The Use of Real-Time Polymerase Chain Reaction Combined with Specific-Species Primer for Analysis of Dog Meat DNA in Meatball

Abstract

The presence of dog meat is a crucial issue because dog meat is non-halal meat for Muslims. The objective of this study was to design and validate species-specific primer for the identification of dog meat DNA in meatball using real-time polymerase chain reaction (real-time PCR). The specific primer targeting mitochondrial cytochrome c oxidase subunit 1 (CO1) was validated. The specific primers used were designed using Integrated DNA Technologies (IDT) software and subjected to NCBI BLAST procedure. The candidate primers were tested for specificity study using several DNAs from fresh meat of pork, chicken, beef, lamb, and rat. The method was also validated by determining several parameters of linearity, sensitivity, precision, and efficiency. The results showed that primer could amplify specifically DNA target at an optimized annealing temperature of 56.6 °C. The limit of detection (LoD) obtained was 5 ng DNA, corresponding to 2.5% of dog meat in a meatball. The repeatability evaluation, expressed with relative standard deviation (RSD), and efficiency value was in the acceptable range (RSD < 25% and efficiency (90–105%). This method was successfully used for the analysis of marketed samples. Real-time PCR can be used as a standard method in halal authentication analysis through DNA analysis.

The employment of real-time polymerase chain reaction for analysis of canine meat in meatball products for halal authentication analysis

“…In recent years, the utilization of metabolomics methodologies using high-resolution liquid chromatography-mass spectrometry has been a potential technology for the detection and identification of non-halal meats; however, the metabolites obtained are very large, which makes the data interpretation difficult [ 15 ]. For this reason, DNA-based methods are the preferred technique for analyzing non-halal meats due to their specificity and sensitivity [ 16 , 17 , 18 ].…”

Authentication of Rattus Norvegicus Fat and Other Animal Fats Using Gas Chromatography-Mass Spectrometry (Gc-Ms) and Principal Component Analysis (Pca)

“…The adulteration of meat by replacing or mixing halal meat with non-halal meat can cause various problems such as (1) religious and belief problems due to the substitution of halal meat, such as beef with non-halal meat such as rat meat, dog meat, wild boar meat and pork. [4][5][6], (2) health-related problems [3] and (3) allergenic reactions [7]. The nutrition and safety of meat are directly related to people's health and quality of life [8].…”

Detection of Pork in Beef Meatballs Using LC-HRMS Based Untargeted Metabolomics and Chemometrics for Halal Authentication

“…Numerous analytical techniques using various instrumental methods have been developed for meat products authentication, such as gas chromatography-flame ionization detector (GC-FID) [ 7 ], gas chromatography-mass spectrometry (GC-MS) [ 8 ], high-performance liquid chromatography with UV-Vis and fluorescence detector, liquid chromatography-mass spectrometry (LC-MS) [ 9 , 10 ], vibrational spectroscopy such as Fourier transform infrared (FTIR) spectroscopy [ 11 ], near-infrared (NIR) spectroscopy [ 12 ], Raman spectroscopy [ 13 ], DNA-based methods such as polymerase chain reaction (PCR) and real time-polymerase chain reaction (RT-PCR) as well as protein-based methods using enzyme-linked immunosorbent assay (ELISA) [ 14 , 15 ]. Methods based on RT-PCR have been widely used in most countries for halal authentication of meat products, such as the detection of pork in beef meatballs [ 3 ], analysis of wild boar in beef meatballs [ 16 ], and detection of dog meat in beef meatballs [ 17 ]. However, the RT-PCR technique is not without limitations.…”