Primer rRNA-12S for detection of bovine gelatine DNA in capsule shells using real-time polymerase chain reaction
2018
Fitriani, N. F. | Sudjadi | Rohman, A.
About 90% gelatine used in food and pharmaceuticals comes from porcine gelatine. The presence of porcine gelatine is hindered by Muslim society; therefore the confirmation of halal gelatine like bovine gelatine is a must. In pharmaceuticals, gelatine is used for making capsule shells. Analysis of the capsule shells containing bovine gelatine via DNA analysis is required in order to verify the halalness status of gelatine sources. The objective of this study was to develop real-time polymerase chain reaction (real-time PCR) for identification of bovine gelatine in capsule shells. In this study, a couple of bovine-specific primer, rRNA-12S primers, (forward: 5’-CCC AAG CTA ACA GGA GTA CG-3’ and reverse: 5’-TAG TGC GTC GGC TAT TGT AG-3’) were designed. The annealing temperature of primer was optimized to get optimumamplification of DNA target at 51.1-58.7°C. The primer was subjected to specificity test using DNAs isolated from fresh chicken, pork, wild boar, lamb, and beef as well as commercial porcine gelatine and bovine gelatine and capsule shells made from the mixture of porcine and bovine gelatines. The used primer successfully amplified bovine gelatine DNA. Primers also showed high specify toward capsule shells made from the mixture of porcine-bovine gelatines. The sensitivity test resulted primers enable to amplify up to 0.48 ng of bovine gelatine DNA,and 1.2 ng of DNA extracted from bovine gelatine capsule. The rRNA-12S primers were finally applied to identify bovine DNA in commercial gelatine capsules.
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