[0034] 2) Utilizing a self-made two-color colloidal gold-coupled immunochromatographic test strip, we achieved simultaneous visual rapid qualitative and quantitative detection of MS-222 and MG in water and aquatic products. By visually observing the color development, we rapidly and qualitatively detected tricaine MS-222 and malachite green in water and fish within 10-15 minutes. Using smartphone photography combined with ImageJ, we were able to quantitatively analyze the contents of MS-222 and MG.

[0035] 3) Simple operation, low cost, simple sample pre-treatment requirements, and can meet on-site testing needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the test principle diagram of the MS-222&MG dual test strip of the present invention

[0037] Figure 2 This is the test diagram of the MG blue colloidal gold test strip and the MS-222 red colloidal gold test strip of the present invention

[0038] Figure 3 The specificity diagram of the MS-222&MG dual test strip of the present invention is DETAILED DESCRIPTION

[0039] Example 1 Preparation and Detection of MG Blue Colloidal Gold Kit and MS-222 Red Colloidal Gold Kit

[0040] 1. MS-222 Red Colloidal Gold Kit

[0041] (1) The preparation method of red colloidal gold is as follows:

[0042] 0.8 mL of 2 mM chloroauric acid solution was heated to boiling, and then 1 mL of 30 mmol / L sodium citrate solution was added. The mixture was heated and stirred for 15 min, and then cooled to room temperature to obtain a red colloidal gold solution with a particle size of 20 ± 5 nm.

[0043] (2) The preparation method of MS-222 red probe is as follows:

[0044] To 600 μL of red colloidal gold solution, 8 μg of MS-222 antibody, 8 μg of bovine serum albumin, and 5 μL of 0.15 M potassium carbonate solution were added, and the mixture was mixed on a vertical mixer for 3 h. Subsequently, the mixture was centrifuged at 7000 rpm / min for 25 min, the supernatant was discarded, and the suspension was washed and resuspended in phosphate buffer containing 4% (v / v) Tween-20. The suspension was then centrifuged at 4000 rpm / min for 35 min, and the washing steps were the same as above. Finally, the suspension was washed with 600 μL of phosphate buffer containing 0.05% (m / v) sodium azide and stored at 4°C until use.

[0045] (3) The preparation method of MS-222 immunochromatographic test strips is as follows:

[0046] A membrane streaker was used to spray T lines and C lines onto the nitrocellulose membrane at a rate of 1 μL / cm. The T line was sprayed with 0.1 mg / mL MS-222-BSA complete antigen solution, and the C line was sprayed with 0.6 mg / mL goat anti-mouse solution. The membrane was then dried in an oven at 40°C for 2 h. The sample pad, conjugate pad, nitrocellulose membrane, absorbent pad, and PVC base were assembled in sequence and sealed in an aluminum foil bag for storage.

[0047] The MS-222 red colloidal gold kit includes an MS-222 red probe, an MS-222 immunochromatographic test strip, and a chromatography fluid (containing 30 g / L Tween-20, 100 g / L BSA, and a 0.2 mol / L phosphate solution at pH 7.4).

[0048] The test process and judgment results are as follows:

[0049] Mix 9 μL of MS-222 red colloidal gold probe with 85 μL of sample (MS-222 concentrations of 0, 1, 5, 10, and 20 μg / mL, respectively). Add 14 μL of chromatography buffer and shake for 25 seconds. The mixture is then added dropwise to the sample pad of the test strip. After a 10-minute reaction, qualitative determination is made by visually observing the color development of the T and C lines. After 20 minutes, take a photo with a smartphone and upload it to ImageJ software to extract the grayscale values ​​of the T and C lines for quantitative detection.

[0050] The results are as follows Figure 2 B. The control line is red. At concentrations of 0 and 1 μg / mL, the T line is red. When the concentration is greater than 5 μg / mL, the T line is blank. This indicates that the detection limit of the test strip for MS-222 is 1 μg / mL.

