Digital PCR and Plant Studies

A special issue of Biology (ISSN 2079-7737). This special issue belongs to the section "Biotechnology".

Deadline for manuscript submissions: closed (31 December 2021) | Viewed by 9779

Special Issue Editors

Consiglio Per la Ricerca in Agricoltura e L’analisi Dell’economia Agraria-Centro di Ricerca Genomica e Bioinformatica (CREA-GB), Via San Protaso 302, 29017 Fiorenzuola d’Arda, PC, Italy
Interests: plant genetics; plant genetic resources; quality; molecular traceability; small grain cereals
Special Issues, Collections and Topics in MDPI journals
Grain Research Laboratory, Canadian Grain Commission, Winnipeg, MB R3C3G8, Canada
Interests: genetically modified organisms; detection; quantitative PCR; digital PCR; DNA extraction

Special Issue Information

Dear Colleagues,

Digital PCR (dPCR) technology emerged as a third-generation PCR technique. It is a breakthrough technology able to provide sensitive and absolute nucleic acid quantification. A unique feature of the technique is that of dividing the sample into numerous separate compartments and independent PCR amplification of each partition. Several instrumental platforms have been developed for this purpose, and different statistical approaches are available for analyzing the digital output data.

Up to now, qPCR has had innumerable applications in the field of plant science. Recently, dPCR has been proposed as a new technique applicable to the same targets of qPCR and also able to provide answers to additional, biological questions. In comparison with conventional end-point PCR and qPCR, dPCR has, in fact, a number of advantages. The biggest advantage is the capacity of dPCR for absolute quantification of a target without reference to a standard/calibration curve.

With this Special Issue, we aim to provide an international platform for a broad audience of researchers and stakeholders to present and discuss the role of digital PCR in the field of Plant Science.

Accordingly, we invite the submission of original research articles, scientific reports, communications, and review articles that address the applications and progresses of dPCR in the Genetically Modified Plants detection, phytopathogen diagnostics, transgenic lines characterization, plant species and varietal identification, gene expression and regulation studies, CNV (copy number variation) detection, genetic markers linked to agronomic and quality traits evaluation, soil, food and feed microbial species quantification. Furthermore, contributions focused on advances in the dPCR techniques and on the optimization and standardization of the data produced are welcome.

Dr. Valeria Terzi
Dr. Tigst Demeke
Guest Editors

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Keywords

  • digital PCR
  • detection
  • genetically modified
  • phytopathogen diagnosis
  • transgenic lines characterization
  • varietal identification
  • copy number variation
  • gene expression and regulation

Published Papers (4 papers)

