CN1858232A - Method for screening sulfanilamide medicine residue in edible animal tissue and reagent kit - Google Patents

Abstract

The invention belongs to the technical field of detection of residual antibiotics in agricultural products. Specifically, the invention relates to a method for detecting residues of sulfonamides in animal edible tissues by using a microbiological method and a kit thereof. The method comprises: preparing culture medium I and culture medium II, using Bacillus megaterium as working bacteria liquid, neomycin disc as antibiotic disc for quality control, trimethoprim as supplementary liquid for synergistic effect, and culturing Base I and medium II are used in combination, and a cotton swab is used to stick the animal tissue sample to be tested, and a little trimethoprim supplement is dropped on the cotton swab, and then it is contacted with the medium for culture. The test results were judged according to the presence or absence of a bacteriostatic zone around the cotton swab. The invention also discloses a special kit for applying the above method. Compared with the prior art, the invention has the advantages of simple detection method, high sensitivity, long storage time of the kit, and is suitable for high-throughput sample screening.

Description

A screening method and kit for sulfonamide drug residues in animal edible tissues

Technical field

The invention belongs to the technical field of detection of antibiotic residues in agricultural products, in particular to a method for screening sulfonamide drug residues in animal edible tissues by using a microbiological method and a kit prepared by the method.

Background technique

Sulfonamides are the earliest artificially synthesized antibacterial drugs. They have the advantages of broad antibacterial spectrum and strong curative effect, and are widely used in livestock, poultry, and aquaculture. However, excessive use of sulfonamides will inevitably lead to residues in edible animal products. .

At present, there are physical and chemical analysis methods and biological analysis methods for the determination of sulfonamide drug residues in food. Physical and chemical analysis methods include high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), capillary electrophoresis (CE), fluorescamine derivatized thin-layer chromatography (TLC), enzyme-linked immunosorbent assay (ELISA) wait. The instruments required for these methods are expensive, have high requirements for operators, are difficult to promote, and are not suitable for high-throughput sample screening. Bioanalysis methods include ELISA method and microbiological method. Although the ELISA method has the advantages of rapidity and high sensitivity, the technology of the currently developed sulfonamide multi-residue ELISA kit is not mature enough, and there are problems such as high detection costs.

Application of microbiological methods to detect residues of sulfonamides in animal food has been reported. It is based on the inhibitory effect of antibacterial substances on the physiological functions and metabolism of microorganisms to qualitatively or quantitatively determine the residues of antibacterial drugs in samples. It has the characteristics of high throughput, fast, convenient, and sensitive, and is widely used in the world. The EU four-plate method, the German three-plate method, the New Holland kidney test method and other methods are to add the working bacteria solution to the liquid agar medium at 40-45°C and mix evenly to make a plate, store it at 4°C, and the validity period is 5 days. This method is cumbersome and laborious to operate, has a short validity period, and is not sensitive to sulfonamides (Korsrud GO, Boison JO, Nouws JF, MacNeil JD. Bacterial inhibition tests used to screen for antimicrobial veterinary drug residues in slaughtered animals. J AOAC Int. 1998, 81(1):21-4), thus limiting the application of these methods in the detection of sulfa residues. The U.S. and Canadian swab methods mainly include the U.S. Calf Antibiotic and Sulfonamide Test (CAST for short) (U.S.Department of Agriculture (1984), Performing the Calf Antibiotic and Sulfa Test. A Self Instructional Guide , USDA/FSIS, Washington, DC;) and Fast Antimicrobial Screening Test (FAST for short) (Fast Antimicrobial Screen Test, (FAST): for detection of antibiotic and sulfonamide residues in Livestock kidney tissue-(1994) Performing the CalfAntibiotic and Sulfa Test.A Self Instructional Guide, USDA/FSIS), both of these methods use Bacillus megaterium as the working bacteria solution, and the components of the culture medium of the two kits are respectively: the culture medium of the CAST kit is Mueller - Hinton's medium (as described in the following documents), the medium of the FAST kit is the above-mentioned Mueller-Hinton medium with glucose and bromocresol purple added. The sensitivity of the FAST kit to antibiotics and sulfonamides is higher than that of the CAST kit. The detection limit of FAST to sulfonamides is 0.125-0.5 μg/g, and that of CAST is 0.25-2.0 μg/g (see: DeyBP, Thaker NH, Bright SA, Thaler AM. Fast antimicrobial screen test (FAST): improved screen test for detecting antimicrobial residues in meat tissue. J AOAC Int., 2005, 88(2): 447-454), the above two kits cannot meet the requirements of sulfa At the same time, the detection time of FAST for antibiotics and sulfonamides is shorter than that of CAST, and the detection time of the two is 6h and 18h, respectively. The formulation of the medium is the main reason for the difference between the above two kits. It is an urgent problem to overcome the long detection time, low sensitivity and short storage period of existing methods and kits.

Contents of Invention

The first object of the present invention is to overcome the defect of prior art, establish a kind of based on microbiology, convenient, sensitive, the screening method that is used for sulfonamide drug residue in edible animal tissue, to overcome prior art low sensitivity, Defects such as long detection time, time-consuming and laborious operation.

The second purpose of the present invention is to develop a test kit used in conjunction with the above purpose to solve the problems of short shelf life and low sensitivity of the existing test kits.

