CN111896738B - Test strip for detecting serpentine bacterial element and application thereof - Google Patents
Test strip for detecting serpentine bacterial element and application thereof Download PDFInfo
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Abstract
The invention discloses a test strip for detecting serpentine bacterial element and application thereof. The test strip comprises a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3), a water absorption pad (4) and a bottom plate (7), wherein the reaction membrane is provided with a detection line (5) coated with a snake fungus hapten-carrier protein conjugate and a quality control line (6) coated with a goat anti-mouse anti-antibody, and the conjugate release pad (2) is sprayed with a snake fungus monoclonal antibody-colloidal gold marker. The invention also provides a method for detecting the residue of the serpentine in the grains such as wheat, corn and the like and the feed by using the serpentine test strip. The test strip provided by the invention has the characteristics of simplicity in operation, high sensitivity, high detection speed, low cost and the like, and is suitable for screening and on-site monitoring of a large number of samples.
Description
Technical Field
The invention relates to a test strip for detecting serpentine and application thereof, in particular to a colloidal gold test strip for detecting serpentine, which is particularly suitable for detecting serpentine residues in grains such as wheat, corn and the like and feeds.
Background
The snake-shaped bacteria, also called diacetyl ribes fusarium enol (DAS), is similar to the T-2 toxin, belongs to trichothecene compounds, mainly pollutes food crops and feeds, has higher toxicity than DON, damages various tissues and organs of human beings and animals, and has similar effect to the T-2 toxin.
In order to reduce the impact on humans and animals, it is important to effectively and rapidly detect DAS in foods and feeds. Compared with the traditional biological detection method, thin layer chromatography, high performance liquid chromatography and the like, the immunochemistry detection method has the advantages of rapidness, specificity, sensitivity, accuracy, batch monitoring, simple sample treatment, automatic operation and the like for mycotoxin detection. The immunochemical detection of DAS requires the presence of antibodies to DAS. The invention is a rapid detection method, has short time, simple operation and low cost, and is suitable for detecting samples in a large scale in a basic unit.
Disclosure of Invention
The invention aims to provide a serpentine residue detection test strip which has high sensitivity, simple operation, low cost and short detection time.
The test strip for detecting the serpentine residue comprises a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3), a water absorption pad (4) and a bottom plate (7); the reaction membrane is provided with a detection line (5) coated with a snake fungus hapten-carrier protein conjugate and a quality control line (6) coated with a goat anti-mouse anti-antibody, and the conjugate release pad (2) is sprayed with a snake fungus monoclonal antibody-colloidal gold marker.
The snake fungus hapten-carrier protein conjugate is obtained by coupling a snake fungus hapten with carrier protein, wherein the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine and human serum albumin.
The snake hormone monoclonal antibody is prepared by taking a snake hormone hapten-carrier protein conjugate as an immunogen, and is secreted by a hybridoma cell strain of the snake hormone monoclonal antibody; the goat anti-mouse antibody is obtained by immunizing a goat with a mouse-derived antibody.
The sample absorbing pad (1), the conjugate releasing pad (2), the reaction membrane (3) and the water absorbing pad (4) are sequentially adhered to the bottom plate (7), and the conjugate releasing pad 1/3-1/2 is covered below the sample absorbing pad.
The bottom plate can be a PVC bottom plate or other hard non-water-absorbing materials; the sample absorption pad can be suction filter paper or oil filter paper; the conjugate release pad may be a glass wool or polyester material; the water absorbing pad is water absorbing paper; the reaction membrane may be a nitrocellulose membrane or a cellulose acetate membrane.
Another object of the present invention is to provide a method for preparing the above test strip, comprising the steps of:
1) Preparing a conjugate release pad sprayed with a serpentine monoclonal antibody-colloidal gold marker;
2) Preparing a reaction membrane with a detection line coated with a snake fungus hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;
3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the bottom plate which are prepared in the steps 1) and 2) into the test strip.
