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 (R 2 [ 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.

Development of molecular markers to distinguish between morphologically similar edible plants and poisonous plants using a real‐time PCR assay

“…To confirm the reproducibility and accuracy of the developed real‐time PCR systems, we performed inter‐laboratory validation in two different laboratories 6 . These inter‐laboratory validation in both laboratories were performed with the same PCR conditions, using a CFX Connect Real‐Time PCR Detection System (Bio‐Rad, Hercules, CA, USA) and an Applied Biosystems 7500 Fast Real‐Time PCR Instrument System (Applied Biosystems).…”

“…DNA‐based polymerase chain reaction (PCR) analyses have been widely used because their economical and timesaving advantages 6 . Especially, the quantitative real‐time PCR (qPCR) assay has high specificity and sensitivity, and can detect very low amounts of target DNA in a food complex.…”

“…Alternatively, SYBR Green I, binding to double‐stranded DNA in a sequence independent manner, can provide more a flexible, convenient and inexpensive method compared to probe‐based methods 8,9 . Indeed, many research studies have used SYBR Green based real‐time PCR assays for the detection of ingredients in commercial foods, such as bovine and poultry, Cynanchum wilfordii , Orostachys species and berry fruits 6,10–12 …”

Development and Validation of a Real-Time PCR Based Assay to Detect Adulteration with Corn in Commercial Turmeric Powder Products

“…DNA is generally believed to be stable enough to withstand various chemical treatments and high temperatures, and small quantities of DNA can be detected with specific primers using PCR-based methods [ 14 ]. DNA-based methods, such as quantitative real-time PCR (real-time PCR), multiplex PCR, and PCR-RFLP have been successfully applied to detect food fraud and adulteration due to their economical and time-saving advantages over other approaches [ 15 , 16 ].…”

Development of DNA-based species-specific real-time PCR markers for four berry fruits and their application in commercial berry fruit foods

“…The United States Grocery Manufacturers Association estimates that food fraud costs the global food industry $10-$15 billion annually, affecting about 10% of all sold commercial foods [6]. In response, various technologies incorporating molecular markers and biochemical analysis as two examples have been developed for prevent food fraud involving plants, such as saffron [7], orange [8], Orostachys japonica [9], and Cynanchum wilfordii [10]. However, prevention of food fraud for berry fruits is relatively unexplored.…”