The present invention is realized through the following technical solutions:

A method for screening sulfonamide drug residues in animal edible tissues, comprising preparation medium, working bacteria liquid of Bacillus megaterium, antibacterial drug discs and sterile cotton swabs, the preparation steps of which are as follows:

1) Separately prepare the culture medium, working bacteria solution, antibacterial drug disc and supplement solution containing trimethoprim;

2) making Bacillus megaterium into a working bacterial liquid with a spore number of 1×10 ⁶ /ml;

3) Prepare the antibacterial drug disc into a disc with a diameter of 7 mm and containing 5 μg of neomycin;

4) The culture medium is made into the culture medium as described below, and its components are as follows:

Medium I:

Beef Extract 1.5～3g/L

Casein peptone 8.5～17g/L

Soluble starch 0.75～1.5g/L

Potassium dihydrogen phosphate 0.5～1.0g/L

Agar 15～20g/L

Sterile calf serum 30～50ml/L

Trimethoprim 0.016～0.04mg/L

Add water to 1L;

Medium II:

Beef Extract 1.5～3g/L

Casein peptone 8.5～17g/L

Soluble starch 0.75～1.5g/L

Potassium dihydrogen phosphate 0.5～1.0/L

Agar 15～20g/L

Sterile calf serum 30～50ml/L

Trimethoprim 0.016～0.04mg/L

p-aminobenzoic acid 0.1～0.2g/L

Add water to 1L;

Prepare as follows:

a) Heat and melt the beef extract, casein peptone, soluble starch, potassium dihydrogen phosphate and agar in the above medium, adjust the pH to 7.0-7.5, sterilize at 121°C for 20 minutes, and cool to 50°C;

b) adding inactivated sterile calf serum and trimethoprim to step a) to obtain culture medium I;

c) adding p-aminobenzoic acid to step b) to obtain medium II;

d) distributing the culture medium in step b) or c) into a petri dish, cooling the culture medium, and making the thickness of the culture medium in the petri dish 1.5-2mm;

6) Evenly spread the working bacteria solution on the culture medium I or II;

7) Place the neomycin disc on the culture medium as a positive control;

8) Take a sample of the tissue to be tested with a cotton swab, and place it on culture medium I or II;

9) drip replenishment solution onto the cotton swab; and

10) Place medium I or II at a temperature of 44-45° C., incubate for 5-8 hours, and judge whether the sample to be tested contains sulfonamides according to the appearance of the inhibition zone on medium I and II.

In the present invention, the trimethoprim in the supplement solution is 1.5-2 μg/ml (prepared with 10% NaCl solution).

Based on the above invention, the present invention prepares a kit suitable for screening sulfonamide drug residues in animal edible tissues. The kit includes working bacteria liquid, culture medium, antibacterial drug discs and sterile cotton swabs. The working bacterial liquid is a Bacillus megaterium bacterial liquid with a spore number of 1×10 ⁶ /ml, the antibacterial drug disc is 5 μg neomycin/∮7 mm, and the replenishing liquid is trimethoprim Liquid, the medium is the combination of medium I and medium II, prepared according to the following components:

Medium I:

Beef Extract 1.5～3g/L

Casein peptone 8.5～17g/L

Soluble starch 0.75～1.5g/L

Potassium dihydrogen phosphate 0.5～1.0g/L

Agar 15～20g/L

Sterile calf serum 30～50ml/L

Trimethoprim 0.016～0.04mg/L

Add water to 1L;

Medium II:

Beef Extract 1.5～3g/L

Casein peptone 8.5～17g/L

Soluble starch 0.75～1.5g/L

Potassium dihydrogen phosphate 0.5～1.0/L

Agar 15～20g/L

Sterile calf serum 30～50ml/L

Trimethoprim 0.016～0.04mg/L

p-aminobenzoic acid 0.1～0.2g/L

Add water to 1L;

Prepare as follows:

a) Heat and melt the beef extract, casein peptone, soluble starch, potassium dihydrogen phosphate and agar in the above medium, adjust the pH to 7.0-7.5, sterilize at 121°C for 20 minutes, and cool to 50°C;

b) adding inactivated sterile calf serum and trimethoprim to step a) to obtain culture medium I;

c) adding p-aminobenzoic acid to the medium of step b) to obtain medium II;

d) Divide the culture medium in step b) or c) into petri dishes, cool the culture medium, and make the thickness of the culture medium in the petri dish 1.5-2mm.

In the present invention, the described replenishment liquid solution containing trimethoprim is formulated with 10% NaCl solution, which contains 1.5-2 μg/ml of trimethoprim (this replenishment liquid is specially used in the detection process) The cotton swab is used as a synergist when taking the sample of the tissue to be tested. It is an independent component (core reagent), and the trimethoprim added in the medium is pre-mixed with other above-mentioned components in the prepared medium. joined together).

The kit of the invention can be applied to detect sulfonamide drug residues in animal edible tissues in vitro.

The detection principle of the kit of the present invention is to utilize the synergistic effect of trimethoprim (TMP) and sulfa drugs to enhance the sensitivity of bacteria to sulfa drugs, and to increase the content of TMP in the culture medium and cotton swabs to be tested The ability to detect sulfonamides. Utilizing the antagonism between p-aminobenzoic acid (PABA) and sulfa drugs, the influence of sulfa drugs was eliminated by supplementing the content of PABA in the medium, and whether the residue was sulfonamide was qualitatively analyzed by combining two media with and without PABA drugs.

Advantages and effects of the present invention: the present invention adopts the method of strain and medium separation, and the main reagents are all provided in the form of working fluid, which solves the problem of poor stability of traditional microbial methods; when measuring samples, only the cotton swab Insert the tissue sample, the sample pretreatment is simple, the whole determination process is shortened from 6h to 5h, suitable for high-throughput sample screening; low sensitivity to sulfa drugs, suitable for qualitative detection of various sulfa drug residues in animal tissue samples; operation The method is convenient and easy to implement, has high sensitivity, is simple and fast, and will provide a strong guarantee for the monitoring of sulfonamide drug residues.

Description of drawings

Fig. 1, Fig. 2: are application kit of the present invention to the result judging schematic diagram of sulfonamide drug residue in the sample, the antibacterial zone diameter that neomycin disc produces on the culture medium of Fig. 1 and Fig. 2 is 20～26mm , A: In Figure 1A and Figure 2A, there is no zone of inhibition around the cotton swab, and the result is judged as negative; B: The cotton swab has a clear zone of inhibition in Figure 1B, but there is no zone of inhibition in Figure 1B, If the diameter of the inhibition zone is ≥ 6mm, the result is judged as positive, indicating that there are sulfonamide drug residues; C: The cotton swabs have clear antibacterial zones in Figure 1C and Figure 2C, indicating that there are other drug residues except sulfonamide drugs .