Specifically, the method comprises the following steps:
1) Hapten preparation: the serpentine element hapten is obtained by a series of chemical reactions of serpentine element, DL-homocysteine and other substances;
2) Coupling the snake fungus hapten with carrier protein to obtain a snake fungus hapten-carrier protein conjugate;
3) Immunizing a mouse by using a snake fungus hapten-carrier protein conjugate, and obtaining a snake fungus monoclonal hybridoma cell strain by fusing and screening spleen cells and myeloma cells of the mouse;
4) Extracting mouse IgG to immunize healthy goats to obtain goat anti-mouse anti-antibody;
5) Preparing colloidal gold by the reaction of trisodium citrate and chloroauric acid;
6) Adding the snake fungus element monoclonal antibody prepared in the step 3) into the colloidal gold prepared in the step 5) to obtain a snake fungus element monoclonal antibody-colloidal gold marker;
7) Spraying a serpentine monoclonal antibody-colloidal gold marker on a conjugate release pad, baking at 37 ℃ for 1h, taking out, and storing in a dry environment for standby;
8) Coating a snake-shaped fungus hapten-carrier protein conjugate on a reaction membrane to form a detection line, and coating a goat anti-mouse antibody on the reaction membrane to form a quality control line;
9) Soaking the sample absorption pad in phosphate buffer solution containing 0.5% bovine serum albumin (volume fraction) with pH of 7.2 and 0.1mol/L for 2h, and drying at 37deg.C for 2h;
10 The sample absorbing pad, the conjugate releasing pad, the reaction membrane and the water absorbing pad are sequentially stuck on the bottom plate, the sample absorbing pad covers the conjugate releasing pad, finally, the sample absorbing pad is cut into small strips with the width of 3mm, a plastic box is added, and the sample absorbing pad can be stored for 12 months at the temperature of 4-30 ℃ after vacuum packaging.
The invention also provides a method for detecting the residue of the snake-shaped bacteria in the grains and the feed by using the test strip, which comprises the following steps:
(1) Sample pretreatment;
(2) Detecting by using a test strip;
(3) And analyzing the detection result.
The rapid detection test strip for the snake-shaped bacteria adopts a highly specific antibody antigen reaction and competitive inhibition immunochromatography analysis technology, a snake-shaped bacteria monoclonal antibody-colloidal gold marker is fixed on a conjugate release pad, and the snake-shaped bacteria in a sample is combined with the snake-shaped bacteria monoclonal antibody-colloidal gold marker on the conjugate release pad in the flowing process to form a medicine-antibody-colloidal gold marker. And the medicine in the sample and the snake fungus hapten-carrier protein conjugate on the reaction membrane detection line compete for combining with the snake fungus monoclonal antibody-colloidal gold marker, and whether the sample liquid to be detected contains snake fungus residues is judged according to whether the red strip of the detection line exists or not or whether the color depth is low or not.
During detection, a sample is dripped into a test strip hole after treatment, when the concentration of the snake-shaped bacteria in the sample is lower than the detection limit or zero, the monoclonal antibody-colloidal gold marker is combined with the snake-shaped bacteria hapten-carrier protein conjugate fixed on the reaction membrane in the chromatography process, and a red strip appears in the detection line (T) and the quality control line (C) respectively; if the concentration of the serpentine element in the sample is at or above the limit of detection, the monoclonal antibody-colloidal gold label will bind to all of the serpentine element, such that no red bands will appear at the T-line due to competition reactions without binding to the serpentine element hapten-carrier protein conjugate.
The test strip has the advantages of high sensitivity, strong specificity, low cost, simple operation, short detection time, suitability for various units, simple storage and long shelf life. The method for detecting the residue of the snake-shaped bacterial element by using the test strip is simple, convenient, quick, visual, accurate, wide in application range, low in cost and easy to popularize and use.
Drawings
FIG. 1 is a diagram of the synthesis of a snake biotin hapten.
Fig. 2 is a schematic diagram of a cross-sectional structure of the test strip.
Detailed Description
The invention is further illustrated below in conjunction with specific examples. It is to be understood that these examples are for illustration of the invention only and are not intended to limit the scope of the invention.
Example 1 preparation of a test strip for detection of Serpentis
The preparation method of the test strip mainly comprises the following steps:
1) Preparing a conjugate release pad sprayed with a serpentine monoclonal antibody-colloidal gold marker;
2) Preparing a reaction membrane with a detection line coated with a snake fungus hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;
3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the PVC bottom plate which are prepared in the steps 1) and 2) into the test strip.
The following is a stepwise detailed description:
1. preparation of serpentine fungus hapten
Taking 20mg of snake fungus essence, adding 10ml of DMF for dissolution, adding 0.1ml of triethylamine, fully stirring, adding 17mg of Carbonyl Diimidazole (CDI), reacting for 2 hours at room temperature, adding 14mg of DL-homocysteine, continuously stirring for 12 hours, stopping the reaction, adding 70ml of water and 80ml of ethyl acetate, fully oscillating, standing, separating out water phase, drying organic phase anhydrous sodium sulfate, evaporating to dryness to obtain yellow oily matter, adding 25.2mg of DL-homocysteine-snake fungus essence hapten product by acetonitrile/n-hexane (v/v, 1/10) and recrystallizing to obtain 73% yield.
