Dataset Information

Duplex droplet digital PCR (ddPCR) for simultaneous quantification of bovine and porcine gelatin in capsules.

ABSTRACT: Detection of bovine and porcine in gelatin-based products is important as species fraud and product mislabeling may have a detrimental impact on customers who have health, ethical, and religious concerns about animal products. The duplex droplet digital PCR (ddPCR) assay using double-quenched probes has been developed for quantification and detection of porcine and bovine DNA in gelatin capsules. A DNA mixture derived from gelatin was found to have a limit of detection as low as 0.001 ng/µl for porcine samples and 0.01 ng/µl for bovine samples. DNA from 12 other distinct species was tested with the bovine and porcine probes, showing high specificity for this method. The test was validated using fifty-five commercial supplement and pharmaceutical capsules, of which 17 were positive for bovine and/or porcine DNA. This study shows that the duplex ddPCR is reliable for routine analysis in the identification of bovine and porcine origins for gelatin capsules.

Supplementary information

The online version contains supplementary material available at 10.1007/s10068-022-01204-x.

SUBMITTER: Mahamad P

PROVIDER: S-EPMC10082860 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature

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Duplex droplet digital PCR (ddPCR) for simultaneous quantification of bovine and porcine gelatin in capsules.

Mahamad Pornpimol P Dahlan Winai W Kahong Saveeyah S So-Audon Sukanya S Munaowaroh Wila W Nopponpunth Vanida V

Food science and biotechnology 20221216 6

Detection of bovine and porcine in gelatin-based products is important as species fraud and product mislabeling may have a detrimental impact on customers who have health, ethical, and religious concerns about animal products. The duplex droplet digital PCR (ddPCR) assay using double-quenched probes has been developed for quantification and detection of porcine and bovine DNA in gelatin capsules. A DNA mixture derived from gelatin was found to have a limit of detection as low as 0.001 ng/µl for ...[more]

PMID: 37041814

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Project description: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.

Dataset Information

Duplex droplet digital PCR (ddPCR) for simultaneous quantification of bovine and porcine gelatin in capsules.

Supplementary information

Publications

Duplex droplet digital PCR (ddPCR) for simultaneous quantification of bovine and porcine gelatin in capsules.

Similar Datasets

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