Ring trial validation of single and multiplex real-time PCR methods for the detection and quantification of the allergenic food ingredients mustard, celery, soy, wheat and rye

We describe an interlaboratory ring trial validation for quantification methods of 3 food allergens using real-time PCR: a real-time PCR method for the joint simultaneous detection and quantification of brown and black mustard, a realtime PCR method for the detection and quantification of wheat and rye, and a multiplex real-time PCR method combining the detection and quantification of brown/black mustard, white mustard, celery and soybean. Mixtures with defined copy numbers of the target sequences from brown mustard, white mustard, celery, soybean and wheat were applied as calibrants for the real-time PCR-based quantification. Boiled sausages incurred with these allergenic ingredients in the range of 0-100 mg/kg were used as ring trial samples. Within the ring trial, we demonstrated that 40 mg/kg of the respective allergens (wheat/rye 80 mg/kg) can reliably be detected and quantified by all PCR systems even in boiled sausage meat. Brown/black mustard was detectable at the lowest concentration level of 10 mg/ kg even after autoclaving. Results for brown/black mustard in terms of sensitivity, trueness and precision were comparable in single-and multiplex PCR. The results of the study revealed the importance of matrix-matched control samples for allergen analysis in terms of both conversion to mg/kg and of normalization variations caused by different extraction procedures.