Analysis of Allergen Genes in Food Using a Microchip Electrophoresis System

Significance of the topic

Food allergen testing is a critical component of consumer safety and regulatory compliance. Molecular methods that target allergen-specific DNA complement protein-based assays by providing improved specificity for closely related species and greater robustness for processed foods in which proteins may be degraded. Automated microchip electrophoresis platforms can streamline downstream analysis of PCR products, increasing throughput, reproducibility, and objectivity of interpretation for routine food quality control and regulatory screening.

Objectives and overview of the study

This application note demonstrates analysis of allergen genes in confectionery products using PCR amplification followed by automated microchip electrophoresis on the MultiNA II (MCE-301). The study follows methods recommended by the Japan Consumer Affairs Agency (CAA) to detect genes of wheat, buckwheat, peanut, walnut, shrimp, and crab. Key aims were to evaluate MultiNA II performance for separation and detection of target amplicons and to apply an automated fingerprinting analysis to determine presence/absence of allergen-specific fragments in processed food samples.

Methodology

• Sample material: Positive controls of raw ingredient materials (wheat, buckwheat, peanut, walnut, shrimp, crab) and confectionery samples.
• DNA extraction: Homogenized samples (1 g) processed with the GM quicker 3 extraction kit following supplier protocol.
• PCR amplification: AmpliTaq Gold master mix used with primer sets specified by the CAA for shrimp, wheat, buckwheat, peanut and crab. A commercial Allergen Checker [Walnut] kit provided primers and conditions for walnut amplification.
• Electrophoresis and detection: PCR products analyzed on the Shimadzu MultiNA II microchip electrophoresis system using the DNA-500 reagent kit and SYBR Gold fluorescent dye.
• Fingerprinting analysis: Automated comparison of detected fragment sizes to defined positive-control base pair lengths with a ±5% tolerance for presence/absence calls.

Used instrumentation

• MultiNA II MCE-301 microchip electrophoresis system (Shimadzu).
• DNA-500 reagent kit for nucleic acid separation and SYBR Gold fluorescent stain.
• AmpliTaq Gold polymerase master mix for PCR.
• GM quicker 3 DNA extraction kit (Nippon Gene).
• Allergen Checker [Walnut] primer kit (Oriental Yeast) for walnut target.

Main results and discussion

• Target amplicon detection: Electrophoresis profiles clearly resolved the expected amplicon sizes specified by the CAA and the walnut kit. Reported characteristic fragment lengths used in the study were: shrimp 187 bp, wheat 141 bp, buckwheat 127 bp, peanut 95 bp, crab 62 bp, and walnut 120 bp.
• Sample outcomes: Two confectionery samples were highlighted. Sample A6 yielded the wheat-specific fragment and sample E6 yielded the walnut-specific fragment. Detected amplicons matched the ingredient declarations on product labels.
• Fingerprinting automation: The MultiNA II fingerprinting function automatically matched sample peaks to reference fragment sizes within a 5% tolerance, simplifying presence/absence determination and reducing subjective interpretation inherent to manual gel reading.
• Analytical performance: The system provided superior separation and reproducibility compared with conventional agarose gel electrophoresis due to microchip format, fluorescence detection, and automated handling of gel preparation, injection, run and detection steps.
• Method scope and limitations: PCR-based detection excels for ingredients with species-specific DNA markers and retains sensitivity in processed matrices where proteins may be denatured. However, PCR cannot discriminate allergenic proteins that share the same genes across food types (e.g., egg, milk), for which protein-based methods such as Western blot or ELISA remain necessary. The authors note that, per CAA guidance, walnut detection may alternatively be performed by real-time PCR or PCR-DNA chromatography; the MultiNA II approach is presented as a screening-level method for walnut.

Benefits and practical applications

• Workflow automation: MultiNA II automates gel preparation, sample injection, separation and detection, reducing hands-on time and variability.
• Objective interpretation: Automated fingerprinting removes subjective band-calling and supports consistent pass/fail screening decisions in QA/QC environments.
• Processed food testing: DNA-targeted assays maintain detectability after heat or fermentation, improving sensitivity for many processed matrices relative to protein assays.
• Regulatory alignment: The method follows national inspection recommendations (CAA), making it suitable for compliance testing and routine screening in food manufacturing and inspection laboratories.

Future trends and potential applications

• Integration with multiplex PCR and higher-throughput microchip platforms could enable parallel detection of multiple allergens in a single run, increasing efficiency for routine screening.
• Coupling automated electrophoresis with quantitative real-time PCR or digital PCR would extend capabilities from screening to quantitation and improved limit-of-detection reporting required for threshold-based labeling policies.
• Development of standardized, validated workflows and certified reference materials for processed foods will strengthen inter-laboratory comparability and regulatory acceptance of microchip electrophoresis–based workflows.
• Advances in on-chip sample preparation and automation could enable near real-time in-line or at-line monitoring within food production environments.

Conclusion

The study demonstrates that the MultiNA II microchip electrophoresis system effectively separates and detects PCR amplicons corresponding to prioritized food allergen genes, with automated fingerprinting enabling rapid presence/absence decisions. The approach offers clear advantages over manual agarose gels in reproducibility, throughput and objectivity, making it a practical screening tool for food quality laboratories. Limitations remain for allergens not amenable to gene-based discrimination, and confirmatory or quantitative analyses (e.g., real-time PCR, protein assays) may be required depending on regulatory or diagnostic needs.

References

1. Handbook on food allergy labeling of processed foods, Consumer Affairs Agency, March 2023 Edition.
2. Appendix: Inspection methods for foods containing allergenic substances, Consumer Affairs Agency, Notification concerning food labeling standards.