dc.description.abstract
The overall objective of this thesis was the development of DNA-based techniques for the rapid analysis of Fusarium species and aflatoxins in maize. The focus was laid on the mycotoxin classes trichothecenes and fumonisins as the most important mycotoxins produced by Fusarium sp. as well as aflatoxin B1, produced mainly by Aspergillus sp., which is considered to be the most potent natural carcinogen known. Although tests for such analytes are available, many of these methods are time-consuming, cost-intensive and difficult to transfer into a more field-applicable approach. The first part of the thesis addresses the development of a multiplex real-time PCR to quantify Fusarium DNA of trichothecene and fumonisin producing strains in maize. The plant pathogenic fungus Fusarium causes considerable economic impact worldwide. The kernel size and weight are usually reduced but even more important are the numerous toxic metabolites produced by these fungi during the colonization of the plant. These mycotoxins have been related to toxic effects upon ingestion by humans and animals. Mycotoxin concentrations in infected plants can vary significantly between maize cultivars and are usually higher in susceptible plants than in more resistant cultivars. Hence, the determination of the resistance of new maize varieties is of high importance and usually combines anaylsis of mycotoxins by enzyme linked immunosorbant assay (ELISA), high performance liquid chromatography (HPLC) coupled either to ultra-violet (UV) and/or mass spectrometry (MS) detection methods and the visual scoring of disease symptoms. However, these methods are time-consuming, cost-intensive (especially HPLC-MS) and only indirect, because they provide no information about the biomass of a fungus in the sample. A new method to gain direct information about the DNA biomass of a fungus is the quantitative polymerase chain reaction (qPCR) technique, which is based on the quantification of the amount of organism specific DNA. Hence, an infection can be detected before any symptoms are visible. Besides the evaluation of the resistance of varieties, qPCR can be applied for Fusarium monitoring projects and as a screening method for food and feed contamination. In this study a sensitive quantification method for trichothecene and fumonisin producing Fusarium species in maize has been developed. This method enables the high-throughput screening of a large number of samples for Fusarium infection in relatively short time due to simultaneous quantification of the mycotoxin-related genes tri5 (encoding for the fungal trichodiene synthase) and fum1 (encoding for a polyketide synthase), responsible for the production of trichothecenes and fumonisins, respectively. The newly developed multiplex method was applied to 24 maize field samples collected in Austria. Results obtained with the triplex assay were compared to the three singleplex qPCR runs to ensure that no loss of sensitivity occurs by using the simplified multiplex method. The new assay was found to be specific for either fumonisin or trichothecene producing Fusarium species. A limit of quantification (LOQ) of 0.32 pg DNA/µl for both Fusarium strains was found. Considering a genome size of 41.7 Mb for the fumonisin producing Fusarium strain F. verticillioides and a genome size of 36.2 Mb for the trichothecene producing Fusarium strain F. graminearium this represents approximately seven or eight genome equivalents, respectively. All samples were further analyzed for the trichothecenes deoxynivalenol (DON), DON-3-glucoside (D3G), nivalenol (NIV), 3-acetyl-DON (3-ADON), T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS), and neosolaniol (NEO) and the fumonisins fumonisin B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3) by liquid chromatography tandem mass spectrometry (LC-MS/MS) and compared with the qPCR results. This assay is the first report of the use of a multiplex qPCR for the quantification of trichothecene and fumonisin producing Fusarium species and was published in Analytical Methods (Appendix publication #1). Aflatoxins represent another important class of mycotoxins. They are mainly produced by the fungal strains Aspergillus flavus and Aspergillus parasiticus. Aflatoxin B1 (AFB1) is the most prevalent and potent one in the class of aflatoxins. It possesses high toxicity and was classified as carcinogenic to humans by the International Agency for Research on Cancer (IARC). Regulatory limits have been introduced for food and feed safety reasons in many countries and range from 1 µg/kg to 20 µg/kg. Therefore, rapid, sensitive and inexpensive analytical techniques are essential to detect and quantify AFB1. Procedures based on chromatographic methods combined with MS and rapid screening approaches with immunoassays have been developed. However, these methods show some major drawbacks such as the requirement of skilled personnel, expensive equipment and sample pre-treatment, and the use of antibodies. Due to the limitations of antibodies used in immunoassays such as thermal instability, laborious and expensive production, aptamer-based analytical methods have been developed offering a promising alternative to antibodies. Aptamers with high affinity to AFB1 have been developed in part two of this study. They have been selected using an iterative selection procedure named Systematic Evolution of Ligands by EXponential enrichment (SELEX) for their ability to bind to AFB1 with high affinity and specificity. Sequences were obtained after four rounds of in vitro selection and were furthermore screened for their ability to bind AFB1. Five unique sequences were obtained and additionally characterized. The dissociation constant for each aptamer was determined by a fluorescence binding assay and was found to be 1.30 µM ± 0.14 µM for aptamer 1 (named Lib5_1e), 5.70 µM ± 0.54 µM for aptamer 2 (named Lib5_6e), 2.34 µM ± 0.13 µM for aptamer 3 (named Lib5_7e), 2.56 µM ± 0.13 µM for aptamer 4 (named Lib5_9e), and 4.95 µM ± 0.95 µM for aptamer 5 (named Lib5_10e). For AFB1 biosensor development approaches a DNA fragment designed by Neoventures Biotechnology Inc. (Canada) in 2009 was used. Until now, a few aptasensors, based on this aptamer, for the rapid detection of AFB1 have been developed. These assays are currently based on an indirect target detection format where a signal is produced if no target is present and vice versa. This study reports a new direct detection format for the target molecule AFB1 with a nucleic acid lateral flow test strip. The idea claims AFB1 induced unwinding of the aptamer-s distinctive structure exposing a binding site for a complementary signaling DNA probe enabling the hybridization of the probe with the aptamer. Different incubation temperatures responsible for hybridization/dehybridization have been evaluated (room temperature, 37 °C, and 40 °C). The test results revealed that a temperature of 37 °C worked best. The developed aptasensor model demonstrates the potential of the structure switching ability of aptamers in terms of direct target detection. To the best of our knowledge, a dipstick assay based on an aptamer enabling an AFB1 direct detection format has not yet been reported. A manuscript has been submitted to the journal Analytical Methods (Appendix publication #2). In conclusion, within this thesis a multiplex qPCR method for the rapid screening of maize samples for all Fusarium species producing the most prevalent mycotoxins in maize, trichothecenes and fumonisins, have been developed. Furthermore, a rapid and simple aptamer dipstick assay using a direct detection format for AFB1 has been designed. However, further optimization to increase the sensitivity and to speed-up the testing procedure is required.
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