Overview
ISO/TS 20224-5:2020 specifies a real-time PCR method for the qualitative detection of goat (Capra hircus) DNA in foodstuffs and feedstuffs. The method targets a single‑copy fragment of goat chromosome 9 (GenBank accession NC_030816.1) and is designed to reliably detect low copy numbers of goat DNA extracted from relevant matrices. The validated assay has an absolute limit of detection of five copies per reaction with ≥95% replicability (LOD95%).
Key topics and technical requirements
- Target and assay performance
- Single‑copy genomic target: partial fragment of goat chromosome 9 (NC_030816.1).
- Absolute LOD: 5 copies/reaction with ≥95% replicability (LOD95%).
- TaqMan‑style probe chemistry (fluorophore/quencher format; FAM/TAMRA used in the validation).
- Sample preparation and DNA extraction
- Representative, homogenized test portion required.
- Extraction and purification of PCR‑amplifiable DNA per ISO 21571 recommended.
- Extracted DNA quality/amplifiability must be verified before species testing.
- PCR setup and controls
- Typical reaction: 25 µl total volume; 5 µl sample DNA (recommended 20–200 ng/µl) and 2× PCR master mix.
- Specific primer/probe set and final concentrations defined in the specification.
- Mandatory controls: positive control, extraction blank, no‑template control and an inhibition/control gene assay (e.g., 18S rRNA or myostatin) to exclude false negatives.
- Instrumentation
- Requires a real‑time thermocycler capable of detecting the chosen fluorophore and supporting TaqMan assays.
- Acceptance criteria and validation
- Guidance on accept/reject criteria, robustness, reproducibility, sensitivity and specificity are provided; laboratories are expected to follow ISO 20813 and ISO 24276 for general quality requirements.
Applications and users
- Practical uses
- Detection of goat material in processed and raw food or feed to prevent species adulteration and mislabeling.
- Verification of declared ingredients for regulatory compliance and market surveillance.
- Support for religious dietary compliance (halal/kosher), supply‑chain integrity, and forensic identification of animal content.
- Primary users
- Food and feed testing laboratories, national regulatory agencies, customs and border laboratories, quality assurance teams in food manufacturing, and research laboratories focused on food authenticity and safety.
Related standards
- ISO 21571 - nucleic acid extraction methods for foodstuffs
- ISO 20813 - general requirements for nucleic acid–based animal species detection
- ISO 24276 - general requirements and controls for nucleic acid testing
- ISO 16577 - terms and definitions for molecular biomarker analysis
Keywords: ISO/TS 20224-5:2020, goat DNA detection, real-time PCR, Capra hircus, molecular biomarker analysis, food testing, feed testing, LOD95, TaqMan.