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Detection and quantification of salep with real time PCR utilizing the nr‐ITS2 region

Kabacaoğlu, Eda, Budak, Barçın Karakaş
Journal of the science of food and agriculture 2019 v.99 no.5 pp. 2447-2454
additives, adulterants, adulterated products, corn flour, dyes, molecular cloning, oligodeoxyribonucleotides, potato starch, quantitative polymerase chain reaction, ribosomal DNA, sequence analysis, starch
BACKGROUND: Salep is a highly valued food product, susceptible to adulteration with less costly additives such as starch. The aim of this study is to develop a real‐time polymerase chain reaction (PCR) method for the detection and quantification of authentic salep using primers designed from the nr‐ITS2 region. RESULTS: The designed primers were shown to amplify selectively with salep DNA and cloning/sequencing of end‐point PCR products obtained with authentic salep (Orchideceae) confirmed amplimers to be copies of the intended target region. Detection of salep DNA using the designed primer set was possible at levels as low as 20 pg. After initial evaluation of the real‐time PCR method with binary DNA mixtures of salep and cornflour, quantification of salep was conducted in binary mixtures of potato starch (a more likely adulterant) and salep, showing that quantification within the range 25%–100% could be achieved. The real‐time assay was performed using the 18S rDNA region co‐amplified as an internal standard and evaluated using the ΔΔCt method. The calibration curves obtained as a result of real‐time PCR runs showed linearity, with R² values above 99%. CONCLUSION: A real‐time PCR method using SybreGreen intercalating dye was established for salep detection and quantification based on the specific amplification of salep DNA. Method validation was performed utilizing blind and commercial samples. © 2018 Society of Chemical Industry