Fig. 3: is the comparison column chart of the minimum detection limit of the kit of the present invention and similar kits CAST, FAST kits to the sulfonamide standard solution. Among the figure: SST represents the kit of the present invention, CAST represents the CAST kit, legend FAST represents the FAST kit; SD is sulfadiazine, SM ₂ is sulfamethazine, SQ is sulfaquinoxaline, and SMP is sulfamethoxine oxazine, SMM is sulfamethoxine, and SMZ is sulfamethoxazole.

Figure 4: is the effect of storage time on the diameter of the neomycin disk inhibition zone.

Figure 5: It is the effect of storage time on the colony concentration of Bacillus megaterium application working bacterial solution.

Fig. 6: is the screening method of the present invention and its kit - schematic diagram of swabbing inoculation, in the figure:

A. Starting from the marked point (X), wipe up and down, from right to left;

B. Turn the petri dish clockwise for 1/4 turn, repeat 1 to wipe;

C. Turn the petri dish 1/4 turn again, repeat the 1-smearing process;

D. Turn the petri dish 1/4 turn again, and repeat the 1-smearing process;

E. Finally, turn the petri dish clockwise for 1/2 turn, and repeat the swabbing process of A.

Figure 7: It is a schematic diagram of the placement of swabs and neomycin discs.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. These examples are only used to illustrate the present invention and are not intended to limit the scope of protection of the present invention. Specific experimental conditions and methods are not indicated in the following examples, usually operated according to conventional methods, referring to Dey B.P., White C.A. and Thakre N.H..Detection of antimicrobial residues by fastantimicrobial screen test(FAST).USDA/FSIS.Microbiology Laboratory Guidebook.3rd edition, 1998. In view of the interdependence between the development of the method and the kit of the present invention, in order to save space, the following descriptions describe the establishment of the detection method together with the kit.

Embodiment 1, establishment of screening method

(1) Design of optimal culture medium

On the composition of basic medium (beef extract 1.5g/L, casein peptone 8.5g/L, soluble starch 0.75g/L), three kinds of factor tests affecting drug sensitivity are measured, respectively A: the concentration of TMP: TMP4μg /ml, TMP1.6μg/ml, TMP0.2μg/ml, added according to 1% of the medium volume; B: the volume of the medium added to the culture dish: 5.5ml, 7ml and 8.5ml; C: serum accounted for the total medium , designed to be 3%, 5%, 7% in turn, with sulfadiazine 0.1 μg/ml as the object, and the number of spores was 1× ¹⁰⁶ /ml Bacillus megaterium working bacteria liquid (Bacillus megaterium, the strain number is ATCC9885 , see: USDepartment of Agriculture (1984) Performing the Calf Antibiotic and Sulfa Test. A Self Instructional Guide, USDA/FSIS, Washington, DC, purchased from: American Type Culture Collection, Rockville, Md. (American Type Culture Collection, Rockville, MD)) (the following examples are all strains from the same source), determine the optimal medium. The results show that: by weight/volume, beef extract 1.5g/L, casein peptone 8.5g/L, soluble starch 0.75g/L, potassium dihydrogen phosphate 0.5g/L, agar powder 15g/L, add distilled water to volume To 1L, adjust the pH to 7.0. Sterilize at 121°C for 20 minutes, cool to 50°C, add sterile 5% calf serum and 1% TMP1.6μg/ml as an optimized medium.

Table 1 The influence of medium formula on the diameter of sulfadiazine inhibition zone Number of trials A (μg/ml) B(ml) C(%) Average diameter of inhibition zone (mm, n=6) 123456789K1K2K3 extremely poor optimal sequence factor primary and secondary arrangement 4441.61.61.60.20.20.211.7612.4410.521.92A21 5.578.55.578.55.578.511.9011.3511.470.55B12 35753773511.5711.7111.440.25C23 12.7311.3511.2012.5812.7312.0210.389.9711.2

Note: A: TMP concentration; B: volume of culture medium added to the culture dish; C: serum accounted for the total amount of culture medium

(2) Determination of the optimal concentration of replenishing solution

Taking sulfadiazine 0.1 μg/ml as the object, compare three 2 μg/ml TMP aqueous solutions, 10% NaCl solution, and 10% NaCl dissolved 2 μg/ml TMP to add different doses of the inhibition zone diameters, as shown in Table 2. From the results, it can be known that 2 μg/ml TMP trimethoprim dissolved in 50 μL of 10% NaCl has a good effect.

Table 2 Effects of different supplements and doses on the diameter of the inhibition zone (unit: mm) Drug concentration (μg/ml) Trimethoprim dose (μL) Trimethoprim (TMP) 2 μg/ml TMP 10%NaCl 10%NaCl+2μg/mlTMP 0 0.20.10.0500.20.10.0500.20.10.050 503010 12.56±0.169.97±0.38--10.15±0.287.60±0.08--9.72±0.30--- 9.35±0.18---8.23±0.06---8.38±0.17--- 13.3±0.21 12.7±0.12--12.37±0.328.70±0.30--10.32±0.23--- ------------

"-" indicates no inhibition zone

(3) Determination of p-aminobenzoic acid concentration

Take 1ml of 1μg/ml sulfadiazine, put it into a flat plate, dilute 1× ¹⁰⁶ spores of Bacillus megaterium to 10 ^-4 , then add 1ml of the diluted Bacillus megaterium solution to the plate, inject 15ml containing Different concentrations (0.05g/L, 0.1g/L, 0.2g/L, 0.4g/L and 1g/L) of PABA were used as agar medium for detection, and a blank control group without PABA was established simultaneously. 5 plates for each concentration, repeated 3 times. After repeated cultivation, the results of 5 plates of the same sampling were added. The measurement results are shown in Table 3.

The results showed that the optimal concentration of PABA in the culture medium was 0.2g/L.

Table 3 Effects of different concentrations of p-aminobenzoic acid on the number of Bacillus megaterium bacteria affected by sulfadiazine Plate number Sulfadiazine group Concentration of para-aminobenzoic acid (PABA) blank control 0.05g/L 0.1g/L 0.2g/L 0.4g/L 1g/L 123 average 9355.6 54695559.3 85696171.7 87979894 84728580.3 44605653.3 88799487

Embodiment 2, the preparation of kit

(1) Composition of the kit

The kit of the present invention comprises a petri dish containing a culture medium, a Bacillus megaterium bacterial liquid with a spore number of 1× ¹⁰⁶ /ml, a 5 μg/diameter 7mm neomycin filter paper, and a 2 μg/ml trimethoprim supplement solution And sterile cotton swabs (specifications: cotton swab head length 26-30mm, diameter 4.2-4.8mm. Sterilize at 121°C for 15 minutes, dry at 37°C, and store in a sealed container).

