CN113030474A - Test strip for detecting golden orange II drug, preparation method and application thereof - Google Patents
Test strip for detecting golden orange II drug, preparation method and application thereof Download PDFInfo
- Publication number
- CN113030474A CN113030474A CN201911348490.3A CN201911348490A CN113030474A CN 113030474 A CN113030474 A CN 113030474A CN 201911348490 A CN201911348490 A CN 201911348490A CN 113030474 A CN113030474 A CN 113030474A
- Authority
- CN
- China
- Prior art keywords
- colloidal gold
- solution
- immune
- paper
- golden orange
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention discloses a test strip for detecting a golden orange II medicament, a preparation method and application thereof, wherein the test strip comprises a substrate, a sample filtering paper, an immune colloidal gold paper sheet, an immune nitrocellulose membrane and absorbent paper, wherein the sample filtering paper, the immune colloidal gold paper sheet, the immune nitrocellulose membrane and the absorbent paper are sequentially connected end to end and are fixed on the substrate; the immune colloidal gold paper sheet is provided with a colloidal gold labeled anti-golden orange II monoclonal antibody, and the immune nitrocellulose membrane is provided with a detection line coated with a golden orange II-bovine serum albumin compound and a quality control line coated with goat anti-rabbit IgG. The test strip disclosed by the invention needs the environment temperature of 4-35 ℃ for use, is suitable for rapid detection of the component II of the golden orange in individuals, factories, food sanitation and quality inspection departments and the like, and has the advantages of strong specificity, high sensitivity, simplicity and convenience in operation and the like.
Description
Technical Field
Belongs to the field of food safety detection, and particularly relates to a test strip for detecting a golden orange II medicament, a preparation method and application thereof.
Background
The golden orange II, also known as acid orange II, second orange, orange yellow orange or acid golden yellow II, is commonly known as golden yellow powder, has a chemical name of 2-naphthol azo sodium p-benzenesulfonate, is a non-biosynthesized colorant, and is easily soluble in ethanol, acetonitrile and the like. Is a chemical dye, is mainly used for dyeing leather and textiles, and is forbidden to be used as a food additive. But the red brown rice has the characteristics of bright color, stable coloring, low price and the like, and illegal vendors use the red brown rice for producing and processing medicinal materials, thereby seriously harming the health of consumers.
The golden yellow powder has strong toxicity and carcinogenicity, and is strictly prohibited to be used for food coloring in relevant regulations at home and abroad. However, in recent years, some manufacturers have been reported to abuse the dye in foods such as baked goods, and the like, and have serious food safety hazards. At present, most of the gold orange II detection methods are high performance liquid chromatography, liquid chromatography-tandem mass spectrometry, reversed phase high performance liquid chromatography and the like. Methods such as high performance liquid chromatography and liquid chromatography-mass spectrometry are the most advanced modern analysis means, can well separate and detect the golden orange II, and reach a very low detection limit, and the sensitivity is very high. By adopting a standard substance comparison method, the content of the golden orange II in the sample can be detected very accurately.
In addition, no quick detection reagent box of golden orange II is found in the market, and if the experimental method is adopted, the operation process is complicated, the experimental time is long, the experimental cost is high, and the method is not suitable for field detection. The method has obvious advantages in detection steps and detection cost.
Disclosure of Invention
The invention aims to provide a test strip for detecting a golden orange II medicament, a preparation method and application thereof.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
The invention provides a test strip for detecting a golden orange II medicament, which comprises a substrate, a piece of filter paper, an immune colloidal gold paper piece, an immune nitrocellulose membrane and a piece of absorbent paper, wherein the filter paper, the immune colloidal gold paper piece, the immune nitrocellulose membrane and the absorbent paper are sequentially connected end to end and are fixed on the substrate; the immune colloidal gold paper sheet is provided with a colloidal gold labeled anti-golden orange II monoclonal antibody, and the immune nitrocellulose membrane is provided with a detection line coated with a golden orange II-bovine serum albumin compound and a quality control line coated with goat anti-rabbit IgG.
The invention also provides a preparation method of the test strip, which comprises the following steps: and sequentially sticking the filter sample paper, the immune colloidal gold paper sheet, the immune nitrocellulose membrane and the absorbent paper on the substrate to prepare the test paper strip.
The invention also provides a kit for rapidly detecting the medicine of the golden orange II, which comprises a box body and a detection card, wherein the detection card comprises a shell and the test strip.
