Backgroud: Probiotics have been shown to benefit human health through several mechanisms, including their role in improving the health of our gastrointestinal tracts. The health benefits of probiotics are strain specific, and therefore it is critical to include the correct strains in probiotic products when claiming specific health benefits. Several studies have reported issues concerning the accuracy of labeling of commercial probiotic products, including inaccurate taxonomy, missing species, or undeclared species. Consequently, there is a growing need to develop and validate assays to reliably verify strain identity in commercial probiotic products. PCR-based methods are the most commonly used methods for food species ingredient diagnostics because they are simple, fast, sensitive, and can be validated. Objective: The aim of this paper is to set the guidelines for validating targeted qualitative real-time PCR assays to verify the presence of specific strains in a probiotic supplement. Methods and Results: Qualitative real-time PCR assays are validated to evaluate the assay performance in terms of specificity, sensitivity, repeatability, and reproducibility in detecting target strains. Conclusions and Highlights: Setting these guidelines will facilitate and streamline the validation process for qualitative real-time PCR-based assays for probiotic identity authentication in support of quality assurance systems.

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.

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