Food Safety Masters Theses

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Browsing Food Safety Masters Theses by Subject "Polymerase chain reaction -- Diagnostic use."

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    Development of Real-Time PCR Method for Detection, Identification and Quantification of Five Different Fusarium Species
    (North Dakota State University, 2010) Goswami, Kakolie
    Fusarium head blight (FHB) is a fungal disease affecting cereal crops worldwide. FHB involves multiple Fusarium species that create food safety concerns by producing mycotoxins such as trichothecenes, zearalenone and moniliformin. Quantitative methods allowing rapid risk assessment of mycotoxigenic Fusarium species are needed to detect Fusarium spp. Real time quantitative PCR (qPCR) is a fast, sensitive and reliable alternative to conventional culture methods. An objective of the study was to develop TaqMan® based qPCR methods to detect, identify and quantify five different Fusarium species associated with FHB, namely F. graminearum, F culmorum, F avenceum, F poae and F sporotrichioides. The project was initiated with the selection of a protocol for Fusarium DNA extraction. Three different commercially available DNA extraction kits, including FastDNA ®, Qiagen® Blood & Tissue kit and Qiagen® DNeasy Plant kit were evaluated for speed, DNA yield and quality. The results showed that FastDNA ® kit gave the highest DNA yield in least time. TaqMan® Minor Groove Binder (MGB) probes for F culmorum, F poae and F sporotrichioides were developed using qPCR primers from a previous study. For F avenaceum and F. graminearum, TaqMan® based methods already available were used with modified conditions for improved detection. The qPCR methods were tested on a wheat system and the result was a qPCR system that rapidly identified and quantified Fusarium species associated with FHB.