Method, test strip and kit for detecting chloramphenicol in livestock hair based on quantum dot microsphere immunochromatography

Abstract

The invention discloses a method, a test strip and a kit for detecting chloramphenicol in livestock hair based on quantum dot microsphere immunochromatography, and relates to the technical field of biology. The test strip comprises a bottom plate, a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad, the nitrocellulose membrane is attached to the bottom plate, the water absorption pad and the combination pad are tightly connected with the two ends of the nitrocellulose membrane in a pressing mode respectively, and the sample pad is tightly connected with the end, away from the nitrocellulose membrane, of the combination pad in a pressing mode; a detection line and a quality control line are sequentially arranged on the nitrocellulose membrane from one end close to the conjugate pad to one end far away from the conjugate pad; the detection line is coated with an antigen; the quality control line is coated with a goat anti-mouse second antibody; the combination pad is provided with a CAP-QM monoclonal antibody probe; the CAP-QM monoclonal antibody probe is obtained by coupling a quantum dot microsphere and a chloramphenicol monoclonal antibody. The test strip can be used for detecting chloramphenicol in animal hair simply, conveniently, quickly and highly sensitively.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method, a test strip and a kit for detecting chloramphenicol in livestock hair based on quantum dot microsphere immunochromatography. Background Art

[0002] Chloramphenicol (CAP) was isolated from the culture medium of soil microorganisms by the University of Illinois in the United States in 1947. CAP has broad-spectrum antibacterial properties, stable properties, low price, and is sensitive to a variety of bacterial pathogens. It was once widely used in livestock and aquaculture, and can effectively treat animal-related diseases such as swine fever, avian influenza, duck cholera and other diseases. Studies have shown that CAP is difficult to degrade in nature and can be enriched in the human body through the food chain, causing serious adverse reactions, such as inhibiting the synthesis of mitochondrial proteins in bone marrow cells, allergic reactions, reproductive system damage, neurotoxicity, and easy to cause "gray baby syndrome". Long-term consumption of foods with CAP residues can cause intestinal flora imbalance and induce digestive dysfunction. The United States, Japan, the European Union, as well as the World Health Organization and the United Nations Organization (WHO) have all prohibited the use of CAP in animal husbandry and aquaculture. On December 27, 2019, the Ministry of Agriculture and Rural Affairs of the People's Republic of China issued Announcement No. 250, and chloramphenicol was included in the list of drugs and other compounds prohibited for use in food animals. On October 27, 2017, the World Health Organization's International Agency for Research on Cancer published a preliminary list of carcinogens for reference, and chloramphenicol was on the list of Class 2A carcinogens.

[0003] In order to effectively regulate CAP residues in food, the country has issued more than 20 national standards and industry standards for CAP detection. The detection objects include milk, livestock meat, honey, aquatic products, etc.; the detection methods include liquid chromatography tandem mass spectrometry, gas chromatography mass spectrometry, gas chromatography, sensor technology, enzyme-linked immunosorbent assay, etc. Due to the complex operation and high cost, these methods cannot be used for on-site live detection of animals during breeding (especially large animals such as pigs, cattle, and sheep) and simple and rapid analysis of a large number of samples. Quantum dot microsphere fluorescent immunochromatography technology is a new type of immunofluorescence labeling technology. It is a highly sensitive rapid detection method established by combining the high specificity of immunology with the strong fluorescence characteristics of quantum dot microspheres. It not only has the quantitative advantages of enzyme-linked immunosorbent assay, but also has the advantages of rapid, simple and easy operation of colloidal gold method, and can be used for on-site live animal detection. The present invention intends to develop a quantum dot microsphere immunochromatography method to achieve low-cost, convenient, rapid, large-scale and live detection and analysis of CAP residues in animal breeding.