2. Preparation of immunogens
Taking 19mg of DL-homocysteine-serpentine hapten, adding 1ml of DMF for dissolution to obtain solution A; taking 50mg of Bovine Serum Albumin (BSA), adding 0.1M PB buffer solution for dissolution, adding 12mg of 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester, reacting at room temperature for 6 hours to obtain solution B, slowly dripping solution A into solution B, reacting at room temperature for 7 hours, dialyzing with 0.02M PBS for 3 days, changing the solution for 3 times each day to obtain the snake-like bacteria-BSA conjugate, namely the immunogen, subpackaging at-20 ℃ for later use.
3. Preparation of coating Material
Taking 6 mgDL-homocysteine-snake fungus hapten, adding 1ml of DMSO for dissolution, adding 0.1ml of N-methylmorpholine, fully and uniformly mixing, adding 0.11ml of isobutyl chloroformate, and reacting for 3 hours at 0-5 ℃ to obtain hapten activating solution A; taking 50mg of Ovalbumin (OVA), adding 0.1M PB buffer solution for dissolution to obtain solution B, slowly dripping solution A into solution B, reacting at room temperature for 7h, dialyzing and purifying with 0.02M PBS for 3 days, changing the solution 3 times a day to obtain the snakegourd fungus-OVA conjugate, namely the coating source, sub-packaging, and preserving at-20 ℃ for later use.
4. Preparation of Serpentis monoclonal antibodies
(1) Immunization of animals
The immunogen obtained in the step 2 is injected into Balb/c mice, and the immune dose is 150 mug/mouse, so that antisera are generated.
(2) Cell fusion and cloning
Spleen cells of an immunized Balb/c mouse are fused with SP2/0 myeloma cells according to the proportion of 8:1 (quantitative proportion), cell supernatant is measured by adopting an indirect competition ELISA method, and positive holes are screened. Cloning the positive hole by limiting dilution method until obtaining hybridoma cell strain for stably secreting monoclonal antibody.
(3) Cell cryopreservation and resuscitation
The hybridoma cells were prepared into 1X 10 by using a frozen stock solution 6 Cell suspensions/ml were stored in liquid nitrogen for a long period of time. And (3) taking out the frozen storage tube during recovery, immediately putting into a 37 ℃ water bath for medium-speed thawing, centrifuging to remove frozen storage liquid, and transferring into a culture flask for culture.
(4) Preparation and purification of monoclonal antibodies
Incremental culture method: the hybridoma cells are placed in a cell culture medium, cultured at 37 ℃, and the obtained culture solution is purified by an octanoic acid-saturated ammonium sulfate method to obtain monoclonal antibodies, and the monoclonal antibodies are preserved at-20 ℃.
The cell culture medium is prepared by adding calf serum and sodium bicarbonate into RPMI1640 culture medium, so that the final concentration of the calf serum in the cell culture medium is 20% (mass fraction), and the final concentration of the sodium bicarbonate in the cell culture medium is 0.2% (mass fraction); the pH of the cell culture medium was 7.4.
5. Preparation of goat anti-mouse antibody
Sheep is used as immune animals, and a murine antibody is used as immunogen to immunize pathogen-free sheep, so that the goat anti-mouse antibody is obtained.
6. Preparation of Serpentis monoclonal antibody-colloidal gold marker
(1) Preparation of colloidal gold
Diluting 1% chloroauric acid into 0.01% (mass fraction) with double distilled deionized water, placing 100ml in a conical flask, heating to boil with a constant temperature electromagnetic stirrer, adding 2.5ml 1% trisodium citrate under continuous high temperature and continuous stirring, stopping stirring and heating until the solution is transparent red, cooling to room temperature, recovering original volume with deionized water, and preserving at 4deg.C. The prepared colloidal gold has pure appearance, is transparent, and has no sediment or floaters.
