A suitable method for the detection of a potential fraud of bringing macaque monkey meat into the food chain

Being the third-largest primate population has not made macaque (Macaca fascicularis sp.) monkeys less exposed to threats and dangers. Despite wildlife protection, they have been widely hunted and consumed in several countries because of their purported nutritional values. In addition to trading as pure bush meats in several places, monkey meat has been sold in meatball and soup products in Indonesia. Thus the possibility of macaque meat trafficking under the label of common meats is quite high. This paper reports the development of a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay with the shortest amplicon length for the confirmed detection of monkey meat under compromised states which are known to degrade DNA. We amplified a 120-bp region of d-loop gene using a pair of macaque-specific primers and confirmed their specificity for the target species through cross-challenging against 17 different species using a 141-bp site of an 18 S rRNA gene as an endogenous control for eukaryotes. This eliminated the possibilities of any false-negative detection with complex matrices or degraded specimens. The detection limit was 0.00001 ng DNA in a pure state and 0.1% of meat in mixed matrices and commercial meatball products. RFLP analysis further authenticated the originality of the PCR product and distinctive restriction patterns were found upon AluI and CViKI-1 digestion. A micro-fluidic lab-on-a-chip automated electrophoretic system separated the fragments with high resolution. The assay was validated for screening commercial meatball products with sufficient internal control. Graphical Abstract

[1]  A. Sazili,et al.  Porcine-Specific Polymerase Chain Reaction Assay Based on Mitochondrial D-Loop Gene for Identification of Pork in Raw Meat , 2012 .

[2]  A. Tiwari,et al.  Beef specific polymerase chain reaction assay for authentication of meat and meat products , 2012 .

[3]  M. C. Mahani,et al.  Phylogenetic relationships of Malaysian monkeys, Cercopithecidae, based on mitochondrial cytochrome c sequences. , 2010, Genetics and molecular research : GMR.

[4]  J. Lenstra,et al.  Differentiation of cattle species in beef by PCR-RFLP of mitochondrial and satellite DNA. , 2002, Meat science.

[5]  S. Singh,et al.  Detection of pork adulteration by highly-specific PCR assay of mitochondrial D-loop. , 2014, Food chemistry.

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

[7]  L. Hoffman,et al.  Game and venison - meat for the modern consumer. , 2006, Meat science.

[8]  N. Saitou,et al.  Mitochondrial DNA sequence phylogeny of 4 populations of the widely distributed cynomolgus macaque (Macaca fascicularis fascicularis). , 2008, The Journal of heredity.

[9]  Ashok Kumar,et al.  Identification of chicken, duck, pigeon and pig meat by species-specific markers of mitochondrial origin. , 2009, Meat science.

[10]  B. Al-Safadi,et al.  Identification of meat species by PCR-RFLP of the mitochondrial COI gene. , 2012, Meat science.

[11]  A. Syvänen,et al.  First generation microarray-system for identification of primate species subject to bushmeat trade , 2009 .

[12]  P S Girish,et al.  Sequence analysis of mitochondrial 12S rRNA gene can identify meat species. , 2004, Meat science.

[13]  F. Meslin,et al.  Wildlife, Exotic Pets, and Emerging Zoonoses , 2007, Emerging infectious diseases.

[14]  C. Roos,et al.  Phylogenetic relationships of Malaysia’s long-tailed macaques, Macaca fascicularis, based on cytochrome b sequences , 2014, ZooKeys.

[15]  E. Bennett.  Is There a Link between Wild Meat and Food Security? , 2002 .

[16]  T. García,et al.  Novel TaqMan real-time polymerase chain reaction assay for verifying the authenticity of meat and commercial meat products from game birds , 2010, Food additives & contaminants. Part A, Chemistry, analysis, control, exposure & risk assessment.

[17]  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.

[18]  P S Girish,et al.  Meat species identification by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of mitochondrial 12S rRNA gene. , 2005, Meat science.

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

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

[21]  Yong-Gang Yao,et al.  Species authentication of commercial beef jerky based on PCR-RFLP analysis of the mitochondrial 12S rRNA gene. , 2010, Journal of genetics and genomics = Yi chuan xue bao.

[22]  O. Olivieri,et al.  Meat from wild ungulates: ensuring quality and hygiene of an increasing resource , 2010 .

[23]  H. Senyuva,et al.  Meat species identification and Halal authentication using PCR analysis of raw and cooked traditional Turkish foods. , 2013, Meat science.

[24]  S. Garrett,et al.  Detection of meat species using TaqMan real-time PCR assays. , 2004, Meat science.

[25]  S. Horai,et al.  Molecular phylogeny of macaques: implications of nucleotide sequences from an 896-base pair region of mitochondrial DNA. , 1996, Molecular biology and evolution.

[26]  A. Eudey,et al.  The Crab-Eating Macaque (Macaca fascicularis): Widespread and Rapidly Declining , 2008 .

[27]  A. Arslan,et al.  Effect of method of cooking on identification of heat processed beef using polymerase chain reaction (PCR) technique. , 2006, Meat science.

[28]  Isabel Mafra,et al.  Food authentication by PCR-based methods , 2008 .

[29]  D. Cawthorn,et al.  What is the role and contribution of meat from wildlife in providing high quality protein for consumption , 2012 .

[30]  M. Nei,et al.  MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. , 2011, Molecular biology and evolution.

[31]  Wen-Tso Liu,et al.  Quantitative effects of position and type of single mismatch on single base primer extension. , 2009, Journal of microbiological methods.

[32]  T. García,et al.  A review of current PCR-based methodologies for the authentication of meats from game animal species. , 2010 .

[33]  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 .

[34]  Md. Al Amin,et al.  Multiplex PCR assay for the detection of five meat species forbidden in Islamic foods. , 2015, Food chemistry.

[35]  J. Lüthy,et al.  PCR-RFLP Analysis of Mitochondrial DNA: Differentiation of Fish Species , 2000 .

[36]  Y. B. Che Man,et al.  Identification of pork derivatives in food products by species-specific polymerase chain reaction (PCR) for halal verification , 2007 .

[37]  U. Hashim,et al.  Swine-Specific PCR-RFLP Assay Targeting Mitochondrial Cytochrome B Gene for Semiquantitative Detection of Pork in Commercial Meat Products , 2012, Food Analytical Methods.

[38]  F. M. Ghazali,et al.  Meat molecular detection: Sensitivity of polymerase chain reaction-restriction fragment length polymorphism in species differentiation of meat from animal origin , 2007 .

[39]  T. García,et al.  PCR identification of meats from chamois (Rupicapra rupicapra), pyrenean ibex (Capra pyrenaica), and mouflon (Ovis ammon) targeting specific sequences from the mitochondrial D-loop region. , 2007, Meat science.

[40]  Sharifah Bee Abd Hamid,et al.  Multiplex PCR in Species Authentication: Probability and Prospects—A Review , 2014, Food Analytical Methods.

[41]  I. González,et al.  Differentiation of European wild boar (Sus scrofa scrofa) and domestic swine (Sus scrofa domestica) meats by PCR analysis targeting the mitochondrial D-loop and the nuclear melanocortin receptor 1 (MC1R) genes. , 2008, Meat science.