[0004] Developing a fast, efficient and accurate CAP residue detection method and realizing efficient supervision of CAP are of great significance for effectively ensuring food safety. Large animals have high monomer value (such as pigs, cattle, sheep, etc.), so it is very important to monitor their living drug use during the breeding process. Studies have shown that CAP residues are easily stored in animal hair, and hair can be used as a detection tissue for CAP in livestock. Therefore, taking hair as a monitoring tissue can not only avoid slaughtering animals, but also reduce sampling costs, and is convenient and fast, achieving the effect of non-destructive sampling, and breaking through the problem of difficult animal sample sampling in traditional detection, and can effectively reflect the illegal use of CAP in animals and supervise the occurrence of illegal drug use. For this reason, the present invention intends to develop a detection method and product that can simply, quickly and highly sensitively detect chloramphenicol residues in animal hair, and judge whether there is illegal drug use in its breeding process by detecting the accumulation of drugs in livestock hair. Summary of the invention

[0005] The purpose of the present invention is to provide a method, test strip and kit for detecting chloramphenicol in livestock hair based on quantum dot microsphere immunochromatography to solve the problems existing in the above-mentioned prior art. The method is suitable for rapid and large-scale non-destructive detection of chloramphenicol in livestock hair, with a quantitative detection range of 0.01-0.09 ng / mg, a cross-reaction rate with common chloramphenicol drugs thiamphenicol and florfenicol of 0.0%, a spike recovery rate of 86%-109%, and an RSD of 2.79%-13.89%.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a test strip for detecting chloramphenicol in livestock hair based on quantum dot microsphere immunochromatography, comprising a bottom plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad; the nitrocellulose membrane is attached to the bottom plate, the water-absorbing pad and the conjugation pad are respectively tightly pressed against two ends of the nitrocellulose membrane, and the sample pad is tightly pressed against one end of the conjugation pad away from the nitrocellulose membrane;

[0008] The nitrocellulose membrane is provided with a detection line and a quality control line in sequence from one end close to the conjugate pad to the end far from the conjugate pad;

[0009] The detection line is coated with antigen; the quality control line is coated with goat anti-mouse secondary antibody;

[0010] The conjugate pad carries a CAP-QM monoclonal antibody probe;

[0011] The CAP-QM monoclonal antibody probe is obtained by coupling quantum dot microspheres and chloramphenicol monoclonal antibodies.

[0012] Furthermore, the quantum dot microspheres and the chloramphenicol monoclonal antibody are coupled in a buffer solution, and the pH of the buffer solution is 6.0.

[0013] Furthermore, the quantum dot microspheres and the chloramphenicol monoclonal antibody are coupled using a coupling agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide in the coupling system is 20 μg / mg.

[0014] Furthermore, the concentration of the chloramphenicol monoclonal antibody in the coupling system is 20 μg / mg.

[0015] Furthermore, the detection line is obtained by spraying a chloramphenicol antigen solution onto the nitrocellulose membrane; the concentration of the chloramphenicol antigen solution is 1.0 mg / mL.

[0016] The present invention also provides the use of the above-mentioned test strip in preparing a kit for detecting chloramphenicol in livestock hair based on quantum dot microsphere immunochromatography.

[0017] The present invention also provides a kit for detecting chloramphenicol in livestock hair based on quantum dot microsphere immunochromatography, comprising the above-mentioned test strip.

[0018] The present invention also provides the use of the above-mentioned test strip or test kit in detecting chloramphenicol in livestock hair.

[0019] The present invention also provides a method for qualitatively detecting chloramphenicol in livestock hair, comprising the following steps:

[0020] The livestock hair sample to be tested is ground, dissolved in a hair lysis solution, and then allowed to stand to collect the supernatant to obtain a liquid sample to be tested;

[0021] The liquid sample to be tested is dripped into the sample pad of the above-mentioned test strip, and after chromatography treatment, the fluorescent signal is observed, and the following judgment is made based on the observation results:

[0022] If fluorescent bands appear on both the quality control line and the test line, it is determined that chloramphenicol is not detected;

[0023] If the control line shows a fluorescent band, but the test line shows no fluorescent band, it is determined that chloramphenicol is detected;

[0024] If the quality control line has no fluorescent strip, the test result is judged to be invalid regardless of whether the test line has a fluorescent strip.

[0025] The present invention also provides a method for quantitatively detecting chloramphenicol in livestock hair, comprising the following steps:

[0026] The livestock hair sample to be tested is ground, dissolved in a hair lysis solution, and then allowed to stand to collect the supernatant to obtain a liquid sample to be tested;

[0027] The liquid sample to be tested is dripped into the sample pad of the above-mentioned test strip, and after chromatography treatment, the fluorescent signal is read by a fluorescent immunoassay analyzer to obtain the signal intensity of the quality control line and the test line, and the T / C value is calculated;

[0028] Substitute the T / C value into the standard curve to calculate the chloramphenicol concentration;

[0029] The standard curve is constructed by using hair lysate containing chloramphenicol standard substances of different concentrations.

