Species-Specific Polymerase Chain Reaction for the Detection of Anethum graveolens Adulteration in Cuminum cyminum Seeds

Abstract Cuminum cyminum (cumin) is a widely used culinary spice valued for its distinct flavor, aromatic profile, and nutritional benefits, making it a staple in global cuisine and traditional medicine. However, due to its higher market value, cumin is often a target for economic adulteration. One of the most reported adulterants is Anethum graveolens (dill), a lower-cost herb known as sathakuppa in Ayurveda. While dill seeds closely resemble cumin morphologically, they pose a significant health risk due to their abortifacient properties, which have been historically documented in Sri Lanka and India. Dill has been traditionally used for treating menstrual disorders due to its bioactive compounds that influence endometrial contractions, making its unintentional consumption particularly concerning for pregnant women.This study aimed to develop a reliable molecular technique to differentiate A. graveolens from C. cyminum to detect dill adulteration in cumin samples, ensuring food authenticity and preventing economic fraud. Species-specific primers were designed targeting the Internal Transcribed Spacer (ITS) region of A. graveolens to ensure specificity without cross-reactivity with C. cyminum. Polymerase Chain Reaction (PCR) assays were performed, followed by agarose gel electrophoresis to visualize amplified DNA fragments and confirm the presence of A. graveolens in cumin samples. For further confirmation, amplified DNA products have been followed by sanger sequencing, providing additional molecular evidence of species differentiation.The molecular approach successfully detected A. graveolens adulteration in cumin samples at levels as low as 1%, demonstrating high sensitivity and specificity. The PCR-based method effectively distinguished between the two species, overcoming the limitations of conventional morphological identification techniques. PCR-based detection, complemented by sequencing, provides a robust tool for identifying A. graveolens adulteration in cumin, ensuring food authenticity and product integrity. The findings highlight the importance of molecular authentication techniques in preventing spice adulteration and mitigating associated health risks.