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

PLoS One. 2017 Aug 10;12(8):e0182872. doi: 10.1371/journal.pone.0182872. eCollection 2017.

Abstract

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.

MeSH terms

  • Animal Feed / analysis*
  • Animals
  • Cattle
  • Chickens
  • DNA, Mitochondrial / analysis*
  • Food Contamination / analysis*
  • Meat Products / analysis*
  • Polymerase Chain Reaction / methods*
  • Swine
  • Turkeys

Substances

  • DNA, Mitochondrial

Grants and funding

The authors gratefully acknowledge the financial support (for preparation of the manuscript) provided by the Canadian Food Inspection Agency (CFIA) (via the Federal Assistance Program) to RHH for HS, and in-kind contributions from Laboratory Services Division, University of Guelph, for the development and validation of the methods.