A multiplex real-time PCR method applied to detect eight pollen species in food for the prevention of allergies

Pollen-induced anaphylaxis is recognized as the most common allergic disease. Methods for pollen species identification are thus regarded as important and urgent for the protection of sensitive consumers. In our study, a multiplex real-time PCR assay was established targeting eight pollens that have been reported recently as potential allergens or were uncommon species that have been newly exploited for marketing purposes (pine pollen, canola bee pollens, kiwi bee pollens, willow bee pollens, corn poppy bee pollens, rose bee pollens, lotus bee pollens, and camellia bee pollens). This method proved to be highly specific and efficient, with four pollen species being identified simultaneously from a single reaction. The absolute sensitivity was 1 pg/mu L to 1 ng/mu L for DNA from the eight pollen species, and as low as 0.5% (w/w) mixed bee pollen powder could be discriminated. A microscopy method using SEM was compared with our multiplex real-time PCR method. Results demonstrated that SEM was much less sensitive than multiplex real-time PCR, with the sensitivity of the former being approximately 10-50% (w/w).