Overview
ISO/TS 21872-2:2020 - "Microbiology of the food chain - Horizontal method for the determination of Vibrio spp. - Part 2" specifies a laboratory method for the direct enumeration of total Vibrio parahaemolyticus (toxR gene) and potentially enteropathogenic V. parahaemolyticus (tdh and/or trh positive) in seafood using nucleic acid hybridization. The technique combines traditional plating with nylon membrane transfer and hybridization using digoxigenin‑labelled oligoprobes, followed by colorimetric detection to enumerate target colonies.
Key topics and technical requirements
- Purpose: Direct enumeration of total and potentially enteropathogenic V. parahaemolyticus in seafood samples.
- Core principle: Plate appropriate dilutions on non‑selective nutrient medium → transfer colonies to membranes → lyse cells and cross‑link DNA → hybridize with labelled oligoprobes (tdh, trh, toxR) → colorimetric detection and colony counting.
- Probes and targets: Digoxigenin‑labelled oligoprobes for tdh, trh (enteropathogenic markers) and toxR (species marker). Annex C contains probe and buffer information.
- Controls: Process control, hybridization control and detection control are required to validate each stage.
- Culture media & reagents: Composition and preparation specified (see Annex B); commercial reagents allowed if conformant. Follow ISO 11133 for media production and performance testing.
- Recommended equipment: rotary blender, pipettes, incubator (37 °C ±1 °C), UV cross‑linker or transilluminator, hybridization oven/water bath, rocking platform, washing containers.
- Procedure steps: Sampling and sample preparation (per ISO 6887 series), plating, colony lift, DNA cross‑linking, proteinase K treatment (if applicable), hybridization, colorimetric detection, optional isolation of positive colonies for confirmation.
- Interpretation & reporting: Enumeration results, detection of tdh/trh and toxR positives, and test report elements are defined in the specification.
Applications and users
- Who uses it: Food microbiology laboratories, seafood processors and quality control teams, public‑health laboratories, regulatory agencies, and research groups monitoring seafood safety.
- Practical applications:
- Routine surveillance of shellfish and other seafood for V. parahaemolyticus contamination.
- Differentiation between total V. parahaemolyticus and potentially enteropathogenic strains (tdh/trh), improving risk assessment.
- Supporting outbreak investigations by enumerating pathogenic subpopulations and guiding isolation for confirmatory testing.
- Method standardization across labs to ensure consistent, comparable results for surveillance and compliance.
Related standards
- ISO 7218 - General requirements for microbiological examinations
- ISO 6887‑1 / ISO 6887‑3 - Sample preparation and dilutions for food and fish products
- ISO 11133 - Preparation and performance testing of culture media
Keywords: ISO/TS 21872-2:2020, Vibrio parahaemolyticus, nucleic acid hybridization, seafood testing, tdh, trh, toxR, enumeration, microbiology of the food chain.