[0030] The present invention discloses the following technical effects:

[0031] The present invention selects quantum dot fluorescent microspheres with high luminescence intensity as signal markers, and establishes a quantum dot microsphere immunochromatography method for rapid, sensitive and nondestructive detection of chloramphenicol in livestock hair by optimizing the coupling conditions of fluorescent immunoprobes. A standard curve is established by the fluorescence signal intensity of the detection line and the quality control line on the test strip, with the chloramphenicol concentration as the horizontal coordinate and the ratio (T / C) of the fluorescence signal intensity of the detection line (T) and the quality control line (C) as the vertical coordinate. The results show that the quantitative detection range of this method is 0.01-0.09 ng / mg, and the cross-reaction rate with common chloramphenicol drugs thiamphenicol and florfenicol is 0.0%. The constructed test strip is applied to the detection of chloramphenicol in pig hair, cattle hair and wool, and the spike recovery rate is 86%-109%, and the RSD is 2.79%-13.89%. This method is suitable for rapid, large-scale and nondestructive detection of chloramphenicol in livestock hair. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 This is the structure and principle diagram of the quantum dot microsphere immunochromatographic test strip;

[0034] Figure 2 Characterization diagrams of QM and CAP-QM monoclonal antibody probe (QM-mAb); A is the TEM image of QM; B is the particle size statistics of QM and QM-mAb; C is the Zeta potential image of QM and QM-mAb; the scale in A is 200 nm;

[0035] Figure 3This is a statistical chart of the optimization results of the pH conditions for coupling antibodies to the QM surface;

[0036] Figure 4 This is a statistical graph of experimental results showing the effect of the amount of EDC used to couple antibodies to the QM surface on the T / C value. DETAILED DESCRIPTION

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] Example 1

[0043] 1. Experimental Methods

[0044] 1.1 Instruments and reagents

[0045] XYZ3000 film dot analyzer (Bio-Dot, USA); Millipore ultrapure water system (Millipore, USA); FD-2000 test card constant temperature incubator (Shanghai Femdetection Company); JEM-2100 transmission electron microscope (TEM, JEOL, Japan); Zetasizer Nano ZS particle size analyzer (Malvern, UK); AF-100 dry fluorescent immunoassay analyzer (Jiangxi Weibang Biotechnology Company); FS-1200 ultrasonic processor (Shanghai Shengxi Ultrasonic Instrument Co., Ltd.); analytical balance (Mettler-Toli, USA); vacuum drying oven (Shanghai Senxin Experimental Instrument Co., Ltd.).

[0046] Quantum dot microspheres (QM, 15 mg / mL, particle size 200 nm, excitation wavelength 365 nm, emission wavelength 610 nm); chloramphenicol antibody, chloramphenicol-conjugated antigen and goat anti-mouse secondary antibody (Jiangxi Weibang Biotechnology Co., Ltd.); 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, Sigma, USA); bovine serum albumin (BSA, Sigma, USA); CN-95 nitrocellulose membrane (NC membrane, Sartorius, USA); absorbent paper, conjugate pad and sample pad (Shanghai Goldbio Co., Ltd.); chloramphenicol, thiamphenicol and florfenicol standards (Sigma, USA); phosphate buffer (PB, self-prepared); phosphate buffered saline (PBS, self-prepared); hair lysis buffer (1 mg / mL proteinase K, 0.5% Tween-20, 0.5% sodium chloride dissolved in Tris buffer, pH 8.0).

[0047] 1.2 Experimental methods

[0048] 1.2.1 Preparation of CAP-QM monoclonal antibody probe

[0049] Take 10 μL quantum dot microspheres (QM, 0.15 mg; purchased from Jiangxi Weibang Biotechnology Co., Ltd., item number: AQM-250) and add 1.5 mL PB buffer (0.01 mol / L, pH 6.0), and ultrasonicate for 2 min; add 9 μL of freshly prepared 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) solution (0.5 mg / mL EDC, 30 μg / mg QM), and activate at 37°C 150 r / min for 15 min. Then add the diluted chloramphenicol antibody to make the final concentration of the antibody in the reaction system 20 μg / mg, and oscillate at 37°C 150 r / min for 1 h; add 150 μL BSA blocking solution dropwise, and react at 37°C 150 r / min for 1 h. After the reaction is completed, centrifuge at 14000 r / min for 15 min, and take the precipitate and re-dissolve it to 200 μL with QM re-solution, and ultrasonicate for 15 min. The reconstituted quantum dot antibody probe is evenly sprayed onto the conjugate pad, dried overnight, and set aside.

