Shehata Hanan R, Li Jiping, Chen Shu, Redda Helen, Cheng Shumei, Tabujara Nicole, Li Honghong, Warriner Keith, Hanner Robert
Department of Integrative Biology, University of Guelph, Guelph, Ontario, Canada.
Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada.
PLoS One. 2017 Aug 10;12(8):e0182872. doi: 10.1371/journal.pone.0182872. eCollection 2017.
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.
食品掺假和饲料污染是食品/饲料行业中的重大问题，尤其是对于肉类产品而言。需要可靠的技术来监测这些问题。已开发并评估了液滴数字PCR（ddPCR）测定法，用于检测和定量食品和饲料样品中的牛、猪、鸡和火鸡DNA。ddPCR方法基于线粒体DNA序列设计，并与人工重组质粒DNA整合，以控制PCR过程中的变异性。通过对目标物种和另外18种非目标物种进行测试，确认了ddPCR测定法的特异性。线性回归确定了从0.26至176 pg新鲜动物组织DNA中目标分子的检测范围为79至33200拷贝，测定系数（R2）为0.997 - 0.999。检测强化热处理食品和饲料样品的方法的定量范围，对于牛和火鸡目标为0.05 - 3.0%（重量/重量），对于猪肉和鸡肉目标为0.01 - 1.0%（重量/重量）。我们的方法在食品和饲料样品的分析过程中显示出可接受的重复性和再现性。使用控制图对74次连续的ddPCR运行进行监测，以对牛DNA进行定量，从而对PCR过程进行内部验证。在用测试中的基质类型建立校准曲线时观察到基质效应，并且在DNA提取中加入内标提供了克服这种效应的有用方法。发现由加热、超声处理或Taq I限制性酶消化引起的DNA降解可使ddPCR读数降低多达4.5倍。结果表明这些方法适用于在无需标准曲线的情况下对食品和饲料样品中的肉类物种进行定量，并有可能支持食品认证和饲料控制的执法活动。未来需要与典型制造工艺匹配的标准参考物质，以验证ddPCR测定法对肉类物种进行绝对定量的能力。
