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Research Project:
Development and Evaluation of Real-Time Pcr Primers and Probes Targeting Twelve Fungal and Bacterial Pathogens of POTATO...QUANTIFICATION
Location:
Project Number: 500-42-001-55
Project Type:
Grant
Start Date: Apr 06, 2004
End Date: Apr 05, 2005
Objective:
Sequence six single-copy genes from twelve potato tuber pathogens, from various geographic locations; sequence the same six genes from closely and distantly related organisms; design primers from pathogen-specific real-time PCR primers and probes from sequenced single-copy genes; and test for uniqueness of the gene copy in the target organism; test primers and probes on inoculated tubers, field derived samples and DNA mixtures.
Approach:
Sequence a number of single-copy genes from isolate collections; use these sequences to design primers and probes conserved among isolates within the same species; amplify various races, varieties, subspecies or strains of each of the potato pathogens; carry out tests to ascertain the selectivity of the developed primers, as well as the number of copies of the selected genes in the organism; perform inoculations with the various pathogens under experimental conditions, in the laboratory and the greenhouse; accomplish quantification of gene copy numbers with Southern blots following the digestion of the DNA of each organism with a number of restriction enzymes; transfer Agarose gels of the digested DNA to blotting membranes and hybridize with the developed primers and probes, turned into chemiluminescing probes.
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