Development of Automated Animal Species Identification for Halal Foods Using Multiplex PCR and Microchip Electrophoresis

Significance of the topic

Accurate and objective identification of animal species in food products is critical for food safety, regulatory compliance, quality control, and consumer trust—especially for religiously mandated foods such as halal products and for preventing undeclared pork contamination. DNA-based assays are the gold standard for species identification in processed foods, but traditional gel electrophoresis requires subjective interpretation. Automated microchip electrophoresis coupled with digital fingerprinting promises reproducible, high-throughput, and objective species verification suitable for routine laboratory workflows.

Objectives and study overview

This study evaluated an automated workflow combining multiplex PCR and the MultiNA II microchip electrophoresis system to (1) identify animal species in commercially available halal-certified and processed meat products, and (2) determine the analytical sensitivity for detecting pork contamination in beef using model admixtures down to very low levels.

Methods and experimental workflow

The workflow comprised sample homogenization and crude lysis, PCR amplification targeting mitochondrial cytochrome b, and automated fragment analysis with digital fingerprinting. Key steps:

• Samples: commercially available halal-certified foods, domestic pork, and processed beef products; model admixtures of pork in beef prepared at 1%, 0.1%, 0.01%, 0.001%, and 0.0001% (w/w).
• Sample preparation: ~20 mg tissue homogenized with 100 µL lysis buffer and five 2 mm zirconia beads using bead beating (5,000 rpm, 30 s). Lysates heated at 95 °C for 5 min to inactivate Proteinase K and used directly as PCR template.
• PCR: Ampdirect plus PCR kit with BIOTAQ polymerase. Reaction setup: 10 µL 2x Ampdirect plus, BIOTAQ 0.5 U, primers 2 µM each, final volume up to 20 µL. Thermocycling: 95 °C 10 min; 35 cycles of 94 °C 30 s, 60 °C 60 s, 72 °C 90 s; final 72 °C 7 min.
• Targets and amplicons: species-specific primers for mitochondrial cytochrome b producing amplicons of 227 bp (chicken), 274 bp (beef), and 398 bp (pork).
• Analysis: MultiNA II microchip electrophoresis for automated sizing and peak detection; fingerprinting analysis software used to assign species-specific patterns objectively and store digital results.

Used instrumentation

• MultiNA II microchip electrophoresis system (Shimadzu) for automated DNA fragment analysis and digital fingerprinting.
• Bead-smashing device for homogenization (5,000 rpm, 30 s) with 2 mm zirconia beads.
• Thermal cycler compatible with the Ampdirect plus PCR protocol.

Results and discussion

Key findings:

• Species identification: PCR generated clear, species-specific fragments (227 bp chicken, 274 bp beef, 398 bp pork). In halal-certified products, chicken or beef fragments were detected where expected and pork fragments were not observed, consistent with labeling. Processed product A contained beef, chicken, and pork signals; product B contained beef and chicken signals only—again matching labels.
• Analytical sensitivity: In model admixtures of pork in beef, pork-specific amplicons were detected in all four replicates at each contamination level down to 0.0001% (w/w). Positive (100% pork) and negative (100% beef) controls performed as expected. Thus, the combined multiplex PCR and MultiNA II analysis demonstrated very high sensitivity for pork detection.
• Automation and data handling: Use of microchip electrophoresis and fingerprinting software removed subjective visual band calling, produced reproducible digital records, and facilitated objective species assignment—beneficial for traceability and audit-ready recordkeeping.
• Limitations noted: Absence of detectable pork DNA does not definitively prove the absolute absence of pork-derived material due to potential DNA degradation, uneven distribution, or limits of sampling; the assay is offered as Research Use Only and not for in vitro diagnostics.

Benefits and practical applications

• High sensitivity and specificity for species discrimination, enabling detection of trace pork contamination relevant to halal certification and allergen/food-safety concerns.
• Objective, automated interpretation reduces operator variability and improves reproducibility compared with agarose gel analysis.
• Digital storage of electropherograms and fingerprint matches supports quality management, audit trails, and regulatory documentation.
• Suitable for routine raw material verification, incoming QC, and final product testing in food manufacturing and inspection laboratories.

Future trends and applications

Potential developments and opportunities include:

• Expansion of multiplex panels to include a broader range of species, plant ingredients, and processed-matrix controls to increase applicability across food types.
• Integration with upstream automation for high-throughput sample preparation to enable larger-scale screening in industrial QC settings.
• Combining microchip electrophoresis with quantitative PCR or digital PCR approaches to provide quantitative estimates of admixture rather than binary detection.
• Adoption of portable or benchtop microfluidic systems for on-site screening in supply-chain checkpoints, slaughterhouses, or import inspection facilities.
• Complementarity with sequencing-based methods for complex matrices or highly processed foods where short or fragmented targets require sequence confirmation.
• Regulatory harmonization and validation studies to move from RUO toward standardized methods for official food control and halal certification bodies.

Conclusion

The combination of multiplex PCR targeting mitochondrial cytochrome b and automated microchip electrophoresis (MultiNA II) with fingerprinting analysis offers an objective, sensitive, and reproducible approach for animal species identification in processed foods and halal certification testing. The method achieved consistent detection of pork contamination at levels as low as 0.0001% in model mixtures and correctly identified species composition in commercial products. Automated digital analyses improve record-keeping and reduce subjective interpretation, making the approach attractive for routine QC, food authenticity testing, and traceback investigations, while acknowledging limitations related to sampling and the RUO status of the assay.

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.