The invention also provides a method for rapidly detecting the golden orange II medicament by using the kit, which comprises the following steps:
sample preparation: weighing a proper amount of a sample, and carrying out pretreatment;
preparation of a standard solution: taking a proper amount of a golden orange II standard product, and dissolving the golden orange II standard product by using dichloromethane;
preparing a solution to be detected: respectively adding the diluent and the extracting solution into a sample or a standard solution, fully oscillating and centrifuging, sucking a centrifuged supernatant part into a test tube, blow-drying, adding a complex solution into the blow-dried test tube, and fully and uniformly mixing;
and (3) detection: and (4) dropwise adding the liquid to be detected into the gold-labeled micropores, and analyzing the detection result.
The invention also provides application of the kit in detection of residual golden orange II medicines in traditional Chinese medicinal materials, traditional Chinese medicine decoction pieces and foods.
The invention has the beneficial effects that:
the test strip qualitatively detects the components of the golden orange II in traditional Chinese medicinal materials, traditional Chinese medicine decoction pieces and foods by an immune competition inhibition method, and has the advantages of strong specificity, high sensitivity, simple and convenient operation and the like.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic view of a gold orange II colloidal gold test strip.
Reference numerals: 1-sample filtering paper; 2-immune colloidal gold paper sheet; 3-nitrocellulose membrane; 4-absorbent paper.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The following describes the test strip for detecting the golden orange II drug, the preparation method and the application thereof provided by the embodiment of the invention.
The embodiment of the invention provides a test strip for detecting a golden orange II medicament, which comprises a substrate, a piece of filter paper, an immune colloidal gold paper sheet, an immune nitrocellulose membrane and a piece of absorbent paper, wherein the filter paper, the immune colloidal gold paper sheet, the immune nitrocellulose membrane and the absorbent paper are sequentially connected end to end and are fixed on the substrate; the immune colloidal gold paper sheet is provided with a colloidal gold labeled anti-golden orange II monoclonal antibody, and the immune nitrocellulose membrane is provided with a detection line coated with a golden orange II-bovine serum albumin compound and a quality control line coated with goat anti-rabbit IgG.
The detection principle of the test strip provided by the embodiment of the invention is as follows: dropping a sample to be detected into the micropore reagent, uniformly mixing, inserting the test strip into the incubated micropore reagent, and diffusing the sample solution to be detected and the gold-labeled antibody in the micropore to the immunonitrocellulose membrane together after being combined; if the content of the aurantio-II medicament in the sample liquid to be detected is high, the aurantio-II medicament in the sample liquid to be detected can be combined with the gold-labeled antibody in the diffusion process, so that the antigen binding site of the aurantio-II medicament on the gold-labeled antibody is completely sealed, the gold-labeled antibody is prevented from being combined with the aurantio-II medicament hapten-bovine serum albumin on the immune nitrocellulose membrane in a composite way, the detection line is not colored, and goat anti-rabbit IgG can be combined with the aurantio-antibody, and the quality control line is colored; if the content of the gold orange II medicament in the sample liquid to be detected is low or zero, the antigen binding site on the gold-labeled antibody cannot be closed, and then the gold-labeled antibody can be combined with the hapten-bovine serum albumin of the gold orange II medicament on the immune nitrocellulose membrane, the detection line is colored, and meanwhile, the goat anti-rabbit IgG can also be combined with the gold-labeled antibody, and the quality control line is colored. If the quality control line does not develop color, the test paper fails.
The embodiment of the invention also provides a preparation method of the test strip, which comprises the following steps:
and sequentially sticking the filter sample paper, the immune colloidal gold paper sheet, the immune nitrocellulose membrane and the absorbent paper on the substrate to prepare the test paper strip.
In some embodiments, the preparation of the immune colloidal gold paper sheet comprises the following steps:
preparing immune colloidal gold solution; soaking the glass fiber paper in the pretreatment solution, drying, spraying the pretreated glass fiber paper with the immune colloidal gold solution, and drying to obtain the immune colloidal gold paper sheet.
In some embodiments, the preparation of the immune colloidal gold solution comprises the steps of: diluting colloidal gold labeled anti-aurantium II monoclonal antibody with gold spraying buffer solution to obtain OD540The immune colloidal gold solution has a value of 1.9-2.1, the concentration of the marker combined by the anti-aurantii II monoclonal antibody and the colloidal gold is 20-30 mu g/ml, the particle size of the colloidal gold is 30-40nm, and the preferred particle size of the colloidal gold is 35 nm.