(2) Preparation of Serpentis monoclonal antibody-colloidal gold marker
Under the magnetic stirring, the pH value of the colloidal gold is regulated to 7.0 by using 0.2mol/L potassium carbonate solution, 20-50 mug of standard is added into each milliliter of colloidal gold solution, the snake fungus element monoclonal antibody is added into the colloidal gold solution, the stirring and the mixing are continued for 30min, 10% BSA is added, the final concentration of the snake fungus element monoclonal antibody in the colloidal gold solution is 1% (volume fraction), and the solution is kept stand for 10min. Centrifuging at 12000r/min and 4deg.C for 40min, discarding supernatant, washing the precipitate twice with redissolving buffer, re-suspending the precipitate with redissolving buffer with volume 1/10 of that of initial colloidal gold, and standing at 4deg.C for use.
Reconstitution buffer: 0.02 to 0.1 percent (mass fraction) of casein, 0.05 to 0.2 percent (mass fraction) of tween-80 and 0.02mol/L of phosphate buffer solution with pH of 7.2.
7. Preparation of conjugate release pads
The conjugate release pad was soaked in a phosphate buffer containing bovine serum albumin (the concentration of bovine serum albumin in the buffer is 0.5%), having a pH of 7.2 and 0.5mol/L, and was uniformly soaked for 1h and baked at 37℃for 3 h. Uniformly spraying the prepared serpentine monoclonal antibody-colloidal gold marker on a conjugate release pad by using an isolow film spraying instrument, spraying 0.01ml serpentine monoclonal antibody-colloidal gold marker on each 1cm conjugate release pad, placing the conjugate release pad in a 37 ℃ environment (humidity is less than 20%) for 60min, taking out the conjugate release pad, and placing the conjugate release pad in a dry environment (humidity is less than 20%) for storage for later use.
8. Preparation of reaction film
The snake-shaped fungus hapten-ovalbumin conjugate is coated on a reaction membrane to form a detection line, and the goat anti-mouse anti-antibody is coated on the reaction membrane to form a quality control line.
The coating process comprises the following steps: diluting the snake fungus hapten-ovalbumin conjugate to 10mg/ml by using a phosphate buffer solution, and coating the snake fungus hapten-ovalbumin conjugate on a detection line (T line) on a nitrocellulose membrane by using an isolow spot membrane tester, wherein the coating amount is 0.8 mu l/cm; the goat anti-mouse antibody was diluted to 200. Mu.g/ml with 0.01mol/L phosphate buffer at pH7.4, and coated on a quality control line (C line) on a nitrocellulose membrane in an isolow spot film apparatus in an amount of 1.0. Mu.l/cm. And (5) drying the coated reaction film for 2 hours at 37 ℃ for standby.
9. Preparation of sample absorbent pad
The sample absorbing pad is placed in phosphate buffer solution containing 0.5% bovine serum albumin (volume fraction), pH7.2 and 0.1mol/L for 2 hours, and is baked at 37 ℃ for 2 hours for standby.
10. Assembly of test strips
Sequentially adhering a sample absorption pad, a conjugate release pad, a reaction membrane and a water absorption pad on a PVC bottom plate; the 1/3 area of the initial end of the conjugate release pad is covered by the sample absorption pad, the tail end of the conjugate release pad is connected with the initial end of the reaction membrane, the tail end of the reaction membrane is connected with the initial end of the water absorption pad, the initial end of the sample absorption pad is aligned with the initial end of the PVC bottom plate, and the tail end of the water absorption pad is aligned with the tail end of the PVC bottom plate; the reaction film is provided with a detection line and a quality control line, and the detection line (T line) and the quality control line (C line) are strip-shaped strips which are perpendicular to the length of the test strip; the detection line is positioned at one side close to the tail end of the conjugate release pad; the quality control line is positioned at one side far away from the tail end of the conjugate release pad; cutting the test paper strip into small strips with the width of 3mm by a machine, and placing the test paper strip in a special plastic card, wherein the test paper strip can be stored for 12 months at the temperature of 4-30 ℃.
Example 2 detection of Serpentis residue in samples
1. Detection is carried out by using a test strip
Sucking the sample solution to be detected by a suction pipe, vertically dripping 3 drops of the sample solution into a sample adding hole, starting timing when the liquid flows, reacting for 5-10 min, and judging the result.
2. Analyzing the detection result
The results were read by a colloidal gold analyzer (abbreviated as "analyzer"):
negative (-). Indicating that the concentration of the substance to be detected in the sample is lower than the detection limit;
positive (+): indicating that the concentration of the substance to be detected in the sample is equal to or higher than the detection limit;
invalidation: indicating that retesting is required.
