ISO 6579-4:2025

Overview

ISO 6579-4:2025 - Microbiology of the food chain - Horizontal method for the detection, enumeration and serotyping of Salmonella - Part 4 defines a PCR‑based method for molecular identification of the monophasic variant of Salmonella Typhimurium (1,4,[5],12:i:-). The standard specifies procedures starting from a pure Salmonella isolate to detect DNA sequences unique to Salmonella Typhimurium and sequences linked to expression of the second H‑phase flagellar antigen. The method has been validated in single‑laboratory and interlaboratory studies (see Annex E).

Key technical topics and requirements

• Scope of analysis: identification and differentiation of monophasic Salmonella Typhimurium versus (biphasic) Salmonella Typhimurium and monophasic variants of other non‑Typhimurium serovars with the same antigenic formula. Applicable to isolates from food, animal feed, environmental and primary production samples - and other domains (human/animal health).
• Molecular targets: detection of an intergenic region of the first H‑phase flagellin cluster for Typhimurium identity and of genes associated with the second H‑phase antigen for monophasic differentiation.
• PCR methods provided: informative annexes include a probe‑based multiplex real‑time PCR (Annex B), agarose gel‑based multiplex target PCR (Annex C) and single target PCR (Annex D). Primers and probes are listed in those annexes.
• Laboratory workflow:
  • Preparation of well‑isolated colonies on non‑selective agar (34–38 °C, ~24 h).
  • Suspension of a single colony in saline or PCR‑grade water.
  • PCR amplification with appropriate positive, negative and inhibition controls.
• Equipment and consumables: standard microbiology and molecular biology equipment per ISO 7218 and ISO 22174 (incubators, thermal cyclers, sterile loops, water baths, quality reagents).
• Quality and validation: performance characteristics and validation procedures are described (Clause 11, Annex E) and the method follows related ISO requirements (ISO 7218, ISO 11133, ISO 22174).

Practical applications and users

• Food safety and testing laboratories performing routine Salmonella serotyping and confirmation.
• Public health and clinical laboratories tracking Salmonella outbreaks and surveillance.
• Veterinary and feed testing labs monitoring animal feed and farm environments.
• Regulatory bodies and quality assurance teams enforcing legal requirements for absence or specific serovars (e.g., Salmonella Typhimurium). This standard helps distinguish the monophasic Typhimurium variant from other serovars that share an identical antigenic formula, supporting accurate reporting and regulatory compliance.

Related standards

• ISO 6579 series (other parts for detection, enumeration and serotyping)
• ISO 7218 (general microbiology laboratory requirements)
• ISO 11133 (culture media)
• ISO 22174 (PCR general requirements)
• Validation guidance referenced: ISO 17468

Keywords: ISO 6579-4:2025, Salmonella Typhimurium, monophasic Salmonella, PCR identification, microbiology of the food chain, Salmonella detection, serotyping.