The colloidal gold in the embodiment of the present invention may be commercially available or prepared by a two-step reduction method as described below, and the particle size of the colloidal gold is 30 to 40nm, preferably 35 nm. The two-step reduction method in the embodiment of the invention is adopted to obtain the 30nm spherical particles with uniform size of the colloidal gold molecules, and the colloidal gold particles have moderate size and are uniform as a whole, so that the binding efficiency of the colloidal gold particles and the monoclonal antibody is higher, and the effect is more stable.
In some embodiments, the metal spraying buffer comprises the following components in percentage by mass: 10-15 wt% of 1.0M Tris solution, 0.2-0.3 wt% of NaOH, 0.2-0.5 wt% of polyethylene glycol 30000, 0.2-0.5 wt% of polyethylene glycol 6000, 0.1-0.2 wt% of bovine serum albumin, 0.1-0.3 wt% of sucrose, 0.3-0.4 wt% of casein, 0.02-0.08 wt% of trehalose and the balance of water, wherein the pH value of the gold spraying buffer solution is 8.0 +/-0.05;
in some embodiments, the pretreatment solution comprises the following components in percentage by mass: 0.3-0.4 wt% of PVP-30k, 6-10 wt% of protein, disaccharide and polysaccharide, and the balance of water, preferably, the pretreatment solution further comprises or does not comprise one or more of protein fragments, synthetic polypeptide and semi-synthetic polypeptide.
In the preparation process of the immune colloidal gold paper sheet in the embodiment of the invention: soaking the glass fiber paper in the pretreatment solution, drying, spraying the pretreated glass fiber paper with the immune colloidal gold solution, and drying to obtain immune colloidal gold paper sheets, wherein each 30-35ml of the pretreatment solution is used for soaking the glass fiber paper with the diameter of 261mm multiplied by 220mm multiplied by 0.5mm, after drying, the glass fiber paper is sprayed with the immune colloidal gold solution, each 261mm multiplied by 220mm multiplied by 0.5mm is sprayed with 20-25ml of the immune colloidal gold solution, and then drying is carried out to obtain the immune colloidal gold paper sheets.
In some embodiments, the preparation of the immunonitrocellulose membrane comprises the steps of: and respectively spraying 0.3-0.5 mg/ml of golden orange II-bovine serum albumin complex and 0.4-0.6 mg/ml of goat anti-rabbit IgG on the positions of a nitrocellulose membrane detection line and a quality control line, and drying for later use to prepare the immune nitrocellulose membrane.
The immunonitrocellulose membranes prepared in the examples of the present invention: 1ml of goat anti-rabbit IgG and golden orange II-bovine serum albumin complex solution are respectively coated on each 1m of the nitrocellulose membrane, and the distance between the detection line and the quality control line is 5.5 mm.
The embodiment of the invention also provides a kit for rapidly detecting the medicine of the golden orange II, which comprises a box body and a detection card, wherein the detection card comprises a shell and the test strip.
The embodiment of the invention also provides a method for rapidly detecting a golden orange II medicament by using the kit, which comprises the following steps:
sample preparation: weighing a proper amount of a sample, and carrying out pretreatment;
preparation of a standard solution: taking a proper amount of a golden orange II standard product, and dissolving dichloromethane;
preparing a solution to be detected: respectively adding the diluent and the extracting solution into a sample or a standard solution, fully oscillating and centrifuging, sucking a centrifuged supernatant part into a test tube, blow-drying, adding a complex solution into the blow-dried test tube, and fully and uniformly mixing;
and (3) detection: and (4) dropwise adding the liquid to be detected into the gold-labeled micropores, and analyzing the detection result.
In some embodiments, the diluent is acetonitrile, the extracting solution is one or more of trichloroacetic acid aqueous solution, acetic acid aqueous solution and formic acid aqueous solution with the concentration of 0.4-0.6 wt%, the complex solution is 0.8-1.2M PBS buffer solution, the volume ratio of the sample, the diluent and the extracting solution is 5:3:2, the volume ratio of the complex solution to the solution to be detected is 5:2, and the reaction time during detection is 3-5 min.
In the method for detecting by using the kit in the embodiment of the invention, the method for processing the kit sample comprises the following steps: adding a proper amount of solvent (including water, ethanol, PBS and the like) into the egg white liquid of the poultry egg to be detected for extraction, and dissolving the golden orange II into the solvent as much as possible. The solvent may be one or more of water, ethanol, PBS or other solvents. The PBS with the concentration of 0.2M is preferred, because the water system is the safest (protects operators), the extraction effect is ideal, and the volume of the solvent is preferably 1-2 times of that of the egg white, so that the residual golden orange II in the egg white can be fully extracted, and the content of the golden orange II does not need to be diluted too much to reduce the detection effect.
