Analysis of Porcine Gelatin DNA in a Commercial Capsule Shell Using Real-Time Polymerase Chain Reaction for Halal Authentication

The presence of pig derivatives, such as porcine gelatin, in any products is prohibited to be consumed by Muslim community. This study is intended to develop a specific primer from mytochondrial D-loop capable of amplifying DNA from porcine gelatin in commercial capsule shells. Two pairs of primers designed from mitochondrial D-loop region were tested in order to confirm the primer specificity in gelatin sources (pork, beef, and catfish) and fresh tissue (pig, cows, goat, chickens, and rat). Primers were then used to perform sensitivity test of six dilution series (1000, 200, 100, 10, 5, and 1 pg/µL) of porcine gelatin and porcine capsule shell. The amplification was also performed on capsule shell from porcine-bovine mixture gelatin at 0, 10, 20, 30, 40, 50, and 100% concentration. The repeatability test was performed by measuring amplification capsule shells from porcine–bovine gelatin mixture. Real time polymerase chain reaction method using primers designed was further applied to analyze capsule shells purchased from markets. From two primers have been designed specifically, only primer D-Loop 108 (forward: 5’-CGT ATG CAA AAA ACC ACG CCA-3’; reverse: 5’-CTT ACT ATA GGG AGC TGC ATG-3’) had the capability to identify the presence of porcine DNA in fresh tissue and gelatin sources at optimum annealing temperature of 58.4ºC. Sensitivity of the developed method expressed as limit of detection of DNA in gelatin and capsule shells is 5 pg.