| Summary: | Porcine contaminant on nugget have been analyzed using real-time Polymerase Chain Reaction (RTi-PCR). The aim of this study is to develop porcine identification method on nugget based DNA which can be used as halal authentication.
Real-time PCR amplified ND5 target gene in pork�s mithochondria DNA using species-specific primer, namely forward primer ND5 (5�-CATTCGCCTCACTCACATTAACC-3�), and reverse primer ND5 (5�-AAGAGAGAGTTCTACGGTCTGTAG-3�). The steps of this study consisted of determining the annealing temperature (Ta) and the method validation include the specificity, precision and determination of the limit detection. Real-time PCR application used for detection porcine contamination on nuggets. DNA was isolated using phenol KIAA. Amplification of DNA fragments was conducted with PCR conditions : pre-denaturation 95 °C for 30 s, denaturation 95 °C for 2 seconds and annealing at optimal Ta result for 5 seconds.
DNA amplification using real-time PCR with primers ND5 produced an amplicon with a melting point of 79 °C and 227 base pair fragment through agarose gel electrophoresis. The optimal annealing temperature was 58 °C. Validated method had high specificity and sensitivity, specific primers amplified DNA ND5 pigs and the detection limit up to 1% porcine contamination in food products. Seven samples were tested and proved not contain pork.
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