(2) Preparation of strains

Bacillus megaterium strains were inoculated on three brain-heart extract medium agars (purchased from Oxoid Company, UK), and cultured at 37°C for 18 hours. Pick a single colony and inoculate it on blood agar medium, and culture at 37°C for 18h. Pick 3 typical colonies from each culture medium for biochemical identification, and then pick a qualified single colony and inoculate it on the slant of AK-2 spore growth agar test tube (Dey BP, White C A and Thakre N H. Detection of antimicrobial residues by fast antimicrobial screen test(FAST).USDA/FSIS.Microbiology Laboratory Guidebook.3rd edition, 1998) (When cultivating and purifying the strains, they must pass the biochemical identification before proceeding to the next step, otherwise the strains will be re-cultivated) , and cultured at 37°C for 18h. Wash the spores with sterile normal saline, transfer them to Luxe flasks with AK-2 spore growth agar, incubate at 37°C for 18-24h, incubate at room temperature for 6d, and examine under the microscope. When 80% of the spores are formed, wash the spores with sterilized physiological saline into a sterilized Erlenmeyer flask, heat in a water bath at 100°C for 10 minutes, aliquot them, centrifuge at 10,000 r/min for 20 minutes, and discard the supernatant. The precipitate was suspended with sterilized physiological saline, centrifuged, and washed twice, and the precipitate was resuspended with sterilized physiological saline to form a spore mixture.

Weigh 11.8g of polyethylene glycol 4000 into a 100ml graduated glass bottle with a stopper, sterilize at 121°C for 5min; add sterilized phosphate buffer (3mol/L phosphate buffer (pH7.1): hydrogen phosphate Dissolve 306.9g of dipotassium and 168.6g of potassium dihydrogen phosphate into 34.1ml of deionized water (1000ml), shake fully, vortex mix at 5000r/min for 5min, and discard. Add 11.8 g of sterilized polyethylene glycol 4000, 34.1 ml of phosphate buffer solution (the same formula as before, pH 7.1), 25 ml of the aforementioned spore mixture, add to 100 ml with sterilized water, and vortex mix at 5000 r/min for 5 min , divided into centrifuge tubes, and centrifuged at 3000r/min for 2min. Take the upper layer liquid into another sterile 50ml centrifuge tube, centrifuge at 5°C, 10000r/min for 20min, and discard the supernatant. Add 20 ml of sterilized water to the sediment to suspend, centrifuge at 5°C for 20 min at 10,000 r/min, discard the supernatant, and repeat washing 5 times. Suspend the precipitate with 50% ethanol, collect the bacterial suspension in a 100ml sterile glass bottle, seal it, and store it at 4°C, with a validity period of 6 months.

Colony counting: Dilute the suspension of Bacillus megaterium with Butterfield phosphate buffer (0.0425 g of potassium dihydrogen phosphate dissolved in 1000 ml of deionized water, pH7.2) to a bacterial solution of 10 ⁻¹ to 10 ⁻¹⁰ . Take 0.1ml of the diluted bacterial solution into a petri dish, add 10ml of melted petri dish count agar, and mix well. Repeat 3 petri dishes for each concentration, incubate at 37°C for 48h, and count. Calculate the concentration of the spore stock solution with the dilution factor of 30-300 colonies per plate. According to the concentration of the spore stock solution, use sterile 50% ethanol to dilute the spore stock solution to the specified concentration of 1× ¹⁰⁶ /ml. The application solution is stored in a sterile container at 4°C.

(3) Preparation of medium and plates

Preparation of medium I: take beef extract 1.5g/L, casein peptone 8.5g/L, soluble starch 0.75g/L, potassium dihydrogen phosphate 0.5g/L, agar powder 15g/L, heat and melt, adjust pH value To 7.0, sterilize at 121°C for 20min. After cooling to 50°C, add 50ml of sterile calf serum and 10ml of 1.6μg/ml TMP solution. After mixing evenly, add 5.5ml of the above-mentioned medium to each culture dish, spread it evenly, place it on a water platform, cool and solidify, spare.

Preparation of medium II: take beef extract 1.5g/L, casein peptone 8.5g/L, soluble starch 0.75g/L, potassium dihydrogen phosphate 0.5g/L, p-aminobenzoic acid 0.2g/L, agar powder 15g /L, heat to melt, adjust the pH value to 7.0, and sterilize at 121°C for 20min. After cooling to 50°C, add 50ml of sterile calf serum and 10ml of 1.6μg/ml TMP solution. After mixing evenly, add 5.5ml of the above-mentioned medium to each culture dish, spread it evenly, place it on a water platform, cool and solidify, spare.

(4) Preparation of Neomycin Discs

Take 200 pieces of filter paper with a diameter of 7mm and put them into a clean penicillin bottle, sterilize at 121°C for 15min, dry at 37°C, add 500μg/ml neomycin (purchased from China Veterinary Drug Control Institute, the content is 709IU/mg, Production batch number: 3099310) solution 2ml, soak each piece of paper and equilibrate overnight at 4°C. After drying at 37°C, seal and store at room temperature (22-25°C). Each disc contains 5 μg of neomycin.

(5) Preparation of Trimethoprim Supplementary Solution

Accurately weigh 10.01 mg of trimethoprim standard substance (content is 99.87%), add 80 ml of sterile 20% ethanol, fully mix, and set the volume to 100 ml to form a trimethoprim standard stock solution with a concentration of 100 μg/ml. Take 1ml of the above-mentioned trimethoprim standard stock solution, dilute it to 50ml with sterile 10% sodium chloride, make a trimethoprim supplement solution with a concentration of 2 μg/ml, and store it at -20°C.

