Towards specific diagnosis of plant-parasitic nematodes using DNA oligonucleotide microarray technology: A case study with the quarantine species Meloidogyne chitwoodi

In