[0050] 1.2.2 Characterization of QM and probes

[0051] The morphology of QM was characterized by transmission electron microscopy, and the hydrated particle size and Zeta potential of QM and CAP-QM monoclonal antibody probe were characterized by a particle size meter.

[0052] 1.2.3 Preparation of test strips

[0053] The immunochromatographic test strip prepared by the present invention comprises a bottom plate, a sample pad, a conjugation pad, a nitrocellulose (NC) membrane and a water-absorbing pad; the nitrocellulose membrane is attached to the bottom plate, the water-absorbing pad and the conjugation pad are respectively tightly pressed against two ends of the nitrocellulose membrane, and the sample pad is tightly pressed against one end of the conjugation pad away from the nitrocellulose membrane;

[0054] A test line (T line) and a control line (C line) are sequentially arranged on the nitrocellulose membrane from one end close to the conjugate pad to the end far from the conjugate pad;

[0055] The test line is coated with antigen; the quality control line is coated with goat anti-mouse secondary antibody;

[0056] The conjugate pad carries the CAP-QM monoclonal antibody probe.

[0057] T line: Chloramphenicol antigen was diluted to 1.0 mg / mL with 0.01 mol / L PBS buffer (pH 7.4), and the diluted solution was sprayed onto the NC membrane using a film dispenser as the T line, with a spraying amount of 0.74 μL / cm.

[0058] Line C: Dilute the goat anti-mouse secondary antibody to 0.1 mg / mL with 0.01 mol / L PBS buffer (pH 7.4), and spray the diluted solution onto the NC membrane using a film dispenser as line C. The spraying amount was 0.74 μL / cm.

[0059] Place the NC membrane sprayed with T lines and C lines in a 37°C constant temperature drying oven and dry overnight. Then paste the sample pad, conjugate pad, NC membrane, and absorbent pad in sequence, and cut them into test strips using a strip cutter after assembly. Figure 1 As shown, install it into the plastic card shell.

[0060] 1.2.4 Detection methods

[0061] Use a pipette to draw 90 μL of the test solution and add it to the sample well of the test card. Incubate the reaction at 37°C for 10 minutes, then immediately read the fluorescence signal with a dry fluorescent immunoassay analyzer to obtain the signal intensity of the T line and C line, and calculate the T / C value.

[0062] 1.2.5 Optimization of fluorescent immunoprobe coupling conditions

[0063] The fluorescent immunoprobe coupling conditions were optimized with probe coupling buffer pH (5.0, 6.0, 7.0, 8.0), EDC dosage (10, 20, 40, 80 μg / mg), chloramphenicol antibody dosage (10, 20, 50 μg / mg), and T-line antigen concentration (0.2, 0.5, 1.0 mg / mL) as single factors, and the optimal conditions were determined based on the ratio of the fluorescence signal intensities of the T line and C line (T / C value) and the inhibition rate.

[0064] 1.2.6 Establishment of standard curve

[0065] Prepare the test strips under the optimal process conditions, assemble the test cards into finished products, use hair lysis solution to prepare chloramphenicol standards into 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 μg / L, and perform the test according to step 1.2.4. Use the concentration of the standard as the horizontal axis and the T / C value as the vertical axis to establish a standard curve. And by adding the spiked negative animal hair before treatment, the coefficient of the buffer standard curve was corrected as the real sample matrix standard curve.

[0066] 1.3 Quantitative detection of chloramphenicol in livestock hair by quantum dot microsphere immunoassay

[0067] 1.3.1 Sample pretreatment

[0068] Use medical scissors or serrated scissors to closely follow the hair within 3 cm of the animal skin surface to be extracted; for hair longer than 3 cm, 3 cm should be cut from the root end; the extracted hair samples should be stored at room temperature, away from light, dry, ventilated, and clean. Take 10 mg of animal hair and place it in a hair grinding tube, cover the tube tightly, and balance it in the grinder for 3 minutes; add 2 mL of hair lysis solution, mix manually for 30 seconds, and let it stand for 1 minute. The supernatant is the test solution.