The embodiment of the invention also provides application of the kit in detection of residual golden orange II medicines in traditional Chinese medicinal materials, traditional Chinese medicine decoction pieces and foods, in particular application in detection of illegal residual golden orange II in egg foods.
The working principle of the test strip for detecting the golden orange II medicament provided by the embodiment of the invention is as follows: by utilizing a highly specific antibody-antigen specific binding reaction and an immune membrane chromatography technology, the golden orange II component in traditional Chinese medicinal materials, traditional Chinese medicine decoction pieces and food is qualitatively detected by an immune competitive inhibition method, and particularly, the golden orange II component is used for quickly detecting whether the golden orange II component is illegally remained in the poultry egg food, so that the golden orange II component detection method has the advantages of strong specificity, high sensitivity, simplicity and convenience in operation and the like.
The detection test strip in the kit is the key for realizing the detection of the golden orange II, and the golden orange II-bovine serum albumin compound is coated on the test strip detection line (T line); the immune colloidal gold paper of the test paper is fixed with colloidal gold labeled anti-golden orange II monoclonal antibody. When the sample extracting solution is added from the sample adding hole and permeates into the sample adding pad of the test strip, the sample to be detected is firstly combined with the gold II monoclonal antibody marked by the colloidal gold on the glass fiber membrane, and is continuously chromatographed to the immune nitrocellulose membrane through capillary action, and sequentially passes through a detection line which is sprayed with a gold II-bovine serum albumin compound on the immune nitrocellulose membrane and a quality control line which is sprayed with goat anti-rabbit IgG. If the sample extracting solution contains the golden orange II, the golden orange II and the golden orange II-bovine serum albumin complex compete for limited antigen binding sites on the colloidal gold-anti-golden orange II antibody, and when the concentration of the golden orange II reaches a threshold concentration of a product design, the golden orange II and the bovine serum albumin complex occupy all the antigen binding sites on the anti-golden orange II monoclonal antibody, so that the colloidal gold-labeled anti-golden orange II monoclonal antibody is prevented from being combined with the golden orange II-bovine serum albumin complex of the detection line, the detection line cannot capture colloidal gold particles without red color bands, and the detection result is positive. And if the sample extracting solution does not contain the gold orange II or the concentration of the gold orange II is lower than the threshold concentration, the anti-gold orange II monoclonal antibody-colloidal gold runs to a detection line along with the sample extracting solution, the detection line captures colloidal gold particles to present a red color band, and the detection result is negative.
The quality control line (C line) on the test strip is coated with goat anti-rabbit IgG to indicate whether the reaction system of the kit works normally. The appearance of the control line was independent of the presence of golden orange II. The appearance of the quality control line color band indicates: the sample is added in sufficient quantity, and the sample runs normally on the paper slip.
The use method of the test strip provided by the embodiment of the invention comprises the following steps:
1. the kit was placed on a horizontal table.
2. The sample extract was aspirated with a sample application pipette, and 5 drops (about 120ul) of the sample extract were added to the application well of the kit. Each different sample being tested takes care of using a different pipette.
3. And (4) observing results: and judging the result 5-10min after the sample is dripped.
The judgment method of the detection result comprises the following steps:
negative: two color bands appear in the result observation window, namely a red line appears at the position of each of the detection line (T line) and the quality control line (C line), and the red line indicates that no golden orange II exists in the sample.
Positive: only one red line appears at the position of the quality control line (line C) of the result observation window, and no line appears in the detection line (line T), which indicates that the golden orange II exists in the sample.
And (4) invalidation: the control line (line C) did not appear. In any case, the C-line should be formed, indicating that the sample application and operation are correct. The absence of line C indicates that the test results are inconclusive and should be redone.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
A preparation method of a detection kit for a golden orange II medicament comprises the following steps:
1. preparation of colloidal gold
The colloidal gold used in the examples of the present invention may be commercially available or prepared by the following two-step reduction method.
