Morcia Caterina, Tumino Giorgio, Gasparo Giulia, Ceresoli Caterina, Fattorini Chiara, Ghizzoni Roberta, Carnevali Paola, Terzi Valeria
Council for Agricultural Research and Economics, Research Centre for Genomics and Bioinformatics, I-29017 Fiorenzuola d'Arda PC, Italy.
Barilla S.p.A., I-43122 Parma PR, Italy.
Microorganisms. 2020 Aug 27;8(9):1307. doi: 10.3390/microorganisms8091307.
Head Blight (FHB) is one of the major diseases affecting small-grain cereals, worldwide spread and responsible for severe yield and quality losses annually. Diagnostic tools, able to track species even in the early stages of infection, can contribute to mycotoxins' risk control. Among DNA-based technologies for detection, qPCR (single and multiplex assays) is currently the most applied method. However, pathogen diagnostics is now enforced by digital PCR (dPCR), a breakthrough technology that provides ultrasensitive and absolute nucleic acid quantification. In our work, a panel of chip digital PCR assays was developed to quantify , , , and . The primers/probes combinations were evaluated on pure fungal samples with cdPCR technique, in comparison with the qPCR approach. Moreover, the cdPCR assays were applied to quantify in durum wheat and oat samples, naturally contaminated or spiked with fungal DNA. For a better evaluation of infection level in plants, duplex assays were developed, able to co-amplify both plant and fungal DNA. To the best of our knowledge, this is the first study directed to the application of digital PCR to diagnosis in plants.
赤霉病（FHB）是影响小粒谷物的主要病害之一，在全球范围内传播，每年都会导致严重的产量和质量损失。能够在感染早期阶段追踪病原菌的诊断工具，有助于控制霉菌毒素风险。在基于DNA的检测技术中，qPCR（单重和多重检测）是目前应用最广泛的方法。然而，病原体诊断现在通过数字PCR（dPCR）得到了加强，这是一项突破性技术，可提供超灵敏和绝对核酸定量。在我们的工作中，开发了一组芯片数字PCR检测方法来定量检测、、、和。与qPCR方法相比，利用cdPCR技术在纯真菌样本上评估了引物/探针组合。此外，cdPCR检测方法还应用于定量检测硬粒小麦和燕麦样本中的，这些样本是自然污染或添加了真菌DNA的。为了更好地评估植物中的感染水平，开发了双链检测方法，能够同时扩增植物和真菌DNA。据我们所知，这是第一项将数字PCR应用于植物病原菌诊断的研究。
