Development of Technical Specification for Clostridium botulinum Detection That Can Be Used as a Reference Method in...

The committee for the National Institute of Health Sciences Japan-The Methods for the Microbiological Examination of Foods (NIHSJ-MMEF). aims to develop domestic standard methodologies for food microbiological testing that conforms with internationally recognized standards. In this study, we developed a new qualitative detection method for Clostridium botulinum (NIHSJ-20TS) based on ISO/TS 17919: 2013 and validated its performance through interlaboratory study. To facilitate interlaboratory studies under stringent legal regulations, a detailed protocol for inoculum preparation was provided instead of distributing inoculated samples. In addition, we evaluated commercial DNA extraction kits as a simpler alternative to the conventional Cetyltrimethylammonium bromide (CTAB) method. The collaborative study plan was optimized as a minimal yet effective plan to verify the reproducibility and applicability of the method. After completing the validation study, the level of detection 50% (LOD₅₀), a proposed reference value for implementation verification described in ISO 16104-3:2021, was evaluated. We believe that NIHSJ-20TS may facilitate the international acceptance of microbiological testing results generated in Japan.