Development and Validation of a Direct qPCR Method for the Detection of Pork Adulteration in Processed Meat Products

Background and Objective: The number of Muslims in Indonesia is increasing; hence, consumption of halal foods is increasing. This urges halal detection of foods as well. Direct qPCR is a method for DNA-based halal detection using crude DNA samples. One factor that affects the success of direct qPCR is the crude DNA. In this study, DNA extraction process was optimized by modifying the lysis step to achieve high-quality crude DNA, optimizing the qPCR conditions and validating the method. Material and Methods: Materials used for DNA extraction from meats and meat products included lysis buffer containing EDTA, NaCl, SDS, NaOH and tween-20. Materials used in qPCR assay included crude DNA, ddH20, primers and master mix. Genomes from meats and processed products were extracted using lysis buffer by optimizing lysis conditions (temperature and incubation time). The crude DNA was assayed for concentration and purity; then, results were compared to chloroform: isoamyl and the commercial methods. The DNA extract from lysis buffer was amplified using qPCR machine. Operational conditions such as DNA concentration, annealing temperature and primer concentration were optimized. Results were validated using specificity, repeatability, reproducibility, applicability, robustness and limit of detection assays. Results and Conclusion: Temperature for optimum lysis in extraction lysis buffer method included 75 °C for 25 min. Concentrations of DNA from the lysis buffer, chloroform-isoamyl and commercial methods did not significantly vary (p-value=0.094). The optimum conditions for qPCR method were set at DNA concentration of 50 ng µl^-1, annealing temperature of 53 °C and primer concentration of 10 pmol. For the validation result, specificity assay showed that the method could be used to detect pork and wild boar in meats using ND4 primers. In addition, repeatability assay included 1.06% and reproducibility assay included 1.57%. Direct qPCR method can be used for various processed meat products and is resistant to inhibitors (alginate, calcium ions, EDTA and cellulose), except for polysaccharides. The LOD value achieved from the PCR sensitivity, linearity and efficiency assay were 0.001 ng µl^-1, 0.996 and 110%, respectively. Results verify that the direct qPCR method is easy, fast, applicable, accurate and precise for the pork detection.