2
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found
      Is Open Access

      Moving from qPCR to Chip Digital PCR Assays for Tracking of some Fusarium species causing Fusarium Head Blight in Cereals

      research-article

      Read this article at

      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Fusarium 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 Fusarium species even in the early stages of infection, can contribute to mycotoxins’ risk control. Among DNA-based technologies for Fusarium 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 Fusarium graminearum, F.culmorum, F. sporotrichioides, F. poae and F. avenaceum. 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 Fusarium 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 Fusarium diagnosis in plants.

          Related collections

          Most cited references24

          • Record: found
          • Abstract: found
          • Article: found
          Is Open Access

          Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data

          Quantitative PCR (qPCR) has become the gold standard technique to measure cDNA and gDNA levels but the resulting data can be highly variable, artifactual and non-reproducible without appropriate verification and validation of both samples and primers. The root cause of poor quality data is typically associated with inadequate dilution of residual protein and chemical contaminants that variably inhibit Taq polymerase and primer annealing. The most susceptible, frustrating and often most interesting samples are those containing low abundant targets with small expression differences of 2-fold or lower. Here, Droplet Digital PCR (ddPCR) and qPCR platforms were directly compared for gene expression analysis using low amounts of purified, synthetic DNA in well characterized samples under identical reaction conditions. We conclude that for sample/target combinations with low levels of nucleic acids (Cq ≥ 29) and/or variable amounts of chemical and protein contaminants, ddPCR technology will produce more precise, reproducible and statistically significant results required for publication quality data. A stepwise methodology is also described to choose between these complimentary technologies to obtain the best results for any experiment.
            Bookmark
            • Record: found
            • Abstract: found
            • Article: not found

            Emerging fusarium-mycotoxins fusaproliferin, beauvericin, enniatins, and moniliformin: a review.

            The contamination of foods and feed with mycotoxins is a commonly known problem. Intense investigations have been conducted to study the occurrence, toxicity, and recently also the prevention and detoxification strategies of mycotoxins in human and animal food chains. Most of the studies have emphasized on "traditional" mycotoxins, such as aflatoxins, ochratoxin A, and trichothecenes. However, one of the most common grain-contaminating genus of fungi, Fusarium spp., is also capable of producing other toxic secondary metabolites - the so-called emerging mycotoxins such as fusaproliferin, beauvericin, enniatins, and moniliformin. So far, only limited data is available on these metabolites. This is not only due to their late recognition but especially the late understanding of their role as mycotoxins. This paper summarizes the existing data on the chemistry, analytical techniques, biosynthesis, production, toxicity, and occurrence data on fusaproliferin, beauvericin, enniatins, and moniliformin. Based on the available studies, attention should be paid to the studies on the distinct significance of these compounds in the human and animal food chains.
              Bookmark
              • Record: found
              • Abstract: found
              • Article: found
              Is Open Access

              dPCR: A Technology Review

              Digital Polymerase Chain Reaction (dPCR) is a novel method for the absolute quantification of target nucleic acids. Quantification by dPCR hinges on the fact that the random distribution of molecules in many partitions follows a Poisson distribution. Each partition acts as an individual PCR microreactor and partitions containing amplified target sequences are detected by fluorescence. The proportion of PCR-positive partitions suffices to determine the concentration of the target sequence without a need for calibration. Advances in microfluidics enabled the current revolution of digital quantification by providing efficient partitioning methods. In this review, we compare the fundamental concepts behind the quantification of nucleic acids by dPCR and quantitative real-time PCR (qPCR). We detail the underlying statistics of dPCR and explain how it defines its precision and performance metrics. We review the different microfluidic digital PCR formats, present their underlying physical principles, and analyze the technological evolution of dPCR platforms. We present the novel multiplexing strategies enabled by dPCR and examine how isothermal amplification could be an alternative to PCR in digital assays. Finally, we determine whether the theoretical advantages of dPCR over qPCR hold true by perusing studies that directly compare assays implemented with both methods.
                Bookmark

                Author and article information

                Journal
                Microorganisms
                Microorganisms
                microorganisms
                Microorganisms
                MDPI
                2076-2607
                27 August 2020
                September 2020
                : 8
                : 9
                : 1307
                Affiliations
                [1 ]Council for Agricultural Research and Economics, Research Centre for Genomics and Bioinformatics, I-29017 Fiorenzuola d’Arda PC, Italy; caterina.morcia@ 123456crea.gov.it (C.M.); giorgiotumino@ 123456hotmail.it (G.T.); giulia.gasparo@ 123456gmail.com (G.G.); caterinaceresoli@ 123456gmail.com (C.C.); chiarafattorini@ 123456alice.it (C.F.); roberta.ghizzoni@ 123456crea.gov.it (R.G.)
                [2 ]Barilla S.p.A., I-43122 Parma PR, Italy; paola.carnevali@ 123456barilla.com
                Author notes
                [* ]Correspondence: valeria.terzi@ 123456crea.gov.it ; Tel.: +39-0523-983758
                Author information
                https://orcid.org/0000-0002-6458-8021
                https://orcid.org/0000-0002-1165-0274
                https://orcid.org/0000-0002-8006-6805
                Article
                microorganisms-08-01307
                10.3390/microorganisms8091307
                7564955
                32867286
                3c38681f-8f81-4359-973e-3b6b09a0590a
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 31 July 2020
                : 26 August 2020
                Categories
                Communication

                molecular diagnostics,fusarium,chip digital pcr,qpcr
                molecular diagnostics, fusarium, chip digital pcr, qpcr

                Comments

                Comment on this article