The preparation of the colloidal gold by the two-step reduction method comprises the following steps (preparing various aqueous solutions and adopting double-steaming or triple-steaming deionized water):
1.1 first reduction of chloroauric acid solution: preparation of HAuCl at a concentration of 0.01 wt%4Boiling 100ml of the aqueous solution for 25min, adding 1 wt% trisodium citrate (Na) under stirring3C6H5O7·2H2O) 2.00ml of aqueous solution, ultrasonic vibrationCooling to room temperature after 3min (frequency is 25kHz) to obtain colloidal gold stock solution with particle size of about 16 nm;
1.2 second reduction of chloroauric acid solution: 30ml of the gold colloidal solution obtained in the first step was placed in a water bath at 4.0 ℃ to keep the temperature constant, followed by addition of 1 wt% HAuCl pre-cooled to 4.0 ℃460ml of aqueous solution, then immediately dropwise adding a 4.0 ℃ mixed aqueous solution of 3 wt% ascorbic acid and 0.139 wt% polyvinylpyrrolidone (PVP) while stirring, wherein the dropwise adding speed is 1-2 drops/second, stirring until the reaction is complete, and the solution is transparent wine red after about 60min to prepare a colloidal gold solution with the particle size of about 35 nm.
2. Preparation of immune colloidal gold
2.1 anti-aurantii II monoclonal antibody was diluted with 0.1M phosphate buffer pH 7.0 to a concentration of 1.0mg/ml antibody solution.
2.2 1000ml of the colloidal gold solution was added to 1/10 times the volume of the colloidal gold in 0.1M pH 7.5 phosphate buffer and mixed for 3 min. Under rapid stirring, 20ml of the diluted anti-aurantii ii monoclonal antibody solution was slowly added thereto. The reaction was carried out at room temperature for 5min while stirring slowly.
2.3 after the reaction, 20ml of a 10 wt% Bovine Serum Albumin (BSA) solution was added to the reaction solution quickly, and the mixture was reacted at room temperature for 5min with gentle stirring.
2.4 centrifuging the prepared immune colloidal gold at 8000 rpm for 20min, keeping the precipitate, collecting the supernatant, centrifuging at 10000 rpm for 30min, and discarding the supernatant. Two centrifugation precipitates were collected and reconstituted to OD with a borate buffer containing 0.1 wt% BSA540At 13.
3. Preparation of immune colloidal gold paper sheet
3.1 preparation of pretreatment solution: 3ml of PVP-30k and 80g of soluble starch are accurately weighed, and purified water is added to the mixture to reach the constant volume of 1000 ml.
3.2 preparation of gold spraying buffer: adding 100ml of 1.0M Tris solution, 0.28g of sodium hydroxide, 3.0g of Tris, 2.0g of bovine serum albumin, 3.0g of cane sugar, 4.0g of casein solution and 0.6g of trehalose into 800ml of purified water, fully dissolving, uniformly mixing, adding purified water to the total volume of 1000ml, and adjusting the pH value to 7.5 by using hydrochloric acid.
3.3 Dilute the colloidal gold labeled anti-aurantii II monoclonal antibody with gold spraying buffer to OD540The value was 2.0, and an immune colloidal gold solution was obtained.
3.4 soaking the glass fiber paper by using the pretreatment solution, wherein each 28ml of the pretreatment solution is used for soaking the glass fiber paper by 261mm, 220mm and 0.5mm for 30min, and then drying at 37 ℃; and spraying 20ml of immune colloidal gold solution on the glass fiber paper with the thickness of 261mm multiplied by 220mm multiplied by 0.5mm, and drying to obtain the immune colloidal gold paper sheet.
4. Preparation of immunonitrocellulose membranes
4.1 dilution of goat anti-rabbit IgG to 0.6mg/ml with phosphate buffer solution to obtain quality control line (C line) solution.
4.2 diluting the aurantium II-bovine serum albumin complex with phosphate buffer solution until the protein concentration is 0.4mg/ml, and preparing a detection line (T line) solution.
4.3 spraying C, T line solution with a film spotting machine to prepare the immune nitrocellulose film. Every 1m of the nitrocellulose membrane is coated with 1ml of C line and T line solution respectively, and the distance between the detection line and the quality control line is 5.5 mm.
5. The filter paper, the immune colloidal gold paper sheet, the immune nitrocellulose membrane and the absorbent paper are sequentially stuck on a rubber plate and cut into test strips with the width of 4mm (as shown in figure 1).
6. And (5) putting the detection test strip into a plastic box to obtain the detection kit.
The threshold value of the gold orange II colloidal gold detection kit designed by the invention is 0.1 mug/ml.