Embodiment 3, the operating procedure that kit of the present invention uses

3.1 Operation steps of the kit of the present invention

Step 1: Carefully check the temperature of the incubator, set the temperature to 44-45°C, place a wide-mouth bottle in the incubator, and fill it with about 120ml of water. Take out the culture medium and bacteria solution from the refrigerator and let it stand at room temperature.

Step 2: Place the sample to be tested in a clean tray, and use a clean scalpel to quickly cut the tissue sample (kidney, muscle, liver) with a depth of about 2 cm. Note that the cut surface remains flat.

Step 3: After taking out the sterile cotton swab, insert two cotton swabs vertically from the incision into the tissue to be tested, and clamp the cut surface with a little force so that the cotton swab head fully contacts the tissue. Place the cotton swab in the tissue for at least 30 minutes until the cotton swab is full of tissue fluid.

Step 4: Use a marker pen to make a reference mark on the outside of the culture dish with the culture medium, as a starting point for swabbing the Bacillus megaterium strain. Check that the caps of the spore-laden bottles are tight. After sealing, shake the spore suspension vigorously to mix the spores evenly. Open the cap of the spore-containing bottle, insert a sterile cotton swab, and take it out after the cotton swab is completely soaked. Tighten the bottle cap of the spore-containing bottle and seal it again with a parafilm. Be careful not to reuse cotton swabs.

Step 5: The process of wiping the bacteria solution is shown in Figure 6.

Step 6: Open the bottle cap of Sheng Neomycin, take out the paper with tweezers, place a piece of neomycin paper at a position 12-13mm away from the marked edge of the petri dish, gently press the paper with tweezers to make the paper Adhere completely to the medium. Note: After the neomycin disc is placed, do not move the position of the neomycin disc. If the position of the neomycin disc is not suitable, the culture medium should be re-smeared.

Step 7: Check whether the cotton swab head is saturated in the tissue, take it out after it is full, cut off the cotton swab rod with scissors, and leave a length of about 10-15 mm, and place them on the culture medium that has been wiped with the bacterial solution ( On the surface of medium I and medium II) (in the shape of a rabbit ear), the cotton swab head should be kept away from the neomycin disc. Place two cotton swabs on each Petri dish (two samples can be determined), and after placing them, use tweezers to lightly press the cotton swab rod to fix it. See attached drawing 7.

Step 8: Use a pipette to draw 50 μL of trimethoprim solution onto the cotton swab and drop it onto the cotton swab, and place the prepared culture dish in an incubator at a temperature of 44°C. Incubate the kit for 5 hours, not exceeding 12 hours, and judge the result.

Step 9: Carefully remove the Petri dish from the incubator and carefully inspect the surface of Medium I and Medium II for bacterial growth. Observe the surface of the medium for some bacterial growth. If yes, continue to the next step; if not, there may be problems with materials or operations.

Step 10: Carefully inspect the area around the neomycin disk. The transparent and clear area around the disk is called the zone of inhibition. This circle is mainly used as a positive control to indicate whether the kit is effective. If there is no antibacterial zone around the paper, it indicates that there is a problem with the kit material or improper operation. Use a plastic ruler to measure the diameter of the inhibition zone. If the diameter of the inhibition zone is below 20mm, it indicates that there is a problem in the experimental operation, and the experiment should be repeated.

Step 11: Carefully inspect the area around the cotton swab head, there is a transparent and clear area around the cotton swab head called the zone of inhibition. The formation of this zone of inhibition is due to the diffusion of sulfa drugs in the cotton swab head into the culture medium to inhibit the growth of bacteria. A zone of inhibition around the swab tip indicates a positive assay result. The cotton swab heads were covered with bacteria, indicating that there was no residue in the renal interstitial fluid. The absence of a zone of inhibition around the swab tip indicates a negative assay result.

3.2 Judgment of the result of the kit of the present invention

If the diameter of the inhibition zone produced by the neomycin disc on the medium I and II is within 20-26mm, the result can be judged (see Figure 1 and Figure 2).

On medium I and II, there was no zone of inhibition around the cotton swab, and the results were judged as negative.

Cotton swabs have a clear zone of inhibition on medium I, but no zone of inhibition on medium II. If the diameter of the zone of inhibition is ≥6mm, the result is judged as positive, indicating that there are sulfonamide drug residues.

Cotton swabs have clear inhibition zones in medium I and II, indicating that there are other drugs except sulfonamides.

Embodiment 4, sensitivity, quantity-response relationship, accuracy, precision, false negative rate, stability experiment of kit

(1) Sensitivity test of the kit

Accurately weigh the standard sulfonamides (as described in Table 4), dissolve with 0.1moL/L sodium hydroxide solution and dilute with distilled water to a standard stock solution with a concentration of 1000 μg/ml. Select the homogeneous blank pig muscle and kidney tissue of 6 different sources (the following examples are the same), each 5g of tissue, add each 0.5ml of sulfonamides, make the concentration of sulfonamides in the tissue to be tested reach 0.025, 0.05 respectively . Potassium 17.1g dissolves, and is settled to 1000ml distilled water) 9.5ml, vortex mixes 5min, room temperature stands for 30min, centrifuges 10min under 4000r/min, gets the supernatant for medium I detection, takes standard solution (sulfonamides drug) simultaneously Concentrations are 0.025, 0.05, 0.1, 0.2, 0.5, 1, 2, 4 μg/ml in sequence). Five replicates were set for each concentration, and a total of 30 determinations were made. The lowest detection limit was the minimum concentration of those whose results were all positive for 30 determinations. The test results of this test are shown in Table 4. It can be seen from Table 4 that the minimum detection limit of the kit of the present invention for the six sulfa drugs in pig muscle and kidney tissue is 0.025-0.1 μg/g. The comparison of the minimum detection limit of the kit of the present invention with similar kits CAST and FAST kits to the standard solution of sulfonamides is shown in Figure 3.