[0069] 1.3.2 Standard curve drawing

[0070] Set three sample spike concentrations of 0.01, 0.02, and 0.04 ng / mg, test each concentration three times with a test strip, and draw a standard curve.

[0071] 1.3.3 Method accuracy and precision

[0072] Accurately weigh 10mg of negative animal hair (pig hair, cow hair, wool) sample, grind it in a grinder for 3min, spike the sample to 0.01, 0.02, 0.04ng / mg, add 2mL hair lysis solution and shake it manually for 30s, let it stand for 1min, take 90μL of supernatant and add it to the test card, incubate the reaction at 37℃ for 10min, and read the result with a dry fluorescent immunoassay. Each sample was tested 3 times to verify the accuracy and precision of the method.

[0073] 1.3.4 Method Specificity

[0074] Add chloramphenicol drugs thiamphenicol and florfenicol to the blank sample, and prepare them at 0.1, 1.0, and 10.0 μg / L respectively. Chloramphenicol is prepared at 0.1 μg / L as a control. Use the prepared test strips for detection, and repeat each test 3 times. Calculate the cross-reaction rate by the following formula:

[0075] Cross-reaction rate (%) = (chloramphenicol concentration / chloramphenicol structural analog concentration) × 100%

[0076] 2 Results and discussion

[0077] 2.1 Characterization of QM and CAP-QM probes

[0078] The transmission electron microscopy results of quantum dot microspheres are as follows Figure 2 As shown in Figure A, the TEM results reflect the internal structure and surface morphology of the quantum dot microspheres. The prepared QMs are evenly distributed inside the microspheres. They are regular spheres with uniform size. Figure 2 From the particle size distribution diagram of B, it can be seen that the hydrated particle size of QM-mAb is 304nm, and the hydrated particle size of QM is 268nm, which increases by 36nm. Figure 2The Zeta potential diagram of C in the figure shows that the Zeta potential of QM before and after antibody coupling increased from -37mV to -23mV. In summary, the characterization results show that the CAP antibody was successfully coupled to the QM surface, with consistent particle size and uniform distribution, forming a fluorescent probe.

[0079] 2.2 Optimization of fluorescent immunoprobe coupling conditions

[0080] The fluorescent immunoprobe was prepared by activating the carboxyl groups on the surface of fluorescent microspheres by the EDC method and then reacting with the amino groups on the surface of antibodies to form covalent bonds. The effects of probe coupling buffer pH, EDC dosage, chloramphenicol antibody dosage, and T-line antigen concentration on the fluorescence intensity and inhibition rate of the synthetic fluorescent probe C-line and T-line were investigated.

[0081] 2.2.1 Buffer pH

[0082] In the preparation of fluorescent immunoprobes, the antibody coupling efficiency and the biological activity of the antibody are related to the pH of the coupling buffer system. The higher the biological activity of the coupled probe, the more probes are bound at the T line of the test strip, and the higher the fluorescence intensity of the T line. The chloramphenicol antibody labeling amount was set to 20 μg / mg, and the coupling buffer system pH 5.0, 6.0, 7.0, and 8.0 were investigated. The results are as follows: Figure 3 As shown in the figure, when the pH of the buffer solution increases from 5.0 to 6.0, the fluorescence intensity of the T line gradually increases, and the inhibition rate gradually increases. The inhibition rate reaches the highest at pH = 6, and the negative fluorescence intensity signal of the T line reaches the highest at this time. When the pH of the buffer solution further increases to 8.0, the fluorescence intensity of the T line gradually weakens, and the fluorescence intensity of the C line gradually increases; the excessively high pH in the system will affect the activation of the carboxyl group by EDC, thereby reducing the coupling efficiency of the microspheres and antibodies. Combined with the fluorescence intensity signal and the inhibition rate, the optimal coupling buffer pH is selected as 6.0.