Example 2
A method for rapidly detecting residues of golden oranges II in meat products comprises the following steps:
1. sample preparation: weighing 5g of homogenized tissue sample, and putting the tissue sample into a 15ml centrifuge tube;
2. preparing a standard substance: collecting powder of fructus Citri Junoris II (98 wt% content, 0.041g, effective component 0.04g, dissolved in 10ml water, and mixing). 4 parts of solutions with different concentrations of 5ppb, 0.4ppm, 40ppm and 4000ppm are prepared;
3. adding 3ml acetonitrile and 2ml trichloroacetic acid water solution into a 15ml centrifuge tube, fully oscillating and uniformly mixing for 3min, and centrifuging for 5min at 4000 rpm;
4. taking all (about 2.5ml) of the layered supernatant (without absorbing the lower layer) by using a 0.5ml disposable straw, adding the supernatant into a matched 5ml centrifuge tube, and drying by air (or nitrogen) at 65 ℃ (the phenomenon that part of granular yellow oil at the bottom of the centrifuge tube is not dried normally);
5. adding 0.3ml (6 drops of) 1M PBS (phosphate buffer solution) into a blow-dried 5ml centrifuge tube, fully shaking and dissolving solid residues at the bottom and the inner wall of the centrifuge tube, wherein the dissolved solution is the solution to be detected;
6: and (3) detection: and (4) dropwise adding the liquid to be detected into the gold-labeled micropores, and reacting for 3-5 min.
Example 3
Sensitivity experiment of gold orange II colloidal gold detection kit
1. The detection method comprises the following steps:
1.1 the gold orange II colloidal gold test kit prepared in example 1 was taken and placed on a horizontal table.
1.2 aspirate the sample with the sample pipette and then drop 5 drops (about 120ul) of the sample into the well of the kit. Different pipettes were used for each different sample tested.
1.3 observations: and judging the result 5-10min after the sample is dripped.
2. Detecting a sample:
quality control reference substances with the concentrations of the golden oranges II of 0 mu g/ml, 0.01 mu g/ml, 0.05 mu g/ml, 0.1 mu g/ml, 0.2 mu g/ml and 0.5 mu g/ml are prepared as samples, and each concentration is repeated for 3 times.
3. And (3) detection results:
samples at concentrations of 0. mu.g/ml, 0.01. mu.g/ml, 0.05. mu.g/ml were all negative, samples at 0.1. mu.g/ml, 0.2. mu.g/ml, 0.5. mu.g/ml were all positive. The detection sensitivity of the kit is 0.1 mug/ml, and the gold orange II colloidal gold detection kit meets the detection requirement.
Example 4
Specificity experiment of gold orange II colloidal gold detection kit
1. The detection method comprises the following steps:
1.1 the gold orange II colloidal gold test kit prepared in example 1 was taken and placed on a horizontal table.
1.2 draw the test sample for the test cross-reaction assay with the sample application pipette and then drop 5 drops (about 120ul) of the sample into the well of the kit. Different pipettes were used for each different sample tested.
1.3 observations: and judging the result 5-10min after the sample is dripped.
2. Detecting a sample:
the kit provided by the invention is used for detecting ciprofloxacin, enrofloxacin, sarafloxacin and the like with different concentrations, and the cross reaction effect is observed.
3. And (3) detection results:
the result proves that the kit has good detection effect on the golden orange II, namely the golden orange II colloidal gold kit has good detection limit on golden orange II medicaments under a certain concentration and has sensitive reaction.
Example 5
Repeatability and stability experiment of gold orange II colloidal gold detection kit
1. In-batch and inter-batch repeatability experiments of the kit
1.1 Experimental methods:
the gold orange II colloidal gold detection kits of the same batch and different batches are respectively used for detecting 0.05, 0.1, 0.2 and 0.5 mu g/ml standard substances, each concentration is repeated for 3 times, and the repeatability of the kit is observed.
1.2. The experimental results are as follows: the verification proves that the within-batch and within-batch repeatability of the golden orange II colloidal gold detection kit is 100%, and the false positive rate and the false negative rate are both 0.
2. Stability test of kit
2.1 purpose of experiment:
the gold orange II colloidal gold detection kit is stored in a sealed manner and is stored at 4 ℃ and room temperature (about 25 ℃), and the influence of different storage temperatures on the stability of the kit is observed.
2.2 Experimental methods:
taking out the kit stored at 4 ℃ 4 times every 10 days, and detecting the standard substance with the concentration of 0.05, 0.1, 0.2 and 0.5 mu g/ml respectively; the kit stored at room temperature (25 ℃) was taken out 4 times per week and the standards at concentrations of 0.05, 0.1, 0.2, 0.5. mu.g/ml were examined.