[0051] 2. MG Blue Colloidal Gold Kit

[0052] (1) The preparation method of blue colloidal gold is as follows:

[0053] Take 1.8 mL of the above-mentioned red colloidal gold solution with a particle size of 20±5 nm and add it to 50 mL of 2 mM chloroauric acid solution, heat it to 45°C, then add 12 mL of 0.1 mM p-diphenol solution and 1.5 mL of 30 mM sodium citrate solution, maintain heating and stirring at 45°C for 1 hour, and cool it to room temperature to obtain blue colloidal gold with a particle size of 80-90 nm.

[0054] (2) The preparation method of MG blue probe is as follows:

[0055] Take 600 μL of blue colloidal gold solution, add 8 μg of malachite green antibody, 8 μg of bovine serum albumin and 5 μL of 0.15 M potassium carbonate solution (pH about 8), mix on a vertical mixer for 3 hours, then centrifuge at 7000 rpm / min for 25 minutes, discard the supernatant, wash and resuspend with phosphate buffer containing 4% (v / v) Tween-20, and then centrifuge at 4000 rpm / min for 35 minutes. The washing steps are the same as above, and finally wash with 600 μL of phosphate buffer containing 0.05% (m / v) sodium azide and store at 4°C until use.

[0056] (3) The preparation method of MG immunochromatographic test strips is as follows:

[0057] A membrane streaker was used to spray T lines and C lines onto the nitrocellulose membrane at a rate of 1 μL / cm. The T line was sprayed with 0.1 mg / mL carboxymalachite green-BSA complete antigen solution, and the C line was sprayed with 0.6 mg / mL goat anti-mouse solution. The membrane was then placed in an oven at 40°C for 2 h. The sample pad, conjugate pad, nitrocellulose membrane, absorbent pad, and PVC base were assembled in sequence and sealed in an aluminum foil bag for storage.

[0058] The MG Blue Colloidal Gold Kit includes an MG Blue Colloidal Gold probe, an MG immunochromatographic test strip, and a chromatography fluid (containing 30 g / L Tween-20, 100 g / L BSA, and a 0.2 mol / L phosphate solution at pH 7.4).

[0059] The MG blue colloidal gold test strips testing process and judgment results are as follows:

[0060] Mix 9 μL of blue colloidal gold probe with 85 μL of sample, then add 14 μL of chromatography buffer (30 g / L Tween-20, 100 g / L BSA in 0.2 mol / L phosphate, pH 7.4). Vortex and mix for 25 seconds before adding the mixture dropwise to the sample pad of the test strip. After a 10-minute reaction, qualitative determination is made by visually observing the color development of the T and C lines. After 20 minutes, take a photo with a smartphone and upload the image to ImageJ software to extract the grayscale values ​​of the T and C lines for quantitative detection.

[0061] The results are as follows Figure 2 A, the control line is blue. When the MG concentration is 0 and 1 μg / mL, the T line is blue. When the concentration is greater than 2 μg / mL, the T line is not colored. This indicates that the detection limit of the test strip for MG is 1 μg / mL.

[0062] Example 2 Preparation of MS-222 & MG Dual Immunochromatographic Test Strips and Dual-Color Detection Kit

[0063] The preparation method of MS-222&MG dual test strips is as follows:

[0064] A streak was used to spray lines T1, T2, and C onto a nitrocellulose membrane at a rate of 1.2 μL / cm. Line T1 was sprayed with a 0.15 mg / mL carboxymalachite green-BSA complete antigen solution, line T2 was sprayed with a 0.25 mg / mL 3-aminobenzoic acid-BSA complete antigen solution, and line C was sprayed with a 0.8 mg / mL goat anti-mouse solution. The lines T1, T2, and C were each 6 mm apart. The membrane was then dried in an oven at 37°C for 4 h. The sample pad, conjugate pad, nitrocellulose membrane, absorbent pad, and PVC base were then assembled in sequence and sealed in an aluminum foil bag for storage.

[0065] The dual-color detection kit includes an MS-222 red colloidal gold probe, an MG blue colloidal gold probe, an MS-222 & MG dual immunochromatographic test strip, and a chromatography fluid (containing 30 g / L Tween-20, 100 g / L BSA, and a 0.2 mol / L phosphate solution at pH 7.4).

