Overview
ISO 22949-1:2021 - "Molecular biomarker analysis - Methods of analysis for the detection and identification of animal species in food and feed products (nucleotide sequencing‑based methods) - Part 1: General requirements" - specifies general performance and validation requirements for DNA sequencing methods used to detect and identify animal species in food and feed. The standard covers Sanger sequencing and next generation sequencing (NGS) (including second‑ and third‑generation short‑ and long‑read methods) for both single‑species and multispecies products. It applies to DNA from mammals, birds, fish, molluscs, crustaceans, amphibians, reptiles and insects. Methods for DNA species quantification are explicitly excluded.
Key topics and technical requirements
- Scope of methods: Performance requirements limited to Sanger and NGS approaches; guidance on when each method is appropriate (e.g., single‑piece single‑species samples vs. multispecies mixes).
- Performance characteristics: Definitions and criteria for fitness for purpose, scientific basis of sequencing, units of measurement, applicability and genomic considerations.
- Validation frameworks: Requirements for single‑laboratory validation and interlaboratory (collaborative) studies to determine method performance.
- Primer selection and evaluation: In silico and empirical assessment of primers for target coverage and specificity.
- Database construction and evaluation: Building and validating reference sequence databases used for species identification.
- Selectivity and specificity: Inclusivity testing, and evaluation of non‑target DNA interference.
- Sensitivity and limit of detection (LOD): Establishing analytical sensitivity appropriate for intended use.
- Robustness: Determination via interlaboratory studies or multifactorial orthogonal designs.
- Laboratory workflow and sample handling: Guidance on facilities, sample categories (single piece, multiple pieces, mixed processed products), DNA extraction, sequencing workflows (Sanger and NGS) and data analysis.
- Bioinformatics validation: Requirements for validating NGS pipelines, including quality metrics and interpretation procedures.
- Reporting: Expectations for test reports and result expression.
Practical applications and users
ISO 22949-1:2021 is intended for:
- Food and feed testing laboratories implementing or validating DNA sequencing for species identification.
- Regulatory authorities and inspection bodies verifying species authenticity and detecting adulteration or substitution.
- Producers, quality assurance teams and certification bodies seeking standardized methods for species verification.
- Developers of sequencing assays and bioinformatics pipelines who need to meet recognized validation and reporting expectations.
Why it matters: standardized sequencing performance and validation improve comparability of species identification results, support food safety and traceability, and reduce the risk of mislabeling and fraudulent substitution.
Related standards
- ISO 16577 - Terms and definitions for molecular biomarker analysis
- ISO 20813 - Nucleic acid‑based methods for detection/identification of animal species (general requirements)
- ISO 21571 - Nucleic acid extraction methods for foodstuffs
For laboratories and stakeholders implementing species identification by sequencing, ISO 22949-1:2021 provides a foundation for reliable, validated workflows and consistent reporting.