Development Of A Rapid Detection Kit For Antibiotics In Milk By Biological Inhibition Method

At present,the domestic kits can only detect few kinds of antibiotics and with low sensitivity,thus the field of testing antibiotics in milk is mainly monopolized by imported kits.Therefore,it is of great significance to develop a kit with wide detection range,high accuracy and good stability.The method is widely used worldwide because of its high detection speed,simple operation,simultaneous detection of various antibiotics,and low cost.The kit of detection antibiotics in milk is developed according to the microbial inhibition method.In this study,the researches of screening and mutagenesis of strains,optimization of fermentation conditions of spore liquid,optimization of spore germination speed and activity conditions,optimization of the testing of antibiotic type,sensitivity and stability of self-made kits were carried out.And finally,the application verification of self-made kits was carried out,the results are as follows:1?Through the mutagenesis screening of Bacillus stearothermophilus confirmed that the optimal conditions for UV radiation mutagenesis of Bacillus stearothermophilus were mutagenesis time 150s,distance 30cm.A strain of Bacillus thermophilic HY was selected by antibiotic susceptibility test.2?The liquid fermentation conditions of spores were optimized,and the optimal ratio of spore culture medium of Bacillus thermophilus was determined to be:0.8%peptone,0.3%yeast powder,0.3%NaCl,0.9 mmol/L Mn²⁺,10 mmol/L Ca²⁺,pH7.0,inoculation amount 0.1%,liquid volume 100 mL,culture time24h and the temperature 65°C.And through the verification of the combined conditions,the final liquid fermentation sporulation rate can be stabilized at about 48%,which is more than 15%higher than before.3?The spore germination conditions were optimized to improve the spore activity,and the optimal ratio of the medium was determined to be 1.0%peptone,0.5%glucose,0.5%agar,0.2%CMC,40 mg/L cresol purple,pH7.0,spores embedding amount was 1×l07 CFU/mL,and the canned amount was 130?L/hole.4?Determination of the detection limit of the kit.The detection limit of the kit for more than 20 kinds of antibiotics were as follows:Tetracycline 100?g/L?Oxytetracycline 100?g/L?Chlortetracycline 100?g/L?Doxycycline 100?g/L?Sulfadiazine 100?g/L?Sulfadimidine 50-100?g/L?Sulfadimidine Sodium50-100?g/L?Sulfamethoxazole 50?g/L?Sulfathiazole 50-100?g/L?Sulfadoxine 100-200?g/L?Penicillin 1-2?g/L?Amoxicillin 2-4?g/L?Ampicillin 2-4?g/L?Cefalexin 50-100?g/L?Oxacillin 5-10?g/L?Cefquinome 25-100?g/L?Cephazolin 5-10?g/L?Ceftiofur 100?g/L?Gentamicin 200?g/L?Kanamycin 1000-2000?g/L?Neomycin 500-1500?g/L?Erythromycin>40?g/L?Tylosin 50-100?g/L?Tilmicosin 75-100?g/L?Spiramycin 150-200?g/L?Lincomycin 100-150?g/L?Chloramphenicol2000-2500?g/L and etc.5?The validation and application of self-made kits was found to be in good consistency with the Ddvotest?kit for the detection of antibiotic residues in milk.The self-made kit is sensitive to?-lactam antibiotics and can meet the detection limits of the EU and China,especially penicillin,which can reach to1-2?g/L and applied in various milk factory and farm.