¹Institute of Microbiology, Faculty of Veterinary Science, University of Agriculture, Faisalabad, 38000, PAKISTAN
²Department of Parasitology, Faculty of Veterinary Science, University of Agriculture, Faisalabad, 38000, PAKISTAN DOI : 10.9775/kvfd.2025.34907 Milk is a nutrient-dense food that provides essential nutrients to support growth, development, and overall health. Currently, over 80% of food animals receive antibiotics as therapeutics, feed additives, and growth promoters. Significant amounts of antibiotics and their metabolites in milk pose public health risks. Antibiotic residues can be detected using different techniques. Detection of antibiotic residues is limited by complex pretreatment, high cost, time-consuming procedures, and required expertise. To overcome these limitations, the current research was conducted to establish a sensitive and time-saving detection kit containing Bacillus subtilis as an indicator to detect antibiotic residues in milk. The kit was validated using 14 antibiotic-spiked samples by measuring the limit of detection, specificity, and sensitivity. The kit successfully detected the antibiotic residues of ampicillin, oxytetracycline, streptomycin, spectinomycin, sulphadimidine, sulphadiazine, tylosin, lincomycin, enrofloxacin, ciprofloxacin, trimethoprim, spiramycin, chloramphenicol, and florfenicol. The limit of detection of the kit was lower than the maximum residue limit (MRL) for various antibiotics, including oxytetracycline, sulphadiazine, tylosin, enrofloxacin, ciprofloxacin, and trimethoprim. High specificity and sensitivity of 98.2% and 99.2%, respectively, were obtained. The kit showed accurate results for most antibiotics over 4 months, indicating good shelf life and suitability for regular use. The developed kit offers a rapid, cost-effective, and reliable method for detecting antibiotic residues in milk, ensuring routine monitoring and safeguarding public health. Keywords : Antibiotic residues, Bacillus subtilis, public health, food safety, milk