Optimization of Mitochondrial DNA-based Hybridization Assays to Diagnostics in Soil

Bradley C. Hyman, John J. Peloquin, Edward G. Platzer

Abstract


Nucleic acid hybridization among root-knot nematode mitochondrial DNAs can be used to identify several Meloidogyne species. Research was initiated to optimize mitochondrial DNA-based molecular diagnostics for the demanding environments likely to be encountered in field isolates. DNA hybridization using reconstituted DNA-soil mixtures revealed a loss of assay sensitivity ranging from 34% to 92% with four agronomic soils tested. This problem was alleviated by the addition of exogenously added DNA. Variation in nematode egg lysis procedures also affected hybridization efficiency, with NaOC1 treatment most effective at disrupting Meloidogyne eggs. These optimized conditions permit detection of mtDNA released from one to five Meloidogyne eggs using standard nucleic acid hybridization procedures. Key words: DNA hybridization, Meloidogyne spp., mitochondrial DNA, root-knot nematode.

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