ISO/TS 20224-4:2020

Overview

ISO/TS 20224-4:2020 specifies a real-time PCR method for the qualitative detection of chicken DNA (Gallus gallus domesticus and jungle fowl) in foodstuffs and feedstuffs. The assay targets a partial fragment of the chicken TGF-β3 (intron 4) gene (GenBank AY685072.1), a single-copy sequence per haploid genome, and achieves an absolute limit of detection (LOD) of five copies per reaction with ≥95% replicability (LOD95%). The method requires extraction of an adequate amount of PCR-amplifiable DNA and is intended for routine species identification and authenticity testing.

Key Topics and Technical Requirements

• Target and assay performance
  • Single-copy target: TGF-β3 gene (intron 4).
  • Reported sensitivity: absolute LOD = 5 copies/reaction; LOD95% reproducibility.
• Real-time PCR format
  • TaqMan-style probe assay with defined primers and probe, suitable for real-time thermocyclers capable of fluorophore excitation/detection.
  • Typical reaction volume: 25 μL with recommended PCR master mix and internal controls.
• Sample preparation and DNA extraction
  • Requires homogenized, representative test portions and PCR-amplifiable DNA extraction per ISO 21571 methods.
• Controls and quality checks
  • Use of inhibition/control assays (e.g., 18S rRNA or myostatin gene) to verify DNA amplifiability and exclude false negatives.
  • Include positive controls, extraction blanks and no-template controls per ISO guidance.
• Validation and performance
  • The document details robustness, reproducibility, sensitivity and specificity assessments and provides interpretation and accept/reject criteria for results.

Applications and Who Uses It

• Food authenticity and fraud detection: verification of meat origin in processed products to detect undeclared chicken adulteration.
• Feed testing: identification of chicken-derived ingredients in compound feeds.
• Regulatory and border control labs: enforcement of labeling, trade and safety regulations.
• Halal/kosher certification, consumer protection and supply-chain auditing: verifying compliance with dietary and religious requirements.
• Research and commercial testing laboratories: method adoption for routine species identification or as a reference assay in method development.

Target users include analytical laboratories, food safety regulators, quality assurance teams, certification bodies and forensic scientists requiring a validated, ISO-aligned procedure for chicken DNA detection.

Related Standards

• ISO 21571 - nucleic acid extraction for foodstuffs (recommended extraction methods).
• ISO 20813, ISO 24276 - general requirements for nucleic acid-based species detection and PCR method controls.
• ISO 16577 - terms and definitions for molecular biomarker analysis.

Keywords: ISO/TS 20224-4:2020, chicken DNA detection, real-time PCR, TGF-β3, food authenticity, feed testing, species identification, LOD95.