Droplet Digital PCR (ddPCR) for Halal Detection of Commercial Gelatin Products

A droplet digital polymerase chain reaction (ddPCR) assay was developed for the identification and quantification of porcine DNA in commercial gelatin products. The method employed primers targeting the beta-actin gene and demonstrated high specificity against five nontarget species across 18 different products. The ddPCR method showed enhanced sensitivity, detecting porcine DNA at concentrations as low as 0.06 ng/μL in standard gelatin powder, surpassing the detection limit of 0.11 ng/μL achieved by real-time PCR. The robustness of the assay was validated using seven types of commercial porcine gelatin products, including candies, marshmallows, dietary supplements, collagen, and capsule shells. ddPCR analysis revealed that six out of seven commercial product samples tested positive for porcine DNA, compared to only three samples detected by RT-PCR. Sequencing of the ddPCR amplicons further confirmed the accuracy of the assay, with high similarity to the reference porcine species.