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Research

16 pages, 3972 KiB  
Communication
A New Approach: Determining cyt b G143A Allele Frequency in Zymoseptoria tritici by Digital Droplet PCR
by Greta Battistini, Katia Gazzetti and Marina Collina
Biology 2022, 11(2), 240; https://0-doi-org.brum.beds.ac.uk/10.3390/biology11020240 - 04 Feb 2022
Viewed by 1842
Abstract
Z. tritici first appeared in Italy later than in northern-central European countries. QoIs fungicides currently play a role in STB control, used in combination with Demethylation Inhibitors (DMIs) or Succinate dehydrogenase Inhibitors (SDHIs). In this study, we set up a fast, sensitive, and [...] Read more.
Z. tritici first appeared in Italy later than in northern-central European countries. QoIs fungicides currently play a role in STB control, used in combination with Demethylation Inhibitors (DMIs) or Succinate dehydrogenase Inhibitors (SDHIs). In this study, we set up a fast, sensitive, and accurate ddPCR protocol in order to investigate the presence and frequency of G143A substitution, causing a reduction in strobilurins’ efficacy in Z. tritici. The best PCR conditions for the clear separation of positive and negative droplets were identified. The lowest wild-type and resistant alleles frequencies were accurately determined on samples consisting of mixed DNAs from monoconidial cultures of Z. tritici and were expressed as fractional abundance. The protocol was tested by determining the copy number and frequency of alleles on gDNA purified in three Italian Z. tritici field populations representative of different fungicide management strategies. For the first time, the determination of allele concentration and the frequency of a mutation involved in Z. tritici fungicide resistance was carried out by employing digital PCR. This new approach provides a diagnostic tool that is rapid and able to detect very low G143A substitution percentages, which is very useful for fungicide resistance detection at early stages, thus, informing field management strategies for contrasting STB disease. Full article
(This article belongs to the Special Issue Digital PCR and Plant Studies)
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10 pages, 979 KiB  
Article
Increasing the Efficiency of Canola and Soybean GMO Detection and Quantification Using Multiplex Droplet Digital PCR
by Tigst Demeke, Sung-Jong Lee and Monika Eng
Biology 2022, 11(2), 201; https://0-doi-org.brum.beds.ac.uk/10.3390/biology11020201 - 27 Jan 2022
Cited by 8 | Viewed by 3096
Abstract
The number of genetically modified (GM) events for canola, maize, and soybean has been steadily increasing. Real-time PCR is widely used for the detection and quantification of individual GM events. Digital PCR (dPCR) has also been used for absolute quantification of GM events. [...] Read more.
The number of genetically modified (GM) events for canola, maize, and soybean has been steadily increasing. Real-time PCR is widely used for the detection and quantification of individual GM events. Digital PCR (dPCR) has also been used for absolute quantification of GM events. A duplex dPCR assay consisting of one reference gene and one GM event has been carried out in most cases. The detection of more than one GM event in a single assay will increase the efficiency of dPCR. The feasibility of detection and quantification of two, three, and four GM canola and soybean events at the same time was investigated at 0.1%, 1%, and 5% levels using the QX200 Droplet Digital PCR (ddPCR) system. The reference gene assay was carried out on the same plate but in different wells. For some of the assays, optimization of the probe concentrations and labels was needed for successful ddPCR. Results close to the expected result were achieved for duplex, triplex, and tetraplex ddPCR assays for GM canola events. Similar ddPCR results were also achieved for some GM soybean events with some exceptions. Overall, absolute quantification of up to four GM events at the same time improves the efficiency of GM detection. Full article
(This article belongs to the Special Issue Digital PCR and Plant Studies)
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14 pages, 1319 KiB  
Article
A Digital PCR Assay to Quantify the Percentages of Hulled vs. Hulless Wheat in Flours and Flour-Based Products
by Caterina Morcia, Raffaella Bergami, Sonia Scaramagli, Chiara Delogu, Lorella Andreani, Paola Carnevali, Giorgio Tumino, Roberta Ghizzoni and Valeria Terzi
Biology 2021, 10(11), 1138; https://0-doi-org.brum.beds.ac.uk/10.3390/biology10111138 - 05 Nov 2021
Cited by 2 | Viewed by 1699
Abstract
Several food products, made from hulled wheats, are now offered by the market, ranging from grains and pasta to flour and bakery products. The possibility of verifying the authenticity of wheat species used at any point in the production chain is relevant, in [...] Read more.
Several food products, made from hulled wheats, are now offered by the market, ranging from grains and pasta to flour and bakery products. The possibility of verifying the authenticity of wheat species used at any point in the production chain is relevant, in defense of both producers and consumers. A chip digital PCR assay has been developed to detect and quantify percentages of hulless (i.e., common and durum wheat) and hulled (i.e., einkorn, emmer and spelt) wheats in grains, flours and food products. The assay has been designed on a polymorphism in the miRNA172 target site of the AP2-5 transcription factor localized on chromosome 5A and involved in wheat spike morphogenesis and grain threshability. The assay has been evaluated even in a real-time PCR system to assess its applicability and to compare the analytical costs between dPCR and real-time PCR approaches. Full article
(This article belongs to the Special Issue Digital PCR and Plant Studies)
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14 pages, 1504 KiB  
Article
Digital PCR for Genotype Quantification: A Case Study in a Pasta Production Chain
by Caterina Morcia, Valeria Terzi, Roberta Ghizzoni, Chiara Vaiuso, Chiara Delogu, Lorella Andreani, Andrea Venturini, Paola Carnevali, Pier Paolo Pompa and Giorgio Tumino
Biology 2021, 10(5), 419; https://0-doi-org.brum.beds.ac.uk/10.3390/biology10050419 - 09 May 2021
Cited by 3 | Viewed by 2218
Abstract
Digital polymerase chain reaction (dPCR) is a breakthrough technology based on the partitioning of the analytical sample and detection of individual end-point amplifications into the separate compartments. Among the numerous applications of this technology, its suitability in mutation detection is relevant and characterized [...] Read more.
Digital polymerase chain reaction (dPCR) is a breakthrough technology based on the partitioning of the analytical sample and detection of individual end-point amplifications into the separate compartments. Among the numerous applications of this technology, its suitability in mutation detection is relevant and characterized by unprecedented levels of precision. The actual applicability of this analytical technique to quantify the presence of a specific plant genotype, in both raw materials and transformed products, by exploiting a point polymorphism has been evaluated. As proof of concept, an Italian premium pasta production chain was considered and a dPCR assay based on a durum wheat target variety private point mutation was designed and evaluated in supply-chain samples. From the results obtained, the assay can be applied to confirm the presence of a target variety and to quantify it in raw materials and transformed products, such as commercial grain lots and pasta. The performance, costs, and applicability of the assay has been compared to analytical alternatives, namely simple sequence repeats (SSRs) and genotype-by-sequencing based on Diversity Arrays Technology sequencing (DArTseqTM). Full article
(This article belongs to the Special Issue Digital PCR and Plant Studies)
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