3. The experimental results are as follows:
the test paper strip can be stored for 18 months at 4 ℃, can be stored for 12 months at room temperature, and can achieve the detection sensitivity of 0.1 mu g/ml in the storage period.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (10)
1. A test strip for detecting a medicine of golden orange II comprises a substrate, a piece of filter paper, an immune colloidal gold paper piece, an immune nitrocellulose membrane and absorbent paper, and is characterized in that the filter paper, the immune colloidal gold paper piece, the immune nitrocellulose membrane and the absorbent paper are sequentially connected end to end and are fixed on the substrate; the immune colloidal gold paper is provided with a colloidal gold labeled anti-golden orange II monoclonal antibody, and the immune nitrocellulose membrane is provided with a detection line coated with a golden orange II-bovine serum albumin compound and a quality control line coated with goat anti-rabbit IgG.
2. A method for preparing the test strip of claim 1, comprising the steps of:
and sequentially adhering the filter sample paper, the immune colloidal gold paper sheet, the immune nitrocellulose membrane and the absorbent paper to the substrate to prepare the test strip.
3. The method for preparing the immune colloidal gold paper sheet according to claim 2, wherein the preparation of the immune colloidal gold paper sheet comprises the following steps:
preparing immune colloidal gold solution; and soaking the glass fiber paper in the pretreatment solution, drying, spraying the pretreated glass fiber paper with an immune colloidal gold solution, and drying to obtain the immune colloidal gold paper sheet.
4. The method for preparing an immune colloidal gold solution according to claim 3, wherein the preparation of the immune colloidal gold solution comprises the steps of: diluting colloidal gold labeled anti-aurantium II monoclonal antibody with gold spraying buffer solution to obtain OD540The immune colloidal gold solution has the value of 1.9-2.1, the concentration of the marker combined by the anti-golden orange II monoclonal antibody and the colloidal gold is 20-30 mu g/ml, the particle size of the colloidal gold is 30-40nm, and the preferred particle size of the colloidal gold is 35 nm.
5. The preparation method according to claim 3, wherein the gold spraying buffer solution comprises the following components in percentage by mass: 10-15 wt% of 1.0M Tris solution, 0.2-0.3 wt% of NaOH, 0.2-0.5 wt% of polyethylene glycol 30000, 0.2-0.5 wt% of polyethylene glycol 6000, 0.1-0.2 wt% of bovine serum albumin, 0.1-0.3 wt% of sucrose, 0.3-0.4 wt% of casein, 0.02-0.08 wt% of trehalose and the balance of water, wherein the pH value of the gold spraying buffer solution is 8.0 +/-0.05;
the pretreatment liquid comprises the following components in percentage by mass: 0.3-0.4 wt% of PVP-30k, 6-10 wt% of protein, disaccharide and polysaccharide, and the balance of water, preferably, the pretreatment solution further comprises or does not comprise one or more of protein fragments, synthetic polypeptide and semi-synthetic polypeptide.
6. The method for preparing an immunonitrocellulose membrane according to claim 2, wherein the preparation of the immunonitrocellulose membrane comprises the steps of: and respectively spraying 0.3-0.5 mg/ml of golden orange II-bovine serum albumin complex and 0.4-0.6 mg/ml of goat anti-rabbit IgG on the positions of a detection line and a quality control line of the nitrocellulose membrane, and drying for later use to prepare the immune nitrocellulose membrane.
7. A kit for rapidly detecting a medicine of golden orange II, which comprises a box body and a detection card, and is characterized in that the detection card comprises a shell and the test strip of any one of claims 1 to 6.
8. The use of the kit of claim 7 for detecting residual aurantii fructus ii in herbal materials, herbal pieces, and foods.
9. A method for rapidly detecting a golden orange ii drug by using the kit of claim 7, which comprises the following steps:
sample preparation: weighing a proper amount of a sample, and carrying out pretreatment;
preparation of a standard solution: taking a proper amount of a golden orange II standard product, and dissolving dichloromethane;
preparing a solution to be detected: respectively adding a diluent and an extracting solution into the sample or the standard solution, fully oscillating and centrifuging, sucking a centrifuged supernatant part into a test tube, blow-drying, adding a complex solution into the blow-dried test tube, and fully and uniformly mixing;
and (3) detection: and (4) dropwise adding the liquid to be detected into the gold-labeled micropores, and analyzing a detection result.
