Rapid PCR detection method of Listeria monocytogenes in oysters utilizing β-cyclodextrin and bentonite-coated activated charcoal without pre-enrichment

Listeria monocytogenes ( L. monocytogenes ) in raw or undercooked oysters poses a significant risk of listeriosis outbreaks, highlighting the need for rapid detection methods. This study developed a PCR assay using β-cyclodextrin and bentonite-coated activated charcoal to remove PCR inhibitors and concentrate L. monocytogenes . Specificity was assessed using inlB -targeted primers against 193 strains. Sensitivity was evaluated using artificially contaminated oysters at 10 0 to 10 4 CFU/25g. Application was verified comparing with GB 4789.30-2016 using commercial oysters. Reagent stability was confirmed after storage at -20 °C over 3, 6, and 12 months. The developed method demonstrated high specificity (100%) and sensitivity (as low as 10 1 CFU/25g), and took only 4 h to complete detection without pre-enrichment. Moreover, the reagents remained stable for at least one year at -20 °C. Overall, the PCR assay was accurate, sensitive, and specific. It enables rapid testing of oysters against L. monocytogenes , providing new technical support for pathogen surveillance in the seafood industry. • PCR assay for L. monocytogenes in oysters was firstly developed without enrichment. • β-cyclodextrin and bentonite-coated activated charcoal can remove PCR inhibitors. • The assay had 100% specificity, 10 1 CFU/25g sensitivity, and detection within 4 h. • The assay efficiently detects L. monocytogenes contamination in commercial oysters.