Validation of NADH Dehydrogenase 4L (ND4L) Primers Gene for Porcine Detection Using A SYBR Green Real-Time PCR Analysis on Commercial Processed Meat Products

Abstract

Porcine contamination in halal products is known to be found in commercial food markets in Indonesia. Detection methods are needed, one of which is Real-Time PCR with robust biomarkers in various types of processed food. In this study, we focused on designing a novel primer from NADH Dehydrogenase 4L (ND4L) gene that has never been explored as a porcine detection. Primers were assembled using GenBank NCBI (MK251046 Sus scrofa), and their efficacy (repeatability, sensitivity, and specificity) was evaluated. The capability primer performance was also compared with a commercial kit. Results showed that primer effectively amplified porcine DNA with a melting curve (Tm) of 78.17 ± 0.05oC and a Cq value of 11.95 ± 0.48 at a 20 ng/µL DNA concentration. CV values of Cq and Tm repeatability were 4,01 and 0.09%, respectively. The limit of detection primer reached 5 pg/µL DNA and 0.001% w/w binary meat mixture of pork-beef. This primer is highly specific to pig and wild boar species (against 30 species (non-pig). A comparative study on the ND4L primer with a commercial porcine detection kit (FAM label) revealed similar results in detecting porcine DNA in food products. ND4L primer successfully detected porcine DNA in 20 of 52 commercial meat products with various types of processing (according to their claims). Primer performed satisfactorily in all validation parameters with high sensitivity and specificity. Hence, this finding of the specific primers on the ND4L gene could be promising for detecting porcine DNA in food products.