[0066] The detection process and judgment results are as follows:

[0067] Same as Example 1, except that 6 μL MG blue probe and 6 μL MS-222 red probe were added to mix with the sample. Figure 1 shown.

[0068] After the test sample is mixed with the MS-222 red colloidal gold probe and the MG blue probe, it is dropped onto the sample pad for chromatography. If the test sample does not contain MS-222 and MG, the blue probe directly captures the MG antigen on the T1 line and displays blue, and the red probe directly captures the MS-222 antigen on the T2 line and displays red. The C line will capture both the red and blue probes and display purple. If the test sample contains MS-222 and MG, the color development will be weakened because MS-222 and MG compete with the T line antigen for binding to the red probe and blue. As the MS-222 and MG content increases, the color of the T line becomes lighter. When the MS-222 result is positive, the T1 line appears blue, the T2 line does not appear, and the C line appears purple; when the MG result is positive, the T1 line does not appear, the T2 line appears red, and the C line appears purple; when the MS-222 and MG results are positive, the T1 line does not appear, the T2 line does not appear, and the C line appears purple; when the results are negative, the T1 line appears red, the T2 line appears blue, and the C line appears purple.

[0069] After the chromatography in step 1 is completed, the color of T1 line, T2 line and C line is observed with naked eyes. The specific judgment is as follows: Figure 1 As shown (Note: If the C line does not show color, it is invalid); take a photo with a smartphone and upload it to ImageJ software. Through image processing, the gray value is measured to achieve the purpose of quantitative detection.

[0070] Example 3 Detection of MS-222 and MG in aquaculture water

[0071] The MS-222 & MG dual test strip prepared by the present invention was used to detect MS-222 and MG in water samples:

[0072] Artificially contaminated water samples were prepared by diluting MS-222 and MG standards to 0.5, 2.5, and 5.0 μg / mL, respectively, using aquaculture water. After filtration through a 0.22 μm membrane, these samples were tested using HPLC and MS-222 test strips, respectively. The results are shown in Tables 1 and 2. The HPLC and test strip methods showed good consistency, demonstrating that the test strips can accurately and reliably detect MS-222 in real water samples.

[0073] Example 4 Detection of MS-222 and MG in fish

[0074] The MS-222 & MG dual test strip prepared by the present invention was used to detect MS-222 and MG in fish meat:

[0075] Fish meat was added with MS-222 and MG to concentrations of 0.5, 2.5, and 5.0 μg / g. After extraction with acetonitrile and ultrasonication for 10 minutes, the extract was centrifuged at 4000 rpm for 10 minutes. The supernatant was then added with a scavenger (N-propylethylenediamine, sodium chloride, magnesium sulfate, or graphite carbon) and ultrasonicated for 3 minutes. The extract was then centrifuged at 4000 rpm for 10 minutes. The supernatant was then air-dried and reconstituted with pH 7.4 phosphate buffer. The extract was then tested using MS-222 & MG dual-element test strips. The test results are shown in Tables 1 and 2, and were consistent with the HPLC results.

[0076] Example 5 Detection of MS-222 and MG in shrimp meat

[0077] The MS-222 & MG dual test strip prepared by the present invention was used to detect MS-222 and MG in shrimp meat:

[0078] The method was the same as in Example 4, except that the substrate was shrimp meat. The test results are shown in Tables 1 and 2, and the test strip test results were consistent with the HPLC results.

[0079] Example 6 Specificity of MS-222 & MG Dual Test Strip

[0080] The MS-222&MG dual test strip prepared by the present invention was subjected to a specificity test:

[0081] MS-222 and different fishing anesthetics such as bupivacaine hydrochloride, benzocaine, lidocaine, and eugenol were diluted to 10 μg / mL with PBS buffer. MG and different dyes such as recessive malachite green, patent blue, and brilliant green were diluted to 10 μg / mL with PBS buffer. The above solutions were tested with test strips, and the specificity of the test strips was analyzed based on the color development. The results are as follows. Figure 3 Except for MS-222, MG and the mixture of the two, the other test results were negative, indicating that the test strip is not interfered by similar substances and has good specificity.

