| Summary: | 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.
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