Droplet digital polymerase chain reaction (ddPCR) assays integrated with an internal control for quantification of bovine, porcine, chicken and turkey species in food and feed

Food adulteration and feed contamination are significant issues in the food/feed industry, especially for meat products. Reliable techniques are needed to monitor these issues. Droplet Digital PCR (ddPCR) assays were developed and evaluated for detection and quantification of bovine, porcine, chicken and turkey DNA in food and feed samples. The ddPCR methods were designed based on mitochondrial DNA sequences and integrated with an artificial recombinant plasmid DNA to control variabilities in PCR procedures. The specificity of the ddPCR assays was confirmed by testing both target species and additional 18 non-target species. Linear regression established a detection range between 79 and 33200 copies of the target molecule from 0.26 to 176 pg of fresh animal tissue DNA with a coefficient of determination (R2) of 0.997–0.999. The quantification ranges of the methods for testing fortified heat-processed food and feed samples were 0.05–3.0% (wt/wt) for the bovine and turkey targets, and 0.01–1.0% (wt/wt) for pork and chicken targets. Our methods demonstrated acceptable repeatability and reproducibility for the analytical process for food and feed samples. Internal validation of the PCR process was monitored using a control chart for 74 consecutive ddPCR runs for quantifying bovine DNA. A matrix effect was observed while establishing calibration curves with the matrix type under testing, and the inclusion of an internal control in DNA extraction provides a useful means to overcome this effect. DNA degradation caused by heating, sonication or Taq I restriction enzyme digestion was found to reduce ddPCR readings by as much as 4.5 fold. The results illustrated the applicability of the methods to quantify meat species in food and feed samples without the need for a standard curve, and to potentially support enforcement activities for food authentication and feed control. Standard reference materials matching typical manufacturing processes are needed for future validation of ddPCR assays for absolute quantification of meat species.

[1]  Enrique Sentandreu,et al.  Authenticity of meat products: Tools against fraud , 2014 .

[2]  K. Uddin,et al.  Species Authentication Methods in Foods and Feeds: the Present, Past, and Future of Halal Forensics , 2012, Food Analytical Methods.

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

[4]  R. Osta,et al.  Beef- and bovine-derived material identification in processed and unprocessed food and feed by PCR amplification. , 2002, Journal of agricultural and food chemistry.

[5]  Phillip Belgrader,et al.  Detection of Methicillin-Resistant Staphylococcus aureus by a Duplex Droplet Digital PCR Assay , 2013, Journal of Clinical Microbiology.

[6]  B Brenig,et al.  Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR). , 2015, Food chemistry.

[7]  T. Dingle,et al.  Tolerance of droplet-digital PCR vs real-time quantitative PCR to inhibitory substances. , 2013, Clinical chemistry.

[8]  T. Civera,et al.  Development of a multiplex real-time PCR assay for the detection of ruminant DNA. , 2012, Journal of food protection.

[9]  T. García,et al.  PCR identification of beef, sheep, goat, and pork in raw and heat-treated meat mixtures. , 2004, Journal of food protection.

[10]  E. Rencová,et al.  Identification of species-specific DNA in feedstuffs. , 2003, Journal of agricultural and food chemistry.

[11]  Xianhua Dai,et al.  Erratum to “A De Novo Genome Assembly Algorithm for Repeats and Nonrepeats” , 2014, BioMed Research International.

[12]  Edmondo Ceci,et al.  Occurrence of mislabeling in meat products using DNA-based assay , 2015, Journal of Food Science and Technology.

[13]  M. Tartaglia,et al.  Detection of bovine mitochondrial DNA in ruminant feeds: a molecular approach to test for the presence of bovine-derived materials. , 1998, Journal of food protection.

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

[15]  Hanlee P. Ji,et al.  Quantitative and Sensitive Detection of Cancer Genome Amplifications from Formalin Fixed Paraffin Embedded Tumors with Droplet Digital PCR. , 2012, Translational medicine.

[16]  Mojca Milavec,et al.  Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR , 2013, PloS one.

[17]  Yicun Cai,et al.  Quantitative Analysis of Pork and Chicken Products by Droplet Digital PCR , 2014, BioMed research international.

[18]  T. García,et al.  Identification of goose, mule duck, chicken, turkey, and swine in foie gras by species-specific polymerase chain reaction. , 2003, Journal of agricultural and food chemistry.

[19]  J. Spink,et al.  Defining the public health threat of food fraud. , 2011, Journal of food science.

[20]  Tania Nolan,et al.  The digital MIQE guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments. , 2013, Clinical chemistry.

[21]  A. Slater,et al.  A TaqMan real-time PCR system for the identification and quantification of bovine DNA in meats, milks and cheeses , 2007 .

[22]  M. Baker Digital PCR hits its stride , 2012, Nature Methods.

[23]  A. Mottola,et al.  Detection of mislabeling in packaged chicken sausages by PCR , 2014 .

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

[25]  Marta Hernández,et al.  Analytical Application of a Sample Process Control in Detection of Foodborne Viruses , 2011 .

[26]  U. Agrimi,et al.  A competitive polymerase chain reaction-based approach for the identification and semiquantification of mitochondrial DNA in differently heat-treated bovine meat and bone meal. , 2003, Journal of food protection.

[27]  V. Beneš,et al.  Guidelines for Minimum Information for Publication of Quantitative Digital PCR Experiments , 2013 .

[28]  M. Ansfield,et al.  Production of a Sensitive Immunoassay for Detection of Ruminant and Porcine Proteins, Heated to ≫ 130°C at 2.7 bar, in Compound Animal Feedstuffs , 2000 .

[29]  Benjamin J. Hindson,et al.  Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification , 2011, Analytical chemistry.

[30]  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.

[31]  Sebastien Gallien,et al.  Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR. , 2012, Journal of virological methods.

[32]  P. O'mahony Finding horse meat in beef products--a global problem. , 2013, QJM : monthly journal of the Association of Physicians.

[33]  Christoph von Holst,et al.  Detection of ruminant meat and bone meals in animal feed by real-time polymerase chain reaction: result of an interlaboratory study. , 2007, Journal of agricultural and food chemistry.

[34]  Douglas D. Richman,et al.  Highly Precise Measurement of HIV DNA by Droplet Digital PCR , 2013, PloS one.

[35]  J. Yongsawatdigul,et al.  Quantification of viable bacterial starter cultures of Virgibacillus sp. and Tetragenococcus halophilus in fish sauce fermentation by real-time quantitative PCR. , 2016, Food microbiology.

[36]  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.

[37]  H. Yacoub,et al.  Molecular detection of adulteration in chicken products based on mitochondrial 12S rRNA gene , 2015, Mitochondrial DNA.

[38]  F. Vogensen,et al.  Species determination - Can we detect and quantify meat adulteration? , 2009, Meat science.

[39]  Jeff Mellen,et al.  High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number , 2011, Analytical chemistry.

[40]  B. Kiepper,et al.  Quantification of Zoonotic Bacterial Pathogens within Commercial Poultry Processing Water Samples Using Droplet Digital PCR , 2013 .

[41]  E. Rencová,et al.  Identification of bovine-specific DNA in feedstuffs. , 2001, Journal of food protection.

[42]  G. Rusul,et al.  Mislabelling of beef and poultry products sold in Malaysia , 2016 .

[43]  M. Zacharisen Severe allergy to chicken meat. , 2006, WMJ : official publication of the State Medical Society of Wisconsin.