Ces44T as an endogenous reference gene in real-time quantitative PCR detection of tiger nut (Cyperus esculentus) ingredients in food

Abstract

Tiger nut (Cyperus esculentus) is a highly adaptable and nutritious plant that can be used to produce health-enhancing food products, edible oil, and functional ingredients. To ensure transparency, reliability, and accurate labeling throughout the food supply chain, accurate methods for identifying food ingredients are indispensable. In this study, the Ces44T gene was identified and validated as the endogenous reference gene for tiger nut. A real-time quantitative PCR assay targeting the Ces44T gene was developed, demonstrating exceptional specificity in distinguishing tiger nut from other plant species, and exhibiting remarkable stability across different tiger nut cultivars. Standard curves were established to validate the reliability and repeatability of the assay, displaying good linearity (R2 > 0.99) and PCR efficiency (90–110 %). The assay was estimated to have a limit of detection (LOD) of 0.0033 ng of tiger nut genomic DNA per reaction and a limit of quantification (LOQ) of 0.013 ng. We applied this assay to both laboratory-prepared samples and commercial food products, confirming its excellent specificity and robust performance. In conclusion, the developed real-time quantitative PCR assay provides an accurate means for the identification and quantification of tiger nut ingredients in various samples.