Table 4 method of the present invention is to the minimum detection limit test result of sulfonamides in standard solution and pig tissue drug name Maximum residue limit (μg/g) Standard solution (μg/ml) Kidney (μg/g) Muscle (μg/g) Sulfadiazine (SD) Sulfamethazine (SM ₂ ) Sulfaquinoxaline (SQ) Sulfamethoxypyridazine (SMP) Sulfamethoxazole (SMM) Sulfamethoxazole (SMZ) 0.1Et0.1Et01Et0.1Et01Et0.1Et 0.10.10.050.050.0250.05 0.10.10.10.10.050.1 0.10.10.10.10.10.05

(2) Quantity-response relationship test of the kit

Take by weighing homogeneous blank pig muscle, kidney tissue sample 5g, add sulfa drug standard working solution (as above) 0.5ml, make the drug concentration in the tissue be respectively 0.1, 0.2, 0.5, 1, 2, 4 μ g/g, After sample preparation, the supernatant was taken for medium I detection. Each concentration was replicated 5 times and measured 5 times. The measured diameter of the inhibition zone was linearly fitted with the corresponding logarithmic concentration, a standard curve was drawn, and the regression equation and correlation coefficient were obtained. Table 5 shows the linear range and correlation coefficient of the standard curve measured by the kit of the present invention to 6 kinds of sulfonamides, showing that in the concentration range of 0.1～4 μg/ml (g), the kit of the present invention is effective for pig muscle and kidney tissue. The correlation coefficients of the six sulfonamides were all above 0.9, showing a good linear relationship.

Table 5 standard curve linear range and correlation coefficient that kit of the present invention records to 6 kinds of sulfonamides drug Linear range μg/ml(g) correlation coefficient standard solution muscle kidney Sulfadiazine (SD) 0.1～4 0.999 0.991 0.909 Sulfamethazine (SM ₂ ) Sulfaquinoxaline (SQ) Sulfamethoxypyridazine (SMP) Sulfamethoxazole (SMM) Sulfamethoxazole (SMZ) 0.1～40.1～40.1～40.1～40.1～4 0.9890.9880.9950.9920.983 0.9950.9850.9850.9860.991 0.9580.9830.9860.9750.976

Note: The standard solution in this table is the same as that shown in Table 4

(3) Accuracy and precision test of the kit

Take by weighing homogeneous blank pig muscle and kidney tissue samples 5g, add 0.5ml of sulfonamides standard working solution (as mentioned above), so that the drug concentration in the tissue is respectively 0.5, 0.2, 0.1 μg/g, after sample preparation, take The supernatant was used for medium I detection. Five replicates were set for each concentration, and a total of five determinations were made to calculate the recovery rate. The results show that when adding 0.1-0.5 μg/g sulfonamides in pig kidney tissue, the recovery rate is 70%-120%; the recovery rate of sulfonamides in pig muscle tissue is 80%-120%. The test results are shown in the table 6 ~ Table 7.

Table 6 kit of the present invention is to the rate of recovery and coefficient of variation test result of sulfonamides in pig kidney tissue drug Concentration added (μg/g) Recovery rate(%) Coefficient of variation between days (%) Sulfadiazine (SD) Sulfamethazine (SM ₂ ) Sulfaquinoxaline (SQ) Sulfamethoxypyridazine (SMP) Sulfamethoxazole (SMM) Sulfamethoxazole (SMZ) 0.50.20.10.50.20.10.50.20.10.50.20.10.50.20.10.50.20.1 92.2±9.993.2±7.9110.3±9.8101.2±7.3115.5±12.9114.7±15.278.1±6.885.2±6.491.0±7.677.7±6.591.5±6.0101.6±9.673.2± 5.089.9±5.793.2±8.269.7±5.975.8±6.180.9±8.9 10.78.58.97.211.213.38.77.58.38.46.69.46.86.48.88.58.011.0

Table 7 The recovery rate and coefficient of variation test result of kit of the present invention to sulfonamides in pig muscle tissue drug Concentration added (μg/g) Recovery rate(%) Coefficient of variation between days (%) Sulfadiazine (SD) Sulfamethazine (SM ₂ ) 0.50.20.10.50.2 94.9±7.1115.4±8.3110.5±12.8103.3±5.295.6±7.9 7.57.212.05.08.3 Sulfaquinoxaline (SQ) Sulfamethoxypyridazine (SMP) Sulfamethoxine (SMM) Sulfamethoxazole (SMZ) 0.10.50.20.10.50.20.10.50.20.10.50.20.1 104.7±6.980.8±5.091.1±13.9105.6±13.484.9±4.998.9±8.8114.6±15.088.4±9.995.5±16.9104.7±21.980.8±4.091.1±13.9105.6± 2.9 6.66.215.312.75.88.913.111.217.720.95.015.32.7

(4) False negative rate test of the kit

Weigh 5 g of homogeneous (prepared according to the general method) blank pig muscle and kidney tissue samples, add 0.5 ml of sulfa drug standard working solution (as described above), so that the concentration of sulfa drug in the tissue to be tested reaches 0.1 μg/g , after sample preparation, the supernatant was taken for medium I detection. Repeat the measurement 100 times, observe the probability of negative results, and calculate the false negative rate. It can be seen from Table 8 that when sulfamethazine in muscle is 0.1 μg/g, the false negative rate is 2%; the false negative rate of sulfamethoxypyridazine is 2%; Oxypyrimidine and sulfaquinoxaline had a false negative rate of 0%. In pig kidneys, when sulfaquinoxaline was 0.1 μg/g, there was no false negative; the false negative rate of sulfamethoxypyridazine was 2%, the false negative rate of sulfamethoxazole was 0%, , sulfamethazine and sulfaquinoxaline had no false negative rate.

