Real Time PCR to detect hazelnut allergen coding sequences in processed foods

Area of Science:

• Food Science
• Molecular Biology
• Allergen Detection

Background:

• Hazelnut allergens (Cor a 9, 11, 13) pose significant risks in food products.
• Accurate detection methods are crucial for food safety and regulatory compliance.
• Existing methods may lack sensitivity or robustness for processed foods.

Purpose of the Study:

• To develop and validate a highly sensitive quantitative RT-PCR method for detecting specific hazelnut allergen genes (Cor a 9, 11, 13).
• To compare the performance of the developed RT-PCR method with existing techniques like ELISA.
• To investigate the impact of food processing methods on the detectability of hazelnut DNA.

Main Methods:

• Development and validation of quantitative RT-PCR using novel primer sets targeting Cor a 9, Cor a 11, and Cor a 13 sequences.
• Optimization of DNA extraction using CTAB-phenol-chloroform method for hazelnut.
• Testing the method's specificity, sensitivity, and robustness with spiked samples and processed food matrices.
• Evaluation of the effect of thermal processing (roasting, autoclaving) and high-hydrostatic pressure on DNA amplificability.

Main Results:

• The optimized CTAB-phenol-chloroform DNA extraction method proved most effective for hazelnut.
• Quantitative RT-PCR with specific primers enabled accurate amplification of Cor a 9, 11, and 13 sequences.
• The limit of detection was established at 1 ppm of raw hazelnut.
• Thermal processing reduced DNA yield and amplificability, while high-hydrostatic pressure had no significant effect.
• The RT-PCR method demonstrated superior sensitivity in detecting hazelnut traces in commercial foodstuffs compared to ELISA.

Conclusions:

• A validated quantitative RT-PCR method offers high sensitivity and specificity for detecting hazelnut allergens (Cor a 9, 11, 13).
• This method is robust and effective for identifying hazelnut contamination in processed foods, outperforming ELISA.
• The developed RT-PCR assay represents a significant advancement for sensitive hazelnut trace detection in complex food matrices.