10. The detection method according to claim 9, wherein the diluent is acetonitrile, the extract is one or more of trichloroacetic acid aqueous solution, acetic acid aqueous solution and formic acid aqueous solution with a concentration of 0.4-0.6 wt%, the complex solution is 0.8-1.2M PBS buffer solution, the volume ratio of the sample, the diluent and the extract is 5:3:2, the volume ratio of the complex solution to the solution to be detected is 5:2, and the reaction time during detection is 3-5 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911348490.3A CN113030474A (en) | 2019-12-24 | 2019-12-24 | Test strip for detecting golden orange II drug, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911348490.3A CN113030474A (en) | 2019-12-24 | 2019-12-24 | Test strip for detecting golden orange II drug, preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113030474A true CN113030474A (en) | 2021-06-25 |
Family
ID=76452038
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911348490.3A Pending CN113030474A (en) | 2019-12-24 | 2019-12-24 | Test strip for detecting golden orange II drug, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113030474A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116643044A (en) * | 2023-06-15 | 2023-08-25 | 广州贝思奇诊断试剂有限公司 | Kit for detecting HIV-1 and HIV-2 antibodies in urine based on colloidal gold method, and preparation method and application thereof |
-
2019
- 2019-12-24 CN CN201911348490.3A patent/CN113030474A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116643044A (en) * | 2023-06-15 | 2023-08-25 | 广州贝思奇诊断试剂有限公司 | Kit for detecting HIV-1 and HIV-2 antibodies in urine based on colloidal gold method, and preparation method and application thereof |
| CN116643044B (en) * | 2023-06-15 | 2023-12-12 | 广州贝思奇诊断试剂有限公司 | Kit for detecting HIV-1 and HIV-2 antibodies in urine based on colloidal gold method, and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| TWI521206B (en) | Organic colored microparticles, including their diagnostic kit and in vitro diagnostic methods | |
| CN105334319A (en) | Test strip for detecting zearalonone in milk | |
| CN106568965A (en) | Auramine O colloidal gold test card and preparation method thereof | |
| CN102650642A (en) | Antibody compound for detecting human prepalin retinol binding protein 4, as well as immunochromatography test card and kit | |
| CN108918849B (en) | Method for rapidly detecting aflatoxin in medicinal materials and test paper card used by same | |
| KR101451733B1 (en) | Labeling agent for aflatoxin B1 detection and the kit for detecting aflatoxin B1 comprising thereof | |
| CN102539786B (en) | Microscale urinary albumin colloidal gold detection kit and preparation technology thereof | |
| CN113030474A (en) | Test strip for detecting golden orange II drug, preparation method and application thereof | |
| CN106568966A (en) | Lemon yellow colloidal gold detection card and production method thereof | |
| CN100405063C (en) | Fabrication method and application for citrinin immune chromatography detection test paper | |
| CN103412124A (en) | Aflatoxin M1 gold label quick detectiontest card and preparation method and application thereof | |
| CN107238700A (en) | Quantum dot immune chromatograph test strip and preparation method thereof is quenched in a kind of collaurum for detecting zearalenone | |
| CN107703298A (en) | Colloid gold immune detection kit, preparation method and the detection method of a kind of aflatoxin B1 | |
| CN109709322A (en) | A kind of detection method detecting aflatoxin B1 | |
| Marquardt et al. | Herbicide Determination, Determination of 2, 4-Dichlorophenoxyacetic Acid (2, 4-D) in Grain and Seed | |
| CN111458525A (en) | Test strip and kit for detecting human body physiological substance concentration and preparation method | |
| CN105181915B (en) | Pollutant tachysynthesis detection method and application in one vegetable oil | |
| CN113030476A (en) | Test strip for detecting auramine O medicines, preparation method and application thereof | |
| CN103869083A (en) | Preparation of immunizing antigen and envelope antigen for detecting chloramphenicol | |
| CN108802378A (en) | A kind of test strips that the nature controlling line of test strips is coated with solution and is prepared with this solution | |
| CN110687294B (en) | Detection card for rapidly and quantitatively detecting mycotoxin and application thereof | |
| CN112415190A (en) | Time-resolved fluorescence immunochromatography method for detecting nicarbazin and application | |
| CN105954513A (en) | {0><}0{>Preparation and application of Flow-through film carrier qunatum dot detection morphine test paper | |
| CN103792358A (en) | Preparation and detection method of aflatoxin G1 colloidal gold immunochromatograohic assay detection board | |
| CN113125725A (en) | Detection card for simultaneously detecting aflatoxin B1 and zearalenone |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20210625 |
|
| WD01 | Invention patent application deemed withdrawn after publication |