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[Key words] biochip ; protein chip; multi-residue detection; sample detection
The protein chip veterinary drug residue detection kit integrates sample pretreatment and sample detection process. This technology is a new food veterinary drug residue detection technology developed in the 21st century. At present, the kit can achieve semi-quantitative detection of four tissues of pork, pig liver, chicken, chicken liver and enrofloxacin, sulfamethazine, streptomycin and clenbuterol in pig urine, in addition to sensitivity. The advantages of high specificity, strong sample preparation, simple sample preparation, convenient and rapid detection, etc., can also realize the advantages of simultaneous detection of the above multiple veterinary drugs, greatly saving the detection cost, shortening the detection time, and further improving the detection efficiency. The author used the protein chip veterinary drug residue detection kit to detect the residues of several commonly used veterinary drugs in animal tissues, and compared with other methods.
1 Materials and methods
1. 1 material
1. 1. 1 instrument and equipment chip scanner, crystal core mEcoScan-100, Beijing Boao Biochip Co., Ltd.; homogenizer, JZ-II type, China Railway Ministry Electric Power Institute Sifang Electrical Equipment Factory; rotary evaporator, RE252AA Type, Shanghai Yarong Biochemical Instrument Factory; Centrifuge, GL-20G-II, Shanghai Anting Scientific Instrument Factory; Microplate Reader, MK3, Finland Thermo; Gas Chromatograph 2 Mass Spectrometer , TRACEDSQ, Feniggen; High performance liquid chromatography , quaternary gradient pump with 2695 UV detector and 2475 fluorescence detector, Waters company; automatic rapid concentration, DSY-II type, Beijing Jinke Jingyuan Institute of Technology; multi-functional microbial automatic measurement analyzer, ZY2300IV, Beijing Pioneer Weifeng Technology Development Company; constant temperature incubator, SGSP202 type, Huangshi Hengfeng Medical Instrument Co., Ltd.; vortex mixer, TDX-1 type, Tongda Technology. Solid phase extraction column: C18, 100 mg/mL, carbon content ≥16 (for enrofloxacin residue analysis); SupelcleanLC-C18 Sep pak 500 mg/3 mL and SCX cartridge LC-SCXSep pak 500 mg/3 mL, US SUPELCO (for the analysis of clenbuterol residues by GC- MS); RIDA C18, German company.
1. 1. 2 EL ISA detection kit Clenbuterol residue detection kit ( 05073 ) and sulfamethazine residue detection kit (EN5327), Germany r2Biopharm company; protein chip veterinary drug residue detection kit, batch number 20031202, 20040701 , Beijing Boao Biochip Co., Ltd.
1. 1. 3 reagent and reagent streptomycin standard, batch number 0308-200012, titer 712 units / mg; clenbuterol hydrochloride reference, batch number 0072-8501, content 99. 6%; sulfamethoxazole The reference substance, the content of 99.0%; all provided by the China National Institute for the Control of Pharmaceutical and Biological Products. Sulfamethazine standard, batch number 51K1523, content 99.0%; sulfamethoxazole reference substance, content 99%; sulfamethoxine reference substance, content 99.9%; sulfamethoxazole reference substance, content 99%; sulfaquinoxaline reference substance, content 97.2%; salbutamol reference substance, content 99%; all purchased from American Sigma company. Reference substance of streptomycin sulfate and dihydrostreptomycin, content ≥99%, Fluka company; ciprofloxacin reference substance, content ≥99%, B iometical company. Enrofloxacin control, batch number H010102, content 100. 1%, provided by China Veterinary Drug Inspection Institute. Methanol and acetonitrile, chromatographically pure, Fisher.
1. 1. 4 Experimental bacteria and experimental animals The experimental bacteria were Bacillus subtilis, and the strain number was CMCC63501, which was provided by the Chinese Veterinary Drug Supervision Institute. The experimental positive animals were 10 pigs and 29 chickens (see Table 1), which were fed with their own drugs. The samples were slaughtered after 2 days of withdrawal; the negative chickens were provided by the Experimental Animal Research Center, and the samples were obtained from the negative pork and liver supermarkets; Part of the sample comes from the market and the slaughterhouse.
1. 2 methods
1. 2. 1 Protein chip kit sensitivity evaluation
1. 2. 1. 1 Preparation of standard curve solution: Various drug reference products (standard products) are prepared into standard solutions according to the experiment, as shown in Table 2.
1. 2. 1. 2 Chip reaction: The method of operation is carried out according to the instructions of the veterinary drug residual protein chip detection kit.
1. 2. 1. 3 Scanning and data processing: After the chip is completed, image scanning and data processing are performed. The corrected fluorescence signal intensity is used as a standard curve for each reference substance (standard product) solution drug concentration, and each type is determined. The IC50 of the drug (the concentration of the drug whose fluorescence signal intensity is reduced by half).
1. 2. 2 Protein chip kit specific evaluation
1. 2. 2. 1 Preparation of standard curve solution: Various drug reference products (standard products) are prepared into standard solutions according to the experiment, as shown in Tables 3, 4, 5 and 6.
1. 2. 2. 2 Chip reaction: According to the instructions of the veterinary drug residual protein chip detection kit.
1. 2. 2. 3 Scanning and data processing: After the chip is completed, image scanning and data processing are performed. The corrected fluorescence signal intensity is used as a standard curve for each reference substance (standard product) solution drug concentration, and each type is determined. The IC50 value of the drug, and the cross-reaction rate of the target drug detected by the chip is 100%, and the other drugs (the test drug B such as sulfaquinoxaline, etc.) and the chip detection target drug (refer to the drug A, such as sulfamethazine) are calculated according to the following formula. Cross-reaction rate between pyrimidines.
