Analysis of pork adulteration in commercial meatballs targeting porcine-specific mitochondrial cytochrome b gene by TaqMan probe real-time polymerase chain reaction.

A test for assessing pork adulteration in meatballs, using TaqMan probe real-time polymerase chain reaction, was developed. The assay combined porcine-specific primers and TaqMan probe for the detection of a 109 bp fragment of porcine cytochrome b gene. Specificity test with 10 ng DNA of eleven different species yielded a threshold cycle (Ct) of 15.5 ± 0.20 for the pork and negative results for the others. Analysis of beef meatballs with spiked pork showed the assay can determine 100-0.01% contaminated pork with 102% PCR efficiency, high linear regression (r(2) = 0.994) and ≤ 6% relative errors. Residuals analysis revealed a high precision in all determinations. Random analysis of commercial meatballs from pork, beef, chicken, mutton and goat, yielded a Ct between 15.89 ± 0.16 and 16.37 ± 0.22 from pork meatballs and negative results from the others, showing the suitability of the assay to determine pork in commercial meatballs with a high accuracy and precision.

[1]  R. Köppel,et al.  Multiplex real-time PCR for the detection and quantification of DNA from beef, pork, horse and sheep , 2008 .

[2]  J. Regenstein,et al.  The Kosher and Halal Food Laws. , 2003, Comprehensive reviews in food science and food safety.

[3]  E. V. Thomas,et al.  Partial least-squares methods for spectral analyses. 1. Relation to other quantitative calibration methods and the extraction of qualitative information , 1988 .

[4]  A. S. Mohamed,et al.  Rapid identification of pork for halal authentication using the electronic nose and gas chromatography mass spectrometer with headspace analyzer. , 2011, Meat science.

[5]  Jian Deng,et al.  Simultaneous spectrophotometric determination of trace amount of malachite green and crystal violet in water after cloud point extraction using partial least squares regression. , 2010, Journal of hazardous materials.

[6]  Wolfhard Wegscheider,et al.  Spectrophotometric multicomponent analysis applied to trace metal determinations , 1985 .

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

[8]  Sang-Ryoul Park,et al.  Use of multiplex polymerase chain reactions to indicate the accuracy of the annealing temperature of thermal cycling. , 2005, Analytical biochemistry.

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

[10]  A. Omar,et al.  Detection of Raw Pork Targeting Porcine-Specific Mitochondrial Cytochrome B Gene by Molecular Beacon Probe Real-Time Polymerase Chain Reaction , 2012, Food Analytical Methods.

[11]  R. Joozani,et al.  Identification of pork genome in commercial meat extracts for Halal authentication by SYBR green I real‐time PCR , 2011 .

[12]  H. Akiyama,et al.  Quantification of Pork, Chicken and Beef by Using a Novel Reference Molecule , 2011, Bioscience, biotechnology, and biochemistry.

[13]  J. Turňa,et al.  Polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) as a molecular discrimination tool for raw and heat-treated game and domestic animal meats. , 2010 .

[14]  A. S. Rohman,et al.  Analysis of pork adulteration in beef meatball using Fourier transform infrared (FTIR) spectroscopy. , 2011, Meat science.

[15]  Ebrahim Rahimi,et al.  Molecular assay to fraud identification of meat products , 2011, Journal of Food Science and Technology.

[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]  L. Vigilant,et al.  The effects of sequence length and oligonucleotide mismatches on 5' exonuclease assay efficiency. , 2002, Nucleic acids research.

[18]  Md. Eaqub Ali.  Nanobiosensor for detection and quantitation of DNA sequences in degraded mixed meats. , 2011 .

[19]  J. Nevado,et al.  Simultaneous spectrophotometric determination of ethinylestradiol and levonorgestrel by partial least squares and principal component regression multivariate calibration , 1997 .

[20]  T. García,et al.  Real-time PCR for detection and quantification of red deer (Cervus elaphus), fallow deer (Dama dama), and roe deer (Capreolus capreolus) in meat mixtures. , 2008, Meat science.

[21]  Marcus Gassmann,et al.  The development of mini pentameric STR loci for rapid analysis of forensic DNA samples on a microfluidic system , 2010, Electrophoresis.

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

[23]  H. Akiyama,et al.  A Real-Time Quantitative PCR Detection Method for Pork, Chicken, Beef, Mutton, and Horseflesh in Foods , 2007, Bioscience, biotechnology, and biochemistry.

[24]  U. Hashim,et al.  Nanoparticle sensor for label free detection of swine DNA in mixed biological samples , 2011, Nanotechnology.

[25]  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.

[26]  R. Bridgman,et al.  Monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for sensitive detection of prohibited ruminant proteins in feedstuffs. , 2004, Journal of food protection.

[27]  R. Hanner,et al.  DNA barcoding detects market substitution in North American seafood , 2008 .

[28]  T. García,et al.  Determination of food authenticity by enzyme-linked immunosorbent assay (ELISA) , 2008 .

[29]  J. Chisholm,et al.  Effect of heat and pressure processing on DNA fragmentation and implications for the detection of meat using a real-time polymerase chain reaction , 2006, Food additives and contaminants.

[30]  Md. Eaqub Ali,et al.  Gold nanoparticle sensor for the visual detection of pork adulteration in meatball formulation , 2012 .

[31]  A. Raha,et al.  Analysis of raw meats and fats of pigs using polymerase chain reaction for Halal authentication. , 2005, Meat science.

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

[33]  Teresa García,et al.  TaqMan real-time PCR for the detection and quantitation of pork in meat mixtures. , 2005, Meat science.

[34]  A. Califano,et al.  Effect of type of emulsifiers and antioxidants on oxidative stability, colour and fatty acid profile of low-fat beef burgers enriched with unsaturated fatty acids and phytosterols. , 2010, Meat science.