Abstract
Objective: This study was conducted to optimize the genomic deoxyribonucleic acid (DNA) based molecular detection of gelatin derived from porcine by performing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and duplex PCR method employing cyt B gene.Methods: Optimization was carried out for DNA extraction, PCR conditions, and the sensitivity of the PCR-RFLP method. Due to the very low DNA trace in gelatin after the various manufacturing process, the extraction was optimized to obtain sufficient DNA which was visible on the agarose gel. PCR-RFLP was carried out using universal primers and BsaJI restriction enzyme, and duplex PCR was carried out using two sets of porcine-specific primers. Porcine and bovine DNA were mixed in various concentration to confirm sensitivity of both methods, i.e. 100%, 50%, 10%, 1%, 0.5%, 0.1%, 0.05%, and 0.01%Results: Both methods, PCR-RFLP, and Duplex PCR, were able to detect as low as 0.01% porcine DNA, indicated by the presence of porcine DNA amplicon bands (131 bp and 228 bp for PCR-RFLP, 212 bp and 398 bp for duplex PCR). Although DNA bands presented in low intensity, identification of porcine and bovine species and estimation of DNA quantities were possible.Conclusion: Both conventional PCR methods, i.e. PCR-RFLP and Duplex PCR, were sensitive, specific, and suitable as a rapid initial detection method for molecular detection of porcine in gelatin capsule shells.
Highlights
Gelatin is a group of polypeptides produced from collagen hydrolysis, which is extracted from the skin, bones, and connective tissue of animals [1]
Literatures review and in silico analysis were performed to determine the primers used in this study: universal primer pair for polymerase chain reaction (PCR)-RFLP [15] and two sets of primer pairs for duplex PCR [16, 17]
Analysis of primer characteristics was summarized in table 1
Summary
Gelatin is a group of polypeptides produced from collagen hydrolysis, which is extracted from the skin, bones, and connective tissue of animals [1]. There are many sources of gelatin, namely chicken, pork, beef, fish, and poultry. In 2009, gelatin was mostly produced from pig skins (46%), cow hides (29.4%), cow bones (23.1%), and other sources (1.5%) [2]. In the provisions of Islam, consuming non-halal products, including derivative products from porcine, is a prohibition. This made gelatin products which originally from porcine, are included in materials prohibited for consumption by adherents of this religion. G. Indonesia of 87.18% [3], has been designing regulations on halal product assurance, i.e. Law no. Indonesia of 87.18% [3], has been designing regulations on halal product assurance, i.e. Law no. 33 y 2014
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