Real-time PCR Methods for Detection of Crustacean Shellfish Allergens in Challenging Food Matrices and Processing Conditions

Introduction:  Food allergen detection requires methods that are highly sensitive and specific for the target allergen.  For crustacean allergen detection, it is also important that methods be able to distinguish the different types of crustaceans—shrimp, crab, and lobster—from each other.  Real-time PCR meets all of these requirements. 

Purpose: The work described here was carried out to evaluate real-time PCR methods we developed for shrimp and lobster. We used food matrices with inherently low DNA content, acidic pH, and treatment with high temperature and pressure.   

Methods: Group-specific real-time PCR primers and probes were developed for shrimp and lobster using mitochondrial 12S and 16S gene sequences.  Shrimp and lobster meat were spiked into food matrices at 0.1, 1, 10, 100, 1000, 10⁴, and 10⁵ ppm, homogenized, and subject to DNA extraction and PCR using methods optimized for this work.

Results: Results were used to generate standard curves which were analyzed with respect to linear range, statistical R² value, and PCR reaction efficiency.  In neutral pH and untreated acidic conditions, both shrimp and lobster assays had reaction efficiencies of 94%-112%, lower limits of 0.1-1 ppm, and linearity over 6-8 orders of magnitude.  Treatment with high temperature and pressure in an acidic food matrix resulted in a loss of signal. 

Significance: There is no effective treatment for food allergy.  Food allergic people can react to minute amounts of allergen, so they rely on avoidance of offending foods.  This makes sensitive detection methods imperative in protecting the public health.  With the exception of combined high heat, high pressure, and acidic pH, the method is highly sensitive and robust.