Table 8 kit of the present invention to the false negative rate (%) in pig kidney and muscle tissue antibacterial drugs Maximum residue limit (μg/g) Concentration added (μg/g) False negative rate (%) kidney muscle Sulfadiazine (SD) Sulfamethazine (SM ₂ ) Sulfaquinoxaline (SQ) Sulfamethoxypyridazine (SMP) Sulfamethoxazole (SMM) Sulfamethoxazole (SMZ) 0.10.10.10.10.10.1 0.10.10.10.10.10.1 000200 020100

(5) Stability test of the kit

1) Stability test of neomycin disc

The neomycin discs prepared by the present invention are stored at 2-8° C., and the neomycin discs are taken every month to measure the diameter of the inhibition zone. Take the newly prepared medium (the components and contents of the medium are as follows: 6g of peptone, 4g of tryptone, 1.5g of beef extract, 3g of yeast extract, and 1g of glucose, dissolve in 1000ml distilled water, adjust the pH to 7.3, add agar powder 15g, obtained by moist heat sterilization), coated with freshly prepared Bacillus subtilis spore liquid (Bacillus subtilis, ATCC6633, American Type Culture Collection, Rockville, Maryland (American Type Culture Collection ^TM , Rockville, MD)) , the concentration of its spore fluid is 1×10 ⁶ /ml. Take 3 neomycin discs and put them on the surface of the medium, incubate at 29°C for 18 hours, measure whether the diameter of the inhibition zone on the surface of the medium is within the range of 20-26 mm, and use it as a reference standard for identifying the expiration date of the medium. As can be seen from Figure 4, the neomycin disc prepared by the present invention is stored at 2-8°C, and the diameter of the inhibition zone within 8 months is between 21.6-23mm, and its validity period can be set within 6 months.

2) The stability of the working bacteria liquid of Bacillus megaterium

The concentration of the spore liquid prepared by the present invention is 1× ¹⁰⁶ /ml of Bacillus megaterium stored at 2-8°C, the colony concentration of the bacterial liquid is measured every month by the colony counting method, and the trend of the colony concentration over time is observed. The results showed that the concentration of the spore fluid decreased slightly, and the change was small within 6 months (Fig. 5). Place the newly prepared neomycin paper on the surface of the culture medium of the present invention that has been coated with the working bacterial liquid for storage, and the diameter of the antibacterial zone of the neomycin paper is in the range of 20 to 26mm, indicating that the bacterial liquid is 6 months old. inside is valid.

3) Stability test of kit culture medium

A number of culture media of the kit of the present invention are newly prepared, the culture dishes are packaged in aluminum-plastic composite bags, and stored at room temperature of 20-25°C and 2-8°C. Samples were taken every month, and 6 petri dishes were sampled each time, and the sensory indicators of the culture medium in the petri dishes were observed. At the same time, each petri dish was smeared with newly prepared Bacillus megaterium applied working bacteria solution (1×10 ⁶ cells/ml), and assessed with newly prepared 0.1 μg/ml sulfadiazine standard solution and newly prepared neomycin disc respectively. . If the detection medium is free of bacteria contamination, there is no significant change in appearance, and Bacillus megaterium grows vigorously and evenly, and the inhibition zone of the neomycin disc is within the range of 20-26mm, then the medium is determined to be effective. As can be seen from Table 9 and Table 10, no matter whether it is at room temperature or at 2～8 ℃ for the medium tested within 6 months, the sensory index of the medium has no obvious change; within 6 months, medium I can detect 0.1 μg/ml sulfadiazine standard solution, sulfadiazine cannot be detected on medium II. The diameter of the inhibition zone of the neomycin disk fluctuated in the range of 20-26 mm, which met the performance index of the specified kit.

Stability test of medium in the test kit of the present invention in table 9 time (month) Antibacterial zone diameter (mm) Room temperature (22-25°C) 2～8℃ Sulfadiazine neomycin disc Sulfadiazine neomycin disc 012345678 7.4±0.37.2±0.27.6±0.67.2±0.27.3±0.27.2±0.46.8±0.56.0±0.8ND 21.8±0.622.6±0.622.2±1.822.0±0.722.4±1.722.4±1.621.8±1.419.5±1.818.2±1.4 7.2±0.47.2±0.27.5±0.57.3±0.37.2±0.57.4±0.36.7±0.46.1±0.46.2±0.4 22.7±1.024.5±0.422.8±0.823.5±1.022.8±1.023.0±2.122.6±0.620.6±0.620.2±0.8

Note: "ND" has no inhibition zone

Stability test of medium in the test kit of the present invention in table 10 time (month) Antibacterial zone diameter (mm) Room temperature (22-25°C) 2～8℃ Sulfadiazine neomycin disc Sulfadiazine neomycin disc 01 NDND 23.3±0.123.7±1.6 NDND 23.4±0.322.4±0.3 2345678 NDNDNDNDNDNDNDND 23.7±1.023.0±0.523.8±0.724.2±0.721.6±0.920.5±1.018.5±1.5 NDNDNDNDNDNDNDND 24.0±0.523.3±1.622.7±1.324.1±0.523.0±0.521.5±0.621.6±0.8

Note: ND: no inhibition zone

Embodiment 5, animal residue test

Table 11 Kit suitability test of the present invention group organize Withdrawal time repeat animal SST (IZ, mm) Judgment result of the kit HPLC (ng/g) test group control group muscle kidney muscle kidney 0d3d5d7d0d3d5d7d03570357 1234567891011121234567891011121314151613141516 8.67±1.1512.28±2.7010.25±2.05NNNNNNNN17.23±1.7414.52±1.5616.85±1.8715.02±3.0111.73±2.058.92±1.598.08±1.457.76±1.03NNNNNNNNNN +++---------++++++++------------ 564±67721±86641±6841±842±243±3NDNDNDNDNDND1674±701561±81596±24185±3183±4163±4145±841±5NDNDNDNDNDNDNDNDNDNDNDND

Note: SST: the diameter of the inhibition zone produced on the culture medium □; N: no inhibition zone; +: positive; -: negative; ND: not detected;

Select 16 weaned piglets whose breed is "Growing Up", each with a body weight of 15±2.5kg, and randomly divide them into 2 groups, with 4 pigs in the control group and 12 pigs in the test group. For 7 days in the pre-test, feed without any antibacterial drugs. After 7 days, enter the formal test. No sulfadiazine was added to the diet formula of the control group, and 100 mg/kg body weight sulfadiazine was added to the diet formula of the test group. During the experiment, the pigs were allowed to eat and drink water ad libitum. After 5 days of continuous feeding, the experimental group stopped feeding the diet plus sulfadiazine. 0 days, 3 days, 5 days, and 7 days after drug withdrawal, 3 pigs of the drug-adding group and 1 pig of the blank control group were slaughtered respectively, and the longissimus dorsi muscle and kidney were collected, and simultaneously the kit of the present invention and high performance liquid chromatography ( HPLC) measure, investigate the detection ability of kit of the present invention to actual sample.

