Validation of a Multiplex Real-time PCR Method for the Detection of Crustacean Allergens (Shrimp, Crab, and Lobster) in Complex Food Matrices

Introduction:  Crustacean shellfish are identified as one of the eight major allergenic foods and food groups in the Food Allergen Labeling and Consumer Protection Act (FALCPA) and affect approximately 2% of the American population. FALCPA requires that different types of crustacean shellfish (shrimp, crab, and lobster) must be differentiated on food labels. While ELISA methods are unable to distinguish between these different crustacean species, PCR-based detection methods have been shown to detect and differentiate all three.

Purpose:  Previously, our laboratory had validated individual real-time PCR assays for shrimp, crab, and lobster. Here, we describe a multiplex method for the detection of all three crustacean types in one assay.

Methods: Assays targeting the 12S gene were previously validated for shrimp, crab, and lobster. The probes were labeled with separate fluorophores for shrimp, crab, and lobster and the master mix was prepared with all primers and probes in the same reaction. The method was evaluated in complex food matrices for the detection and differentiation of crustacean type.

Results: Assays individually validated for probe performance produced linear standard curves with efficiencies in the range of 86.8 to 91.6% and R² values between 0.997 to 0.999. The multiplex method produced linear standard curves for each target within appropriate efficiency range 94.6 to 111.7% and R² values within appropriate range 0.980 to 0.995. There were no adverse effects from the cooccurring assays. All assays were evaluated from 0.1 ppm to 10⁶ppm DNA equivalence in food matrices.

Significance: This multiplex method allowed for a more efficient way to detect and identify the crustacean allergen or multiple allergens in one assay and will be used for direct comparison with ELISA detection methods.