Impact of amplicon length on the identification of edible oils based on real-time PCR

Edible oil adulteration is a significant food safety concern in today's food fraud landscape. DNA-based detection methods emerging as the most reliable approach for identifying adulterated products. However, the processed oil matrix contains only trace amounts of highly degraded DNA, which significantly increases the difficulty of DNA extraction and greatly reduces the success rate of PCR amplification. To address these technical challenges, we designed species-specific primers for amplicons of varying lengths and systematically evaluated their performance in real-time PCR using DNA extracted from soybean, peanut, and rapeseed oils, followed by application in adulterated oil detection. All primer sets demonstrated high specificity, sensitivity and, most importantly, equal amplification efficiency. Results indicated that shorter amplicons were consistently detectable across all three vegetable oils, while longer amplicons were more likely to yield false negatives in real-time PCR reactions. Soybean oil showed CT values of 29 (74 bp) versus 38 (170 bp), peanut oil exhibited a CT value of 31 (54 bp) with no amplification for 86 bp, and rapeseed oil displayed a CT value of 34 (66 bp) with no signal for 101 bp. The use of shorter primers successfully facilitated the detection of adulterated oil.These findings suggest that amplicon length being a critical determinant for reliable DNA-based identification and authentication of edible oils. Thus, designing amplicons within the 50–80 bp range can significantly enhance the success rate of DNA-based identification and authentication of edible oils.