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Quantitative PCR Cycler


The objective of this proposal is to purchase a multiuser quantitative PCR cycler to support the research program of the investigators of this grant working in food safety related issues and in a number of investigations of food borne pathogens. The PD's (Birgit Pruess)long-term goal is the investigation of global gene regulation by proteins of the flagellar system in Escherichia coli with the ultimate goal to reduce the number of bacteria on the meat surface. The Co-PDs (Charlene Wolf-Hall, Penelope Gibbs, Rubella Goswami) extend the research topics to other organisms, including different species of toxigenic fungi. They will use real-time PCR to identify and quantify different species of pathogenic bacteria and fungi. In summary, the PCR cycler will serve the confirmation of microarray data and the identification and quantification of pathogenic organisms on different crops and other food commodities. The cycler will be located in room 108 in Van Es Hall and will be available to all PDs. In addition, we will provide access for the equipment to other members of the College of Agriculture and the Great Plains Institute of Food Safety and for training of graduate researhers in molecular studies and techniques.

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NON-TECHNICAL SUMMARY: We need to purchase a quantitative PCR cycler to complement the microarray experiments of E. coli (Pruess Lab) and allow semi-high throughput studies in a number of other organisms. We have chosen a cycler that permits the processing of a large number of samples at the same time, while allowing measurements on five different channels (multiplex PCR). This will enable us to test several genes simultaneously under identical PCR conditions in real-time. The PD and several of the Co-PDs have previous experience with this equipment. We believe that the new cycler will facilitate and enhance our research, while intensifying our diagnostic development efforts. In addition to the PD and the Co-PDs, many other researchers will be provided access to the cycler. This means that the equipment will enhance many research projects across the NDSU campus.


APPROACH: PD: Birgit Pruess: We will isolate mRNA and determine the levels of transcript for selected genes, comparing between wild-type bacteria and several flagellar mutants. Primarily, this will serve the confirmation of microarray data. Co-PDs: The Co-PDs extend these studies to different organisms. For all projects, DNA will be isolated and used to identify and quantify pathogenic bacteria and fungi. Charlene Wolf-Hall: The ability to efficiently quantify Fusarium species and evaluate the chemotypes in wheat and barley is important due to the significant number of grain samples and the differences in the toxicity of these mycotoxins. The goals of this project are to develop and to apply a reliable quantitative PCR assay to screen grain samples to accurately assess the distribution of Fusarium spp. within the region. Penelope Gibbs: The Gibbs laboratory has collected and identified (by automated biochemical phenotypes) numerous suspect Enterobacter spp. isolated from clinical cases. The purpose of this proposal is to extend our identification of Enterobacter spp. in fecal samples from clinical cases or for routine screening submitted to the North Dakota Veterinary Diagnostic Laboratory. Rubella Goswami: The objective involves the use of quantitative PCR to develop a comprehensive multiplex method for detection and quantification of toxigenic fungal species (Fusarium, Alternaria, Aspergillus, Penicillium and Botrytis)in seeds and other propagules, infected plant material and harvested products of dry beans and pulses (peas and lentils).

Wolf-Hall, Charlene; Gibbs, Penelope; Pruess, Birgit
North Dakota State University
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