Detection of DNA pork in processed meat products with real-time polymerase chain reaction

Abstract

Real-time polymerase chain reaction (qPCR) technique is a suitable method for identifying animal species in processed meat because of its ability to amplify a few fragments of DNA. A specific fragment of pork (mitochondrial cytochrome b gene (cyt-b)) was used as a DNA ladder. This study aimed to evaluate the use of a cyt-b gene generated primer for detecting the presence of pork in processed meat products by qPCR and determining the threshold cycle cut-off. The evaluation of the primer effectiveness was performed by threshold cycle (Ct) value, amplicon size by electrophoresis and melting curve. Two corned (A, B) and two jerkies (C, D) collected from the market were used as the sample. Genomic DNA from samples, fresh beef (as negative control) and fresh pork (as positive control) were extracted using Qiagen Kits. Amplification condition for 50 cycles of the cyt-b gene was performed as the initial step at 95°C for 10 mins, denaturation step at 95°C for 15 s, annealing step at 55°C for 60 s, extension step at 72°C for 30 s and post-PCR at 72°C for 3 mins. The threshold cycle (Ct) cut-off less than 30 confirmed as pork positive. The result obtained indicated that sample A and D were pork negative, with Ct value respectively 40.73 and 43.59. Melting temperatures of amplicon were ranged from 79.5 to 80.5°C, only differed by 1°C, and the amplicon electrophoresis resulting in a single band of the same size (149 bp). Hence, the melting curve analysis and electrophoresis of PCR products were not able to differentiate between pork and beef.