School of Life Science and Environment, Dept. of Genetics and Biotechnology, Blocos Laboratoriais bdg, Univ. of Trás-os-Montes and Alto Douro, 5000-801, Vila Real, Portugal.
Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Inst. Campo Grande, Univ. of Lisboa, C8 bdg, 1749-016, Lisboa, Portugal.
J Food Sci. 2018 Oct;83(10):2415-2423. doi: 10.1111/1750-3841.14333. Epub 2018 Sep 14.
The need to support food labelling has driven to the development of PCR-based techniques suitable for food analysis. DNA-based markers have been successfully employed for varietal tracing in Protected Designation of Origin (PDO) olive oils. In this study, we report a fast, sensitive, and easy-to-use strategy for PDO olive varietal identification. To achieve this aim four different DNA extraction methods were tested and compared, based on initial volume, extraction time, the gDNA concentration, and quality ratios. The optimized DNA extraction protocol from extra virgin olive oils, based on CTAB-hexane-chloroform extraction, proved to be the most effective. High-resolution melting (HRM) DNA assay was developed based on nuclear microsatellites (gSSR) and plastid DNA (cpDNA) aiming an accurate identification of the olive varieties present in the olive oil samples. After PCR reproducibility evaluation, six molecular markers: three SSRs and three cpDNA loci were chosen based on their discrimination power. The SSR-HRM strategy assays were designed to target UDO99-011, UDO99-039, UDO99-024, and ssrOeUA-DCA16 loci. All SSR-PCR products generated from these primers were analyzed by capillary electrophoresis (CE) for HRM data validation. The SSR coupled with HRM melting curve analysis generated 14 HRM profiles sufficient to genotype all varieties, highlighting their potential use for varietal discrimination. The locus ssrOeUA-DCA16 generated a specific melting curve that allow a high-throughput discrimination of the Picual and Cobrançosa varieties in olive oil samples. Further, the UDO99-024 was also tested by SSR-HRM assay in commercial olive oil samples with promising results. Considering time, cost, and performance SSR-HRM proved to be a reliable method suitable for varietal tracing of olive oils. PRACTICAL APPLICATION: Olive oil authenticity is a form of protecting producers and consumers against fraudulent practices. Herein, we present a DNA barcode suitable for the identification of olive varieties, allowing an accurate identification of the olive varieties in olive oil samples using SSR-HRM assay. Its applicability in commercial olive oil samples is viable. This methodology can be used as a tool for Extra Virgin Olive Oil (EVOO) adulterations detection.
支持食品标签的需求推动了适合食品分析的基于 PCR 的技术的发展。基于 DNA 的标记物已成功用于受保护原产地名称 (PDO) 橄榄油的品种追溯。在这项研究中，我们报告了一种快速、敏感且易于使用的 PDO 橄榄油品种鉴定策略。为了实现这一目标，测试并比较了基于初始体积、提取时间、gDNA 浓度和质量比的四种不同的 DNA 提取方法。基于 CTAB-己烷-氯仿提取的特级初榨橄榄油优化 DNA 提取方案被证明是最有效的。基于核微卫星 (gSSR) 和质体 DNA (cpDNA) 开发了高分辨率熔解 (HRM) DNA 分析，旨在准确鉴定橄榄油样品中的橄榄油品种。在评估 PCR 重现性后，根据其区分能力选择了六个分子标记：三个 SSR 和三个 cpDNA 位点。SSR-HRM 策略分析旨在针对 UDO99-011、UDO99-039、UDO99-024 和 ssrOeUA-DCA16 基因座。从这些引物中产生的所有 SSR-PCR 产物均通过毛细管电泳 (CE) 进行分析，以验证 HRM 数据。SSR 与 HRM 熔解曲线分析相结合，生成了 14 种 HRM 图谱，足以对所有品种进行基因分型，突出了它们在品种鉴别中的潜在用途。ssrOeUA-DCA16 基因座产生的特定熔解曲线允许在橄榄油样品中高通量区分皮夸尔和科布兰萨品种。此外，UDO99-024 也通过 SSR-HRM 分析在商业橄榄油样品中进行了测试，结果令人鼓舞。考虑到时间、成本和性能，SSR-HRM 被证明是一种可靠的方法，适用于橄榄油的品种追溯。实际应用：橄榄油的真实性是保护生产者和消费者免受欺诈行为的一种形式。本文介绍了一种适合鉴定橄榄油品种的 DNA 条码，可通过 SSR-HRM 分析准确鉴定橄榄油样品中的橄榄油品种。其在商业橄榄油样品中的适用性是可行的。该方法可用作特级初榨橄榄油 (EVOO) 掺假检测的工具。