Table 11 lists the detection results of the kit of the present invention and the HPLC method for the actual samples of sulfadiazine. Use kit of the present invention to detect sulfadiazine residues in muscles and kidneys, and the detectable time in muscles is 0 days after drug withdrawal, and the muscle samples collected in 3 to 5 days after drug withdrawal are all negative; the detectable time in kidneys is 5 days after stopping the drug. The kit of the present invention detects 3 positive muscle samples, 9 negative samples, 8 positive kidney samples, and 4 negative samples; the muscle and kidney tissues of the blank group are not detected. As determined by HPLC, the detectable time of sulfadiazine in muscle was 3 days, 3 samples were detected as positive in muscle, 3 samples were lower than the maximum residue limit, and 6 samples were lower than the detection limit; drug withdrawal of sulfadiazine in kidney After 5 days, it can still be detected, and the total detection is 8 samples, of which 7 samples have a concentration higher than the maximum residue limit, 1 sample is lower than the limit, and no drug is detected in the control group. It can be seen from the results that the test kit of the present invention has a coincidence rate of 100% with the HPLC method for sulfadiazine in muscle and a coincidence rate with kidney of 93.75%.

Claims (5)

1, method for screening sulfanilamide medicine residue in a kind of edible animal tissue comprises preparation substratum, bacillus megaterium work bacterium liquid, the antibacterials scraps of paper and aseptic cotton swab, and its step is as follows:

1) substratum, work bacterium liquid, the antibacterials scraps of paper and trimethoprim being replenished liquid prepares separately;

2) bacillus megaterium being made spore count is 1 * 10 ⁶Individual/ml work bacterium liquid;

3) the antibacterials scraps of paper being prepared into diameter is 7mm, contains the scraps of paper of Xin Meisu 5 μ g;

4) described substratum is made following substratum, its component is as follows:

Substratum I:

Beef extract 1.5～3g/L

Casein peptone 8.5～17g/L

Zulkovsky starch 0.75～1.5g/L

Potassium primary phosphate 0.5～1.0g/L

Agar 15～20g/L

Aseptic calf serum 30～50ml/L

Trimethoprim 0.016～0.04mg/L

Keep the skin wet to 1L; And

Medium ii:

Beef extract 1.5～3g/L

Casein peptone 8.5～17g/L

Zulkovsky starch 0.75～1.5g/L

Potassium primary phosphate 0.5～1.0/L

Agar 15～20g/L

Aseptic calf serum 30～50ml/L

Trimethoprim 0.016～0.04mg/L

Para-amino benzoic acid 0.1～0.2g/L

Keep the skin wet to 1L;

Prepare according to following steps:

A) with the beef extract in the above-mentioned substratum, casein peptone, Zulkovsky starch, potassium primary phosphate and agar heating and melting, transfer pH to 7.0～7.5,121 ℃ sterilization 20min, be cooled to 50 ℃;

B) the aseptic calf serum and the trimethoprim of adding deactivation in step a) obtain substratum I;

C) add para-amino benzoic acid to step b), obtain medium ii;

D) with step b) or c) the substratum branch be filled in the culture dish, make substratum cooling, and to make the thickness of substratum in the culture dish be 1.5～2mm;

6) the bacterium liquid of will working is evenly coated on substratum I or the II;

7) the Xin Meisu scraps of paper are placed make positive control on the substratum;

8) get the sample of tissue to be measured with cotton swab, be placed on substratum I or the II;

9) on cotton swab, drip described trimethoprim and replenish liquid; With

10) substratum I or II are placed 44～45 ℃ of temperature, cultivate 5～8h, judge whether contain sulfa drugs in the testing sample according to the appearance situation of substratum I and the last inhibition zone of II.

2, method according to claim 1 is characterized in that, trimethoprim is 1.5～2 μ g/ml in the additional liquid of described trimethoprim.

3, the test kit of method for screening sulfanilamide medicine residue in claim 1 or the 2 described a kind of edible animal tissues, comprise substratum, bacillus megaterium work bacterium liquid, the antibacterials scraps of paper and aseptic cotton swab, it is characterized in that described work bacterium liquid is that spore count is 1 * 10 ⁶The bacillus megaterium bacterium liquid of individual/ml, the described antibacterials scraps of paper are 5 μ g Xin Meisu/∮ 7mm, and described additional liquid is trimethoprim liquid, and described substratum is the substratum of substratum I and medium ii coupling, prepare according to following component:

Substratum I:

Beef extract 1.5～3g/L

Casein peptone 8.5～17g/L

Zulkovsky starch 0.75～1.5g/L

Potassium primary phosphate 0.5～1.0g/L

Agar 15～20g/L

Aseptic calf serum 30～50ml/L

Trimethoprim 0.016～0.04mg/L

Keep the skin wet to 1L;

Medium ii:

Beef extract 1.5～3g/L

Casein peptone 8.5～17g/L

Zulkovsky starch 0.75～1.5g/L

Potassium primary phosphate 0.5～1.0/L

Agar 15～20g/L

Aseptic calf serum 30～50ml/L

Trimethoprim 0.016～0.04mg/L

Para-amino benzoic acid 0.1～0.2g/L

Keep the skin wet to 1L;

Prepare according to following steps:

A) with the beef extract in the above-mentioned substratum, casein peptone, Zulkovsky starch, potassium primary phosphate and agar heating and melting, transfer pH to 7.0～7.5,121 ℃ sterilization 20min, be cooled to 50 ℃;

B) the aseptic calf serum and the trimethoprim of adding deactivation in step a) obtain substratum I;

C) in the substratum of step b), add para-amino benzoic acid, obtain medium ii;

D) with step b) or c) the substratum branch be filled in the culture dish, make substratum cooling, and make that substratum thickness is 1.5～2mm in the culture dish.

4, test kit according to claim 3 is characterized in that the trimethoprim that replenishes in the liquid is 1.5～2 μ g/ml.

5, claim 3 or the application in the sulfa drug residue in the vitro detection edible animal tissue of 4 described test kits.

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