Development Of Microbiological Methods And Their Kits For Screening The Antimicrobial Residues In Milk And Urine

The breeding industry’s development were promoted owing to the antibiotics which were widely used in the animal production and veterinary clinic.Application of antibiotics has brought huge economic efficiency for the breeding industry in our country.But,as a result of veterinary antimicrobials’s illegal application,it always residued in animal derived food,and constituted huge harm to society and environment.The antibiotics such as:penicillins,cefalosporins,aminoglycosides, tetracyclines,macrolides and sulfonamides,which led to antimicrobial residued in animal derived food,were always used in clinical veterinary.The microbiological screening assays,which don’t need complicated treating processe and can screen the mass samples,were considered as simple,quick,economical,high sensitivity,high efficiency,high throughout methods and widely applied in food safe examination fields.In our country there has not been any commercial kits which are only restricted in the methodology for determination of multi-residues of antimicrobials in animal derived food.The imported microbiological kits were too expensive to be used in screening for residues.To monitor the common antibiotics effectively,detect antibiotic residue,protect people’s health and promote the import and export trade,we established and developed the microbiological screening methods and their kits for the common antibiotics residue in milk and urine.The base of the microbiological method for detecting antimicrobial residues in milk is microorganism inhibition test.This indicator bacteria of this method is Bacillus stearothermophilus.Experiments of the kits such as:sensitivity experiment, reproducibility experiment,false positive experiment,false negative experiment, incurred sample experiment,comparison experiment and shelf life of kits experiment were preformed,according to the international standard milk and milk products-guidelines for a standardized description of microbial inhibitor tests (ISO13969).The results indicated that the limits of detection of the kit were as follows:penicillins were 2～15μg/L,cefalosporins were 10～60μg/L,aminoglycosides were 50～1250μg/L,tetracyclines were 100～250μg/L,macrolides were 40～700μg/L, sulfonamides were 50～100μg/L.The coefficient varies between batches of the method are all below 25.1%.In this study,we established a microbiological kit for screening the main antibiotics residues in milk.It’s false positive rate was 1.0%and false negative rate was 0.More over,through incurred sample experiment,blind sample experiment and animal residue test,comparison of the kit and Eclipse 50 showed that sensitivity,accuracy,false negative rate,false positive rate,reproducibility of this kid were the similar to the other,and partial targets surpassed Eclipse 50.The stability test showed that this kid was efficient more than 6 months if the kits were kept between 2～8℃.The indicator bacteria of this method is also Bacillus stearothermophilus. Sensitivity experiment,reproducibility experiment,false positive experiment,false negative experiment,incurred sample experiment,comparison experiment and shelf life of kits experiment were also preformed.The results showed that the limits of detection for penicillins were 2～25μg/L,cefalosporins were 30μg/L,aminoglycosides were 50～2000μg/L,tetracyclines were 75～150μg/L,macrolides were 40～700μg/L, sulfonamides were 75～150μg/L.The coefficient varies between batches of the method were within 25.9%.In this study,we established a microbiological kit for screening the main antibiotics residues in urine.It’s false positive rate was 7.0%and false negative rate was 0.Increases,through incurred sample experiment,blind sample experiment and animal residue test,comparison of the kit and LAST showed that sensitivity,accuracy,false negative rate,false positive rate,reproducibility of this kid are the similar to the other,and partial targets surpass LAST.The stability test showed that this kid can be used correctly after more than 6 months if they were kept within 8℃.In this study we estabished and developed the microbiological screening methods and their kits for detecting the main antibiotics residues in milk and urine.Comparing with the foreign similar kits,this kits’ technical index reached to the international advanced level.Because the two screening kits have so many advantages such as quickness,easiness,high sensitivity,stable property,it will be widely used in the veterinary drug residue sections,food sanitation departments and import-export trade departments.It will provide a powerful technical support for the detection of the comprehensive multi-residues and have a bright future for developing and application.