Specific, semi-quantitative detection of the soybean allergen Gly m Bd 30K DNA by PCR

Abstract

A semi-quantitative PCR-based system has been developed for detection of DNA sequences for the soybean allergen Gly m Bd 30K. The selected primers were highly specific for soybean and did not show amplification from a panel of legume relatives. Repeatability was assessed in a spiking experiment of soybean in wheat flour (0.0001–100% soybean), using known standards for comparison of the amount of output DNA from different PCR reactions. The frequency of PCR reactions with successful amplification of the soybean allergen sequence was highly dependent on initial target DNA concentration, and showed a rapid sigmoidal decrease, when target concentration approached the detection limit (0.01%). For samples with successful amplification there was a good correlation between the initial amount of soybean in the mixture and the output from PCR, when suitable block designs were used to control experimental errors. The simple approach used for quantification in this study proved efficient for assessment of homogeneity of self-prepared soybean bars used for provocation tests of food allergic patients in clinical practice. In addition the method was efficient in detecting soybean allergen sequences in a number of processed foods.