[0082] Table 1 MS-222 spike recovery test results

[0083]

[0084] Table 2 MG spike recovery test results

[0085]

Claims

1. A method for rapid and simultaneous detection of MS-222 and malachite green, characterized in that the steps include: (1) Mix the red probe and blue probe with the sample to be tested and the chromatography fluid, and then drop them onto the sample pad of the immunochromatographic test paper; The preparation method of the red probe and the blue probe is as follows: the red nano-colloidal gold particles are connected to the MS-222 antibody, and the blue nano-colloidal gold particles are connected to the malachite green antibody, the particle size of the red nano-colloidal gold particles is 10-40 nm, and the particle size of the blue nano-colloidal gold particles is 75-100 nm; The immunochromatographic test paper is provided with a test line 1, a test line 2 and a quality control line; the test line 1 is coated with carboxylated malachite green-BSA complete antigen, the test line 2 is coated with 3-aminobenzoic acid-BSA complete antigen; the quality control line is coated with goat anti-mouse IgG; The chromatography fluid is a phosphate buffer, borate buffer or tris-aminomethane buffer containing 20-50 g / L Tween-20 and 0-300 g / L BSA, pH = 7.3-7.5; (2) Observe the color of the test strip: if the test line 1 is blue, the test line 2 is red, and the quality control line is purple, the test sample does not contain MS-222 and malachite green; if the test line 1 is blue, the test line 2 is not colored, and the quality control line is purple, the test sample contains MS-222; if the test line 1 is not colored, the test line 2 is red, and the quality control line is purple, the test sample contains malachite green.

2. The method according to claim 1, characterized in that The preparation method of the red nano colloidal gold particles comprises the following steps: Mix boiling chloroauric acid solution and sodium citrate solution, heat and stir for 5-20 minutes; the concentration of chloroauric acid in the reaction system is 0.5-5 mmol / L, and the concentration of sodium citrate is 3-40 mmol / L; The preparation method of blue colloidal gold nanoparticles comprises the following steps: mixing red colloidal gold particles with a particle size of 10-40 nm with a chloroauric acid solution, a sodium citrate solution and a p-diphenol solution and heating; in the reaction system, the molar ratio of red colloidal gold to chloroauric acid is 1:40-150; The molar ratio of red colloidal gold to chloroauric acid is 1:50-100.

3. The method according to claim 1 or 2, characterized in that The particle size of the red nano-colloidal gold particles is 15-25 nm, and the particle size of the blue nano-colloidal gold particles is 80-90 nm.

4. The method according to claim 1, wherein In step (2), qualitative observation is performed by naked eye, or grayscale values ​​are extracted and quantitatively measured after taking a photo.

5. The method according to claim 1, wherein The preparation method of the blue probe or the red probe is as follows: taking a red colloidal gold solution or a blue colloidal gold solution, adding one of a malachite green antibody or an MS-222 antibody, as well as BSA and a carbonate solution, mixing evenly, and then centrifuging to obtain a precipitate for washing; the carbonate is potassium carbonate or sodium carbonate, and the carbonate concentration in the reaction system is 0.001-0.002 mol / L.

6. The method according to claim 5, characterized in that The pH value of the reaction system is 6-10.

7. A dual-color detection kit for rapid and simultaneous detection of MS-222 and malachite green, characterized in that: Includes immunochromatographic test strips, red probes, and blue probes; The preparation method of the red probe and the blue probe is as follows: the red nano-colloidal gold particles are connected to the MS-222 antibody, and the blue nano-colloidal gold particles are connected to the malachite green antibody, the particle size of the red nano-colloidal gold particles is 10-40 nm, and the particle size of the blue nano-colloidal gold particles is 75-100 nm; The immunochromatographic test paper is provided with a test line 1, a test line 2 and a quality control line; the test line 1 is coated with carboxylated malachite green-BSA complete antigen, the test line 2 is coated with 3-aminobenzoic acid-BSA complete antigen; the quality control line is coated with goat anti-mouse IgG; The method further comprises a chromatography fluid, which is a phosphate buffer, a borate buffer or a tris-aminomethane buffer containing 20-50 g / L Tween-20 and 0-300 g / L BSA and a pH of 5.0-7.5.

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