A Real-time PCR-based Method for Estimatingthe Thermal History of Cockroach in Processed Foods, Resistant to Enzymatic DNA Degradation

We previously developed a real-time PCR-based analytical method to evaluate the thermal history of insects (cockroaches) found in processed foods by measuring the degree of genomic DNA fragmentation. However, in this study, we identified a challenge-postmortem enzymatic DNA degradation occurs in insect bodies, making it difficult to distinguish between heat-induced DNA fragmentation and enzymatic degradation. To address this issue, we developed another approach targeting mitochondrial DNA. The new method may be able to detect DNA fragmentation caused by heating even in the model sample by immersing Periplaneta fuliginosa in curry sauce products at 25℃ for three days, showing resistance to enzymatic degradation. Additionally, considering the limitation of available body parts in practical tests, we divided the bodies of model insects into nine different parts and analyzed them. The improved method showed smaller ΔΔCq values across all body parts, suggesting that the mitochondrial DNA is less prone to enzymatic DNA fragmentation than genomic DNA. The improved method enables the detection of DNA fragmentation at temperatures exceeding 100℃, making it particularly suitable for analyzing insect contaminants in retorted food products.