VALIDASI UJI KONTAMINASI BABI DALAM BAKSO DENGAN METODE REAL TIME-POLYMERASE CHAIN REACTION (RTi-PCR) VALIDATION OF TESTING METHOD FOR PORCINE CONTAMINANT IN MEATBALLS BY REAL TIME-POLYMERASE CHAIN REACTION (RTi-PCR) METHOD

Porcine contamination in the meatballs samples have been analyzed by real time polymerase chain reaction (RTi-PCR) based on the differences of mitochondrial sequence DNA. The research aims to obtained a valid analytical method of porcine contamination that is ready to be accredited. The study consisted of three stages which are optimization, validation, and application of the method. The study was begun with isolation of DNA using phenol- CIAA (chloroform-iso amyl alcohol). RTi-PCR optimization method was performed using gradient temperature (50.5 � 59.5 °C) to obtain the optimal annealing temperature (Ta) of ND5 forward primer (5'-CATTCGCCTCACTCACATTAACC- 3') and ND5 reverse primer (5'-AAGAGAGAGTTCTACGGTCTGTAG-3'). PCR conditions were used pre-denaturation at temperature 95 °C for 30 seconds, denaturation at temperature 95 °C for 2 seconds and annealing and extension at optimal Ta for 5 seconds then melting curve analysis at 65.0 � 95.0 oC. Method was then validated for its specificity, precision and limit of detection (cut off). Application of the methods was performed by testing of commercial meatballs. The results of RTi-PCR with ND5 primer is resulted in DNA fragments with Tm (melting temperature) at 78.50 ºC and various Ct (cycle number threshold) confirmed as 227 bp fragment size using agarose gel electrophoresis. Optimum Ta for PCR is 58.0 ºC. The method show high specificity which ND5 primers are only amplified porcine DNA and no amplicon present in negative control (porcine meatball 0%). The method show good precision with the consistent results after 10 replication of each positive and negative control. Methods were still detected up to 1% contamination of porcine. RTi-PCR analyses of 8 commercial samples succeed to detect one meatball sample from traditional market is positively contaminated by porcine