Identification of Pork Contamination in Meatballs of Indonesia Local Market Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis

Abstract

This research applied and evaluated a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using cytochrome b gene to detect pork contamination in meatballs from local markets in Surabaya and Yogyakarta regions, Indonesia. To confirm the effectiveness and specificity of this fragment, thirty nine DNA samples from different meatball shops were isolated and amplified, and then the PCR amplicon was digested by BseDI restriction enzyme to detect the presence of pork in meatballs. BseDI restriction enzyme was able to cleave porcine cytochrome b gene into two fragments (131 bp and 228 bp). Testing the meatballs from the local market showed that nine of twenty meatball shops in Yogyakarta region were detected to have pork contamination, but there was no pork contamination in meatball shops in Surabaya region. In conclusion, specific PCR amplification of cytochrome b gen and cleaved by BseDI restriction enzymes seems to be a powerful technique for the identification of pork presence in meatball because of its simplicity, specificity and sensitivity. Furthermore, pork contamination intended for commercial products of sausage, nugget, steak and meat burger can be checked. The procedure is also much cheaper than other methods based on PCR, immunodiffusion and other techniques that need expensive equipment.

Keywords: Detection; Meatballs; Polymerase Chain Reaction; Pork Contamination.

Figure 1

Total genomic DNA extracted from local market meatballs. M: marker 100 bp DNA ladder (Invitrogen); A and B: genomic DNA of meatballs from different meatball shops in Yogyakarta (lane 1 to 20); C and D: genomic DNA of meatballs from Surabaya Region (lane 1 to 19); b: Total DNA of raw beef, and p: Total DNA of raw pork.

Figure 2

PCR products of cytochrome b gene fragments 359 bp long in samples from different meatballs products separated by 2% high-resolution agarose gel electrophoresis, M: marker 100 bp DNA ladder (Invitrogen), A–B: PCR products of cytochrome b gene from twenty of meatball samples from Yogyakarta region (1–20), C–D: PCR products of cyt b gene from nineteen of meatball samples from Surabaya region (1–19), b: PCR product of raw beef DNA, and p: PCR product of raw pork DNA. PCR, polymerase chain reaction.

Figure 3

Electrophoretic patterns of cytochrome b gene after digestion by BseDI restriction enzymes. M: marker 100 bp DNA ladder, lane 1 to 20: DNA fragment of different meatball samples from twenty meatball shops in Yogyakarta region, b: DNA fragment after digestion of PCR product of raw beef cytochrome b gene, and p: DNA fragment after digestion of PCR product of raw pork cytochrome b gene. PCR, polymerase chain reaction.

Figure 4

Electrophoretic patterns of cytochrome b gene after digestion by BseDI restriction enzymes. M: marker 100 bp DNA ladder, lane 1 to 19: DNA fragment of different meatball samples from twenty meatball shops in Surabaya region, b: DNA fragment after digestion of PCR product of raw beef cytochrome b gene, and p: DNA fragment after digestion of PCR product of raw pork cyt b gene. PCR, polymerase chain reaction.