Overview
ISO/TS 20224-3:2020 specifies a real-time PCR method for the qualitative detection of porcine DNA in foodstuffs and feedstuffs. The technical specification targets a partial fragment of the Sus scrofa beta actin (ACTB) gene (GenBank DQ452569.1), which is present as a single copy per haploid genome. The assay is applicable to both domestic pig (Sus scrofa domesticus) and wild boar (Sus scrofa) materials and requires extraction of PCR‑amplifiable DNA from the test matrix. The validated assay achieves an absolute limit of detection of five copies per reaction with ≥ 95% replicability (LOD95%).
Key topics and technical requirements
- Scope: Qualitative detection of porcine DNA in foods and feeds using real‑time PCR (TaqMan-style probe assay).
- Target sequence: Partial Sus scrofa beta actin (ACTB) gene - single-copy target (GenBank DQ452569.1).
- Sensitivity: Absolute LOD = 5 copies/reaction with ≥95% replicability.
- DNA extraction: Requires an adequate, amplifiable DNA extract; follow ISO 21571 guidance for extraction/purification.
- Controls: Include inhibition/quality control assays (e.g., 18S rRNA or myostatin) to verify amplifiability and exclude false negatives.
- PCR setup: Typical reaction volume is 25 µL (e.g., 20 µL master mix + 5 µL sample DNA). Use validated PCR master mix (UDG recommended to prevent carry‑over) and fluorescence‑capable real‑time thermocycler.
- Oligonucleotides: The specification provides primer and probe sequences and recommended concentrations for reliable detection.
- Acceptance criteria & validation: Includes robustness, reproducibility, specificity and sensitivity performance criteria and reporting requirements.
Practical applications and users
ISO/TS 20224-3:2020 is designed for practical use in:
- Food and feed testing laboratories performing species identification and adulteration screening.
- Regulatory and inspection bodies enforcing labeling, safety and import/export controls.
- Certification bodies (e.g., halal/kosher) and supply‑chain auditors verifying absence/presence of porcine material.
- Food manufacturers and ingredient suppliers conducting raw material verification and supplier audits.
- Use cases include food fraud detection, verification of ingredient claims, allergen and dietary compliance checks, and traceability investigations.
Related standards
- ISO 21571 - nucleic acid extraction for foodstuffs (recommended extraction methods)
- ISO 20813 - general requirements for nucleic acid‑based animal species detection methods
- ISO 24276 - general requirements and controls for GMO and nucleic acid testing
- ISO 16577 - terms and definitions for molecular biomarker analysis
Keywords: ISO/TS 20224-3:2020, porcine DNA detection, real-time PCR, Sus scrofa, beta actin, LOD95, food fraud detection, molecular biomarker analysis.