Design and optimisation of rapid quadruplex PCR technique to identify plant-derived adulterants in saffron

Due to its beneficial properties, saffron is the costliest spice worldwide. The high price and decreasing availability of high-quality saffron promote the tendency of adulteration in saffron products by incorporating some alternative plant materials with a similar appearance. Current methods for detecting saffron adulteration, such as simplex and duplex PCR, are effective but often require multiple reactions, making them time-consuming and resource-intensive. To address this limitation, we optimized, for the first time, a quadruplex PCR to detect plant-based adulterants in saffron, providing a more efficient solution for simultaneous detection of multiple adulterants. For this purpose, Species-specific primers were selected for 'Crocus sativus L', 'Carthamus tinctorius L', 'Zea mays L' and 'Curcuma longa L' to simultaneously identify these species using a single and reliable method. As a result of the specificity test and with Duplex PCR, we proved the absence of cross-reactivity between the different species primers. The optimized multiplex PCR yields distinct amplicon sizes of 299, 205, 152, and 131 for saffron, Curcuma, Corn and Safflower respectively. Moreover, the developed assay was sensitive enough to detect three target species using only 0.25 ng/µL. The designed quadruplex PCR is fast, specific, cost-effective and useful in detection of adulterants in saffron products.