Wild Boar-Specific PCR Assay and Sequence Analysis Based on Mitochondrial Cytochrome-B Gene for Halal Authentication Studies

Wild boar meat (WBM) is non-halal meat widely abused in Indonesia. The most common case is mixing beef with WBM either in raw or processed foods. Therefore, it is necessary to develop a detection method of WBM contamination. The objective of this study was to employ polymerase chain reaction (PCR) and sequence analysis using species specific primer (SSP) targeting on wild boar mitochondrial cytochrome-b (CYTBWB2-wb) gene for the identification of WBM in a meatball. The specificity of primer was tested, and the amplicon size was confirmed with conventional PCR and agarose electrophoresis. The base sequences were analyzed using GeneStudio software and subjected to BLAST using NCBI. CYTBWB2-wb primer was also used to test the reference meatballs made from beef and WBM using real-time PCR. The result showed that CYTBWB2-wb amplified wild boar Cyt-B mt-DNA gene specifically. The amplicon size was 194 base pair (bp) with a similarity of 93–98% toward gen Cyt-B mt-DNA of several wild boar types. The primer is able to detect WBM on the reference meatballs up to 0.1% wt/wt with efficiency value of 108.0% and coefficient of determination (R 2 ) of 0.970. The CYTBWB2-wb primer proved to be specific and could be used as a standard method to identify the presence of WBM contamination in meatball products for halal authentication studies.

[1]  Md. Eaqub Ali,et al.  The employment of q-PCR using specific primer targeting on mitochondrial cytochrome-b gene for identification of wild boar meat in meatball samples , 2019, Journal of advanced veterinary and animal research.

[2]  R. Pratiwi,et al.  Forgery Detection Beef with Mice Meat (Mus musculus) in Meatballs Using Real-Time Polymerase Chain Reaction (Real-Time PCR) Primer Specific for a Target Mitochondrial DNA ND-1 Gene , 2019, Indonesian Journal of Chemistry.

[3]  Abdul Rohman,et al.  The employment of Fourier transform infrared spectroscopy coupled with chemometrics techniques for traceability and authentication of meat and meat products , 2018, Journal of advanced veterinary and animal research.

[4]  Abdul Rohman,et al.  The use of species-specific primer targeting on D-loop mitochondrial for identification of wild boar meat in meatball formulation - , 2018 .

[5]  E. Rencová,et al.  Counter immunoelectrophoresis: a simple method for the detection of species-specific muscle proteins in heat-processed products , 2018 .

[6]  S. Rahman,et al.  OPTIMIZATION OF COUNTERCURRENT IMMUNOELECTROPHORESIS AND AGAR GEL IMMUNODIFFUSION TESTS FOR THE COMPARATIVE DETECTION OF HORSE AND DONKEY MEAT , 2018 .

[7]  K. Triyana,et al.  Identification of pork in beef meatballs using Fourier transform infrared spectrophotometry and real-time polymerase chain reaction , 2017 .

[8]  Martono,et al.  Analysis of beef meatball adulteration with wild boar meat using real-time polymerase chain reaction , 2017 .

[9]  Deepak Sharma,et al.  Identification of Species Origin of Meat and Meat Products on the DNA Basis: A Review , 2015, Critical reviews in food science and nutrition.

[10]  A. Rohman,et al.  FTIR Spectroscopy in Combination with Chemometrics for Analysis of Wild Boar Meat in Meatball Formulation , 2015 .

[11]  R. Tiwari,et al.  Identification of different animal species in meat and meat products: trends and advances. , 2015 .

[12]  J. Slamecka,et al.  Multilevel D-loop PCR identification of hunting game , 2013, Applied & translational genomics.

[13]  A. Mursyidi.  The Role of Chemical Analysis in the Halal Authentication of Food and Pharmaceutical Products , 2013 .

[14]  B. Mane,et al.  Sequence analysis of mitochondrial 16S rRNA gene to identify meat species , 2013 .

[15]  Nathan D. Olson,et al.  DNA extract characterization process for microbial detection methods development and validation , 2012, BMC Research Notes.

[16]  M. Uddin,et al.  Analysis of pork adulteration in commercial meatballs targeting porcine-specific mitochondrial cytochrome b gene by TaqMan probe real-time polymerase chain reaction. , 2012, Meat science.

[17]  A. Rohman,et al.  PCR-RFLP Using BseDI Enzyme for Pork Authentication in Sausage and Nugget Products , 2011 .

[18]  Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived products — Qualitative nucleic acid based methods , 2011 .

[19]  S. Radu,et al.  Meat species identification and Halal authentication analysis using mitochondrial DNA. , 2009, Meat science.

[20]  João Carneiro,et al.  Identification of species with DNA-based technology: current progress and challenges. , 2008, Recent patents on DNA & gene sequences.

[21]  Dietrich Maede.  A strategy for molecular species detection in meat and meat products by PCR-RFLP and DNA sequencing using mitochondrial and chromosomal genetic sequences , 2006 .

[22]  A. K Lockley,et al.  DNA-based methods for food authentication , 2000 .

[23]  K. Mullis,et al.  Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. , 1985, Science.