Example 3 sample detection example
1. Limit of detection test
Blank wheat and corn samples were taken, serpentine was added to the samples to a final concentration of 5, 10, 20 μg/kg, and test strips were taken for testing, and each sample was assayed three times.
When the test strip is used for detecting wheat and corn samples, the analyzer shows negative when the adding concentration of the snakelike bacteria is 5 mug/kg; the analyzer showed positive when the concentration of snakelike added was 10, 20. Mu.g/kg.
2. False positive rate and false negative rate test
Taking 20 parts of positive samples of wheat and corn with known serpentine content of 10 mug/kg and 2 parts of negative samples of wheat and corn with content of less than 5 mug/kg, detecting by using three batches of test strips, and calculating the negative-positive rate. The results are shown in Table 1 and Table 2.
Table 1 wheat test sample results
TABLE 2 corn test sample results
The results show that: when the test strips produced in 3 batches are used for detecting positive wheat and corn samples, the results are positive, the coincidence rate of the positive samples is 100%, and the false negative rate is 0; when 20 negative wheat and corn samples were tested, the results were all negative, and it was found that the negative coincidence rate was 100% and the false positive rate was 0. The test strip for detecting the serpentine hormone can be used for rapidly detecting the serpentine hormone residues in wheat and corn.
3. Specificity test
A serpentine test strip is used for detecting 500 mug/kg aflatoxin B1, T-2 toxin, ochratoxin and other medicaments. The result shows that the quality control line and the detection line of the test strip are both developed and negative. The test strip has no cross reaction to 500 mug/kg aflatoxin B1, T-2 toxin, ochratoxin and other medicines.
Claims (6)
1. The test strip for detecting the snake fungus essence comprises a sample absorption pad (1), a conjugate release pad (2), a reaction membrane (3), a water absorption pad (4) and a bottom plate (7), and is characterized in that the reaction membrane is provided with a detection line (5) coated with a snake fungus essence hapten-carrier protein conjugate and a quality control line (6) coated with a goat anti-mouse anti-antibody, the conjugate release pad (2) is sprayed with a snake fungus essence monoclonal antibody-colloidal gold marker, the snake fungus essence hapten-carrier protein conjugate is obtained by coupling a snake fungus essence hapten with carrier protein, and the carrier protein is bovine serum albumin, ovalbumin, hemocyanin, thyroxine or human serum albumin, and the preparation method of the snake fungus essence hapten is as follows:
taking 20mg of snake fungus essence, adding 10ml of DMF for dissolution, adding 0.1ml of triethylamine, fully stirring, adding 17mg of carbonyl diimidazole, reacting for 2 hours at room temperature, adding 14mg of DL-homocysteine, continuously stirring for 12 hours, stopping the reaction, adding 70ml of water, 80ml of ethyl acetate, fully oscillating, standing, separating out water phase, drying the organic phase by anhydrous sodium sulfate, evaporating to dryness to obtain yellow oily matter, adding 3ml of acetonitrile/n-hexane with the volume ratio of 1:10, recrystallizing to obtain 25.2mg of DL-homocysteine-snake fungus essence hapten product, and obtaining the yield of 73%;
the molecular structural formula of the serpentine fungus hapten is as follows:
2. the test strip according to claim 1, wherein the sample absorbing pad (1), the conjugate releasing pad (2), the reaction membrane (3) and the water absorbing pad (4) are sequentially adhered to the bottom plate (7).
3. The test strip of claim 1, wherein the conjugate release pad is 1/3 to 1/2 covered under the sample absorbent pad.
4. The test strip of claim 1, wherein said monoclonal antibodies to said serpentine antigen are prepared from a serpentine hapten-carrier protein conjugate as an immunogen, and said anti-murine goat antibodies are obtained by immunizing a goat with a murine antibody.
5. A method of preparing the test strip of any one of claims 1-4, comprising the steps of:
1) Preparing a conjugate release pad sprayed with a serpentine monoclonal antibody-colloidal gold marker;
2) Preparing a reaction membrane with a detection line coated with a snake fungus hapten-carrier protein conjugate and a quality control line coated with a goat anti-mouse anti-antibody;
3) And (3) assembling the conjugate release pad, the reaction membrane, the sample absorption pad, the water absorption pad and the bottom plate which are prepared in the steps 1) and 2) into the test strip.
6. A method for detecting serpentine residues in wheat, corn and other grains, feed comprising the steps of:
1) Sample pretreatment;
2) Detecting with the test strip of any one of claims 1-4;
3) And analyzing the detection result.
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