Development of a real-time PCR method for the detection of penicillin fermentation residue in feed

Abstract

Feed safety is critical to the quality of livestock and poultry products and plays a key role in ensuring food safety and public health. To address the growing issue of illicit addition of antibiotic fermentation residues, such as penicillin mycelial waste, into feed, we developed a real-time PCR method targeting the orf70c gene, a specific marker within the biosynthetic gene cluster of industrial penicillin-producing strains. Specific primers and a probe were designed to enable accurate detection across nine types of feed. The assay requires no complex sample pretreatment and completes the entire process from DNA extraction to result within 2 h. It reliably detects as little as 0.5 % (w/w) of penicillin fermentation residue in feed. The method demonstrated high reproducibility, with inter-assay variation between 0.46∼0.55 % and intra-assay variation between 0.37∼0.69 %. To our knowledge, this is the first real-time PCR assay developed for detecting antibiotic fermentation residues in feed. The method provides a practical tool for feed safety monitoring and supports sustainable practices in the feed and animal production industries.