Validation of NADH dehydrogenase 4 L (ND4L) primers for porcine detection using qPCR analysis on commercial food products

Halal meat products must be free from any ingredients prohibited under Islamic law, such as porcine derivatives. The real-time polymerase chain reaction (qPCR) method is commonly used to detect porcine DNA in food products. Although various genes have been studied to develop porcine-specific primers, the NADH Dehydrogenase 4 L (ND4L) gene remains unexplored. This study aimed to design novel qPCR primers targeting the ND4L gene to detect porcine DNA in commercial food products. The primers were evaluated based on repeatability, sensitivity, and specificity. SYBR Green dye was used for amplification detection, and Ct values were recorded for quantification analysis. Fifty-two commercial food products were tested to assess the method’s robustness. The designed primers had the following sequences: forward, 5′-TCATTATAGCATTCACGATTGCCC-3′, and reverse, 5′-TGTGTGTTTAGGACAATTAGAGTCG-3′, yielding a 133 bp PCR product. These primers effectively amplified porcine DNA (20 ng/µL) with a Ct value of 12.44 ± 0.30 and a melting temperature (Tm) of 78.25 ± 0.05 °C. The limit of detection (LOD) was determined at 5 pg/µL of DNA and 0.001% w/w in a pork-beef binary mixture. The ND4L primers demonstrated high specificity, amplifying only pig and wild boar DNA without cross-reacting with 30 non-target animal species. Among the tested samples, 20 out of 52 were positive for porcine DNA, consistent with their labeling. These findings indicate that ND4L-based primers provide a reliable and sensitive approach for detecting porcine DNA in processed food products.