[0083] 2.2.2 EDC dosage

[0084] The amount of EDC in the coupling system will affect the biological activity of the fluorescent probe. When the amount of antibody is constant, a low amount of EDC will reduce the antibody coupling efficiency, thereby reducing the biological activity of the coupled probe; a high amount of EDC will lead to over-activation of the fluorescent microspheres, thereby causing probe aggregation and affecting the probe coupling effect. The chloramphenicol antibody labeling amount was set to 20 μg / mg, and the effect of EDC dosage of 10, 20, 40, and 80 μg / mg on the system was explored. The results are as follows: Figure 4As shown. When the EDC dosage increased from 10μg / mg to 20μg / mg, the fluorescence intensity of the T line and the C line gradually increased and reached the highest value at 20μg / mg. At this time, the inhibition rate also reached the highest value of 57.02%. However, with the further increase in the EDC dosage, the fluorescence intensity of the T line gradually decreased. When the EDC dosage increased from 20μg / mg to 40μg / mg, the fluorescence intensity of the T line decreased by about 35%, indicating that excessive EDC dosage will reduce the biological activity of the coupled probe. In summary, the optimal dosage of coupled EDC for the fluorescent immunoprobe in the present invention is 20μg / mg.

[0085] 2.2.3 Optimization of antibody dosage and T-line spray volume

[0086] In the covalent coupling reaction of quantum dot microspheres and antibodies, if the amount of antibody is small, the binding rate between antigen and antibody will be reduced; if the amount of antibody is too large, a steric hindrance effect will be produced, which will weaken the competition between CAP and CAP-BSA in the sample and reduce the sensitivity. The effects of antibody dosage and T-line spray volume on the fluorescence intensity and inhibition rate of T-line and C-line were investigated, and the results are shown in Table 1. The optimal dosage combination is: chloramphenicol antibody concentration 20μg / mg, T-line spray volume 1.0mg / mL. Under this condition, the inhibition rate of 0.1ng / mL sample is 59.09%.

[0087] Table 1 Optimization results of antibody dosage and T-line spray volume

[0088]

[0089] 2.3 Standard curve

[0090] Under the optimal conditions, i.e., probe coupling buffer pH 6.0, EDC dosage 20 μg / mg, chloramphenicol antibody concentration 20 μg / mg, T-line antigen concentration 1.0 mg / mL, chloramphenicol concentrations were set to 0, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 μg / L, and the experiment was performed according to the method in Section 1.2.6. The standard curve was established with chloramphenicol concentration as the horizontal axis (x) and the ratio of the fluorescence signal intensity of the T line and the C line (T / C) as the vertical axis (y). The linear regression equation was y=2.44768×exp(-x / 0.11598)+0.15258, and the regression coefficient R 2 The actual animal hair samples were treated with hair lysis buffer and then spiked. The coefficient of the buffer standard curve was corrected. The results are shown in Table 2. The concentration range of 40-90% inhibition rate was selected as the quantitative detection range. The quantitative detection range of this method is 0.01-0.09 ng / mg.

[0091] Table 2 Actual sample standard curve data

[0092]

[0093]

[0094] 2.4 Quantitative detection of chloramphenicol in livestock hair by quantum dot microsphere immunoassay

[0095] Negative pig hair, cow hair and wool samples were used for spike experiments, and the spike concentrations were 0.01, 0.02, and 0.04 ng / mg. As shown in Table 3, the recoveries of the three different spike concentrations of the above samples were between 86% and 109%, and the RSDs were between 2.79% and 13.89%, indicating that the accuracy and precision of the method were good.

[0096] Table 3 Recovery and relative standard deviation of pig hair, cattle hair and wool samples detected by quantum dot microsphere fluorescence immunochromatography

[0097]

[0098] 2.5 Method Specificity

[0099] To verify the specificity of this method for chloramphenicol detection, common chloramphenicol drugs Thiamphenicol and Fluorfenicol were added to the blank sample, tested under optimal conditions, T / C values ​​were calculated, and the standard curve was substituted to calculate the test results and cross-reaction rate. As shown in Table 4, the cross-reaction rate of this quantum dot microsphere immunochromatography method for chloramphenicol was 100%, and the cross-reaction rates of thiamphenicol and florfenicol were both 0.00%, indicating that the specificity of this method is good.

