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.
Currently, along with the development of science and technology, the diversification of food products occurs in the market. Food products can contain non-halal components like porcine gelatine. One of food suspected to use gelatine is soft candy. Gelatin can be made from pork or beef or other animal. The presence of porcine gelatine in any food products is not allowed for Moslem community, therefore an analytical method offering reliable results must be developed. This study is intended to use Real-Time Polymerase Chain Reaction (RT-PCR) for analysis of porcine gelatine in soft candy. Isolation of DNA was performed with mitochondrial DNA Isolation Kit K280-50 (Bio-Vision). The DNA was analyzed by RT-PCR using primer D-Loop 318. Analysis for the primer specificity was performed on fresh tissue (pig, cows, chickens, goats and rats) and gelatin sources (beef, pigs and catfish). Primer D-loop318 can amplify porcine DNA at the optimum temperature 61.4°C. Repeatability test demonstrated amplification of all positive response samples containing porcine DNA in serial dilution of 10000-1 pg). The Coefficient of Variation (CV) is 6.32%. The repeatability test was also performed on soft candy 100% having CV of 1.06%. The commercial soft candy samples evaluated do not contain porcine DNA.
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