RAPID PCR–BASED DETECTION OPTIMIZATION OF PORCINE DNA IN GELATIN CAPSULE SHELL


Amalia Sitti Khayyira, Viktoria Mardhika Estepane, Amarila Malik

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 PCR-RFLP method. Due to very low DNA trace in gelatin after 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 rapid initial detection method for molecular detection of porcine in gelatin capsule shells.


Keywords


capsule shell, duplex PCR, gelatin, PCR-RFLP, porcine

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