Markers for distinguishing Orostachys species by SYBR Green-based real-time PCR and verification of their application in commercial O. japonica food products

Abstract

Human consumption of plant functional foods has been rapidly increasing owing to the health benefits they provide. In particular, in Korea, the plant Orostachys japonica has attracted attention for its anticancer and other effects. Of the 12 established Orostachys species, only three (viz., O. iwarenge, O. malacophyllus, and O. japonica) have been allowed for use as foods in Korea. In this study, 12 species-specific primer sets based on single nucleotide polymorphisms of five chloroplast genes and one nuclear gene were developed to discriminate Orostachys species through quantitative real-time PCR (qPCR) analysis with SYBR Green staining. The efficiencies of the designed primer pairs in amplifying the target species ranged from 80 to 110%, with strong correlation coefficients (R2 > 0.99), whereas no clear correlation coefficient was evident for the non-target species. In order to verify the specificity of the 12 developed Orostachys-specific primers, binary mixtures of the DNAs (tenfold serially diluted samples) from the target species and each of the other non-target species were generated for qPCR analysis, with results suggesting that the primers could clearly discriminate at least 0.1% of O. japonica DNA (10 pg) in the mixtures. With regard to the feasibility of the developed qPCR system for detecting Orostachys species in O. japonica food products, O. japonica DNA was detected in all eight commercial products tested, with low Ct values (< 20), whereas none of the other Orostachys species DNAs were detected, confirming that the tested foods contained only O. japonica. Therefore, developed primers and qPCR conditions would be useful for verifying the authenticity of commercial O. japonica food products.