Application of the Detection of Pork-Derived DNA in Food to Halal Food Management

Importance of the Topic

Verification of animal species in food is essential for food safety, traceability, quality control and for compliance with religious requirements such as halal certification. Highly sensitive, objective and digitally traceable methods for detecting species-specific DNA are needed to replace subjective agarose-gel assessments and to ensure reproducible record-keeping in routine testing and certification workflows.

Objectives and Study Overview

The study evaluated the MultiNA II microchip electrophoresis system (Shimadzu) combined with PCR-based species-specific primers for objective identification of animal species in food matrices and for sensitive detection of pork adulteration. Tests included analysis of commercially available halal-certified foods and processed beef products, and sensitivity assessment using model mixtures of pork in beef at admixture levels from 1% down to 0.0001% (four replicates per level).

Methods and Workflow

Key steps:

• Sample types: halal-certified foods, domestic pork, processed beef products, and model pork-in-beef mixtures at defined admixture rates (1%, 0.1%, 0.01%, 0.001%, 0.0001%).
• Sample preparation: ~20 mg sample mixed with 100 µL lysis buffer (20 mM Tris-HCl pH 8.0, 5 mM EDTA, 400 mM NaCl, 0.3% SDS) and five 2 mm zirconia beads; disrupted with bead-beater at 5,000 rpm for 30 s; heated at 95 °C for 5 min to inactivate Proteinase K; 0.5 µL of resulting suspension used as PCR template.
• Target: mitochondrial cytochrome b gene using species-specific primers (based on Matsunaga et al.). Amplified fragment sizes: chicken 227 bp, beef 274 bp, pork 398 bp.
• PCR reagents and program (as applied): Ampdirect Plus PCR buffer and BIOTAQ polymerase; typical mix composition included 2× Ampdirect buffer, BIOTAQ (~0.5 U), primers at ~2 µM, final volume up to 20 µL. Thermal profile: initial denaturation (95 °C, ~10 min), 35 cycles of 94 °C 30 s / 60 °C 60 s / 72 °C 90 s, final extension 72 °C 7 min.
• Analysis: PCR products separated and detected on MultiNA II microchip electrophoresis; automated fingerprinting analysis by MultiNA II software to recognize species-specific peak patterns and convert results to digital records.

Instrumentation Used

• MultiNA II Microchip Electrophoresis System (MCE-301) with MultiNA II analysis software for automated separation, detection and fingerprinting.
• Ampdirect Plus PCR kit (Shimadzu) and BIOTAQ DNA polymerase (Meridian Bioscience trademark).
• Bead-smash/bead-beating device and 2 mm zirconia beads for mechanical disruption.
• Standard thermal cycler for PCR.

Main Results and Discussion

Species identification in real samples:

• Halal-certified food items: chicken and beef products showed only their respective species-specific peaks; no pork-derived DNA was detected in halal-certified samples.
• Processed beef products: Product A contained beef, chicken and pork DNA; Product B contained beef and chicken DNA. These findings agreed with product labeling that indicated shared production lines handling pork and chicken.

Sensitivity testing with model adulteration series:

• Pork-specific DNA was detected at all contamination levels tested, including the lowest level of 0.0001% pork by weight (which corresponds to detection in 4/4 replicates at that level).
• Positive and negative controls performed as expected (100% pork positive control, 100% beef negative control).

Interpretation:

• The MultiNA II workflow provides objective, software-driven recognition of species-specific amplicons, removing subjectivity inherent to gel-based visualization.
• Digital data output enhances reproducibility and facilitates record-keeping required for QA/QC and certification.
• The reported limit of detection (0.0001% pork) indicates high analytical sensitivity suitable for detecting trace cross-contamination relevant to halal certification and food fraud surveillance. A cautionary note included in the source: failure to detect pork DNA does not absolutely guarantee absence of pork contamination due to sampling, processing, or inhibition factors.

Advantages and Practical Applications

• Objective fingerprinting analysis enables automated species assignment and reduces operator bias.
• High sensitivity allows detection of minute cross-contamination, supporting halal compliance, supply-chain verification, and anti-fraud measures.
• Fast digital reporting simplifies audit trails and archival of analytical records for regulatory or certification audits.
• Method is adaptable to routine QC labs and can be integrated into existing PCR workflows with modest additional instrumentation (microchip electrophoresis system and software).

Future Trends and Potential Uses

• Expansion of the primer panel to cover more species and processed-matrix-optimized assays will broaden applicability across complex food matrices.
• Integration with high-throughput sample preparation and LIMS could enable large-scale screening programs for food fraud and certification compliance.
• Combination with quantification strategies (e.g., qPCR calibration curves or digital PCR cross-validation) would allow more precise estimation of contamination levels where regulatory thresholds exist.
• Continuous improvements in microchip electrophoresis sensitivity and multiplexing may permit consolidated assays for multi-species detection in a single run.

Conclusion

The described method—PCR targeting mitochondrial cytochrome b combined with automated microchip electrophoresis and fingerprinting on the MultiNA II—provides a highly sensitive, objective and digitally traceable approach for animal species identification in foods. It demonstrated concordant species calls for halal-labelled products and routine processed foods, and reliably detected pork contamination down to 0.0001% in model mixtures. The approach is well suited for QC, halal certification support and food fraud countermeasures, while mindful of inherent limitations that prevent declaring absolute absence of contamination from a single negative result.

References

1. Matsunaga T., et al. Effects of processing conditions on species identification of meat products. Nippon Shokuhin Kagaku Kogaku Kaishi. 1999;46(3):187-194.
2. Shimadzu Corporation. Application note: MultiNA II MCE-301 — Detection of Pork-Derived DNA in Food for Halal Food Management. Application News. First Edition: Aug. 2026.