Cross-reaction rate of drug B = IC50 of reference drug A / IC50 of test drug B × 100%
1. 2. 3 Sample preparation
1. 2. 3. 1 Protein chip kit: Weigh 5. 0 g of the pulverized sample, put it into a centrifuge tube, add 1.0 mL of tissue sample preparation solution, tighten the tube cover, and mix vigorously with a vortex mixer. 2 min, 80 ° C water bath for 20 min, remove the centrifuge tube, cool to room temperature, centrifuge at 5 000r / min for 10 min at room temperature, then pipet 200 μL of the supernatant into a 1.5 mL centrifuge tube, add 800 μL tissue sample dilution Cover the tube cover and mix it for testing.
1. 2. 3. 2 Determination of sulfamethazine residue (EL ISA method): Weigh 5. 0 g of the pulverized sample and mix with 20 mL of acetonitrile aqueous solution (86 +16) for 10 min, 15 °C to 6 000 r / Centrifuge for 10 min at min, take 3 mL of supernatant and mix with 3 mL of distilled water, add 4.5 mL of ethyl acetate for 10 min, centrifuge at 6 000 r/min for 10 min at 15 °C, transfer the ethyl acetate layer to another bottle. Completely dry, dissolve the residue with 1.5 mL of diluted buffer, add 1.5 mL of n-hexane for 5 min, centrifuge at 6 000 r/min for 10 min at 15 °C, completely remove the n-hexane phase, and take 50 μL of water phase. Analyze.
1. 2. 3. 3 Determination of sulfamethazine residues ( HPLC method) according to the literature [1] method.
1. 2. 3. 4 Streptomycin residue determination (microbiological method) [4]: ​​Weigh 5.0 g of the pulverized sample, add 20 mL of phosphate buffer (pH 1. 5), homogenize and centrifuge. . The supernatant was adjusted to pH 8.5 with a 10 mol/L sodium hydroxide solution, and the supernatant was taken as a sample solution after centrifugation.
1. 2. 3. 5 Clenbuterol Residue Determination (EL ISA Method): Follow the instructions in the kit instructions.
1. 2. 3. 6 Clenbuterol residue determination (GC2MS method): according to NY/QY468-2001 method [2].
1. 2. 3. 7 Determination of enrofloxacin residues (HPLC method): according to the literature [3] method.
1. 2. 4 Sample determination: Protein chip veterinary drug residue detection method, according to the veterinary drug residual protein chip detection kit instruction manual; EL ISA method, according to sulfamethazine, clenbuterol EL ISA detection kit operation Description: The high performance liquid chromatography for the detection of sulfamethazine and enrofloxacin residues was carried out according to the literature [1, 3]; the microbiological detection method of streptomycin residues in animal tissues, according to the literature [4] [GC2MS] detection method in pig urine; according to the literature [5]; determination of clenbuterol in animal tissues (GC2MS), according to reference [2].
2 results
2. 1 Protein chip kit index test results: This experiment conducted a preliminary study on protein chip related indicators for veterinary drug residue detection. The test results of each index are shown in Tables 7 and 8.
2. 2 Protein chip kit and other test methods sample test results: the same sample was detected by protein chip kit, and then EL ISA kit, antibiotic microbial assay, HPLC method and GC-MS method were used. According to the veterinary drug Zui high residue limit standard [6] in the Ministry of Agriculture No. 235 document, it is determined whether the veterinary drug residue in the sample exceeds the standard. The results are shown in Table 9.
3 Discussion
3. 1 It can be seen from 1.1.2 that the sample preparation method for veterinary drug residue detection using protein chip is simple and fast, and a variety of veterinary drugs are extracted by the same method, which reduces the sample preparation time (and The comparison of other methods is shown in Table 10).
3. 2 From Table 9, it is concluded that the number of positive samples detected by other methods is 38 in the detection of 102 samples, and the number of positive samples detected by the protein chip kit is 40, which has a good coincidence rate. A total of 58 samples were detected by EL ISA kit, of which 22 were positive samples, and 20 samples were positive by protein chip kit, and the coincidence rate was also good. The above results indicate that the protein chip kit results are reliable.
3. 3 Compared with other methods, the protein chip kit has the ability of parallel processing, and can detect more than two kinds of veterinary drugs simultaneously on one chip. The current protein chip kit can simultaneously detect sulfamethazine and kelen. Troll, streptomycin and enrofloxacin can detect a variety of veterinary drugs on a single chip as needed.
4 Conclusions Biochips for veterinary drug residue detection have the advantages of simultaneous detection of multiple veterinary drugs, high accuracy, simple pretreatment, and high speed.
At present, the biochip for veterinary drug residue detection is in the early stage of development, and the number of detectable veterinary drugs is relatively small, but the multi-residue detection is initially realized, showing the parallelism and high throughput of the biochip, and the biochip is easy to realize. Integration and automation have certain advantages in the automated detection of veterinary drug residues. This experiment is the first time at home and abroad to use biochips to detect veterinary drug residues.
PHYTASE
PROTEASE
XYLANASE
AMYLASE
GLUCO OXIDASE
ALPHA GALACTOSIDASE
ACID MANNANASE
SWINE
WHEAT DIET
POULTRY
MISC.GRAIN AND MEAL
REGULAR DIET
Application of protein chip in detection of veterinary drug residues
[Abstract] The application of protein chip detection kit, EL ISA detection kit and confirmation method in veterinary drug residue detection were compared by detecting ketonerol and streptomycin in porcine and chicken tissue samples. The results showed that the protein chip detection kit and the EL ISA kit have the same performance, and have good consistency with the results of the confirmation method, and have the advantages of simultaneous detection of various veterinary drugs, simple pretreatment method and high speed.
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