Overview - ISO/TS 20224-2:2020 (Ovine DNA detection by real-time PCR)
ISO/TS 20224-2:2020 defines a validated real-time PCR (qPCR) method for the qualitative detection of ovine (sheep, Ovis aries) DNA in foodstuffs and feedstuffs. The assay targets a partial fragment of the ovine prolactin receptor short form (PRLR) gene (GenBank AF041979.1), a single-copy nuclear locus. The specified TaqMan-format assay achieves an absolute limit of detection (LOD) of five copies per reaction with ≥95% replicability (LOD95%). The technical specification covers reagent and apparatus requirements, PCR setup, controls, acceptance criteria and basic validation/performance considerations for routine laboratory use.
Key topics and technical requirements
- Target sequence and assay format: PRLR partial fragment; single-copy gene; TaqMan probe labeled (example: FAM/TAMRA) for specific detection.
- Sensitivity: Absolute LOD = 5 copies/reaction with ≥95% replicate detection (LOD95%).
- Reaction setup: 25 µl total volume; typical recipe: 5 µl sample DNA (20–200 ng/µl), 12.5 µl 2× PCR master mix, primers and probe added to specified final concentrations (primers ~400 nmol/l, probe ~200 nmol/l as demonstrated).
- Quality controls: Mandatory PCR controls per ISO 20813/ISO 24276; inhibition control/reference gene assay (e.g., 18S rRNA or myostatin) to verify DNA amplifiability and exclude false negatives.
- Sample prep and DNA extraction: Requires extraction of PCR-amplifiable DNA following ISO 21571 recommendations and general good laboratory practice (aerosol-free tips, nuclease-free reagents).
- Instrumentation: Real-time thermocycler capable of fluorescence excitation/detection for TaqMan assays and consistent thermal profiles.
- Documentation and acceptance criteria: Includes decision rules for positive/negative calls, reproducibility, robustness and reporting requirements.
Applications and who uses it
- Food authenticity testing: Detects undeclared ovine meat/adulteration in processed foods.
- Feed compliance: Verifies animal material content in feed formulations.
- Regulatory and import control: Laboratories in government agencies for species surveillance and enforcement.
- Contract and commercial testing labs: Routine screening for suppliers, certification bodies, and private quality assurance.
- Forensic and supply-chain investigations: Tracing species origin in contamination or fraud cases.
This technical specification is particularly useful where reliable, species-specific molecular biomarker analysis is required for food authenticity, regulatory compliance, and anti-fraud measures.
Related standards
- ISO 20224 series (other species-specific parts)
- ISO 21571 - DNA extraction for foodstuffs (nucleic acid extraction)
- ISO 20813 - General requirements for nucleic acid‑based species identification methods
- ISO 24276 and ISO 16577 - General method and terminology guidance
Keywords: ISO/TS 20224-2:2020, ovine DNA detection, real-time PCR, PRLR gene, food authenticity, feed testing, limit of detection, species identification.