[0100] Table 4 Specificity of quantum dot microsphere immunochromatography

[0101]

[0102] 3 Conclusion

[0103] The present invention uses 200nm quantum dot fluorescent microspheres as markers to establish a new method for rapid detection of chloramphenicol in livestock hair using a fluorescent immunochromatographic test strip. Under optimal process conditions, the quantitative detection range of the chloramphenicol quantitative detection test strip is 0.01-0.09ng / mg, and the cross-reaction rates with common chloramphenicol drugs sulfamphenicol and florfenicol are both 0.0%. The recovery rate of spiked samples of actual samples of pig hair, cattle hair and wool is 86%-109%, and the relative standard deviation (RSD) is 2.79%-13.89%, indicating that the method has good accuracy and precision; it has great application prospects in the rapid and non-destructive detection of chloramphenicol in large livestock.

[0104] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A test strip for detecting chloramphenicol in livestock hair based on quantum dot microsphere immunochromatography, characterized in that: It comprises a bottom plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad; the nitrocellulose membrane is attached to the bottom plate, the water-absorbing pad and the conjugation pad are respectively tightly pressed against two ends of the nitrocellulose membrane, and the sample pad is tightly pressed against one end of the conjugation pad away from the nitrocellulose membrane; The nitrocellulose membrane is provided with a detection line and a quality control line in sequence from one end close to the conjugate pad to the end far from the conjugate pad; The detection line is coated with antigen; the quality control line is coated with goat anti-mouse secondary antibody; The conjugate pad carries a CAP-QM monoclonal antibody probe; The CAP-QM monoclonal antibody probe is obtained by coupling quantum dot microspheres and chloramphenicol monoclonal antibodies.

2. The test strip according to claim 1, characterized in that: The quantum dot microspheres and the chloramphenicol monoclonal antibody are coupled in a buffer solution, and the pH value of the buffer solution is 6.

0.

3. The test strip according to claim 1, characterized in that: The quantum dot microspheres and the chloramphenicol monoclonal antibody are coupled using a coupling agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the concentration of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide in the coupling system is 20 μg / mg.

4. The test strip according to claim 3, characterized in that: The concentration of the chloramphenicol monoclonal antibody in the coupling system is 20 μg / mg.

5. The test strip according to claim 1, characterized in that: The detection line is obtained by spraying a chloramphenicol antigen solution onto the nitrocellulose membrane; the concentration of the chloramphenicol antigen solution is 1.0 mg / mL.

6. Use of the test strip according to any one of claims 1 to 5 in preparing a kit for detecting chloramphenicol in livestock hair based on quantum dot microsphere immunochromatography.

7. A kit for detecting chloramphenicol in livestock hair based on quantum dot microsphere immunochromatography, characterized in that: The invention comprises the test strip according to any one of claims 1 to 5.

8. Use of the test strip according to any one of claims 1 to 5 or the kit according to claim 7 in detecting chloramphenicol in livestock hair.

9. A method for qualitatively detecting chloramphenicol in livestock hair, characterized in that: The following steps are involved: The livestock hair sample to be tested is ground, dissolved in a hair lysis solution, and then allowed to stand to collect the supernatant to obtain a liquid sample to be tested; The liquid sample to be tested is dripped into the sample pad of the test strip according to any one of claims 1 to 5, and after chromatography treatment, the fluorescent signal is observed, and the following judgment is made based on the observation result: If fluorescent bands appear on both the quality control line and the test line, it is determined that chloramphenicol is not detected; If the control line shows a fluorescent band, but the test line shows no fluorescent band, it is determined that chloramphenicol is detected; If the quality control line has no fluorescent strip, the test result is judged to be invalid regardless of whether the test line has a fluorescent strip.

10. A method for quantitatively detecting chloramphenicol in livestock hair, characterized in that: The following steps are involved: The livestock hair sample to be tested is ground, dissolved in a hair lysis solution, and then allowed to stand to collect the supernatant to obtain a liquid sample to be tested; The liquid sample to be tested is added dropwise to the sample pad of the test strip according to any one of claims 1 to 5, and after chromatography, the fluorescent signal is read by a fluorescent immunoassay analyzer to obtain the signal intensity of the quality control line and the test line, and the T / C value is calculated; Substitute the T / C value into the standard curve to calculate the chloramphenicol concentration; The standard curve is constructed by using hair lysate containing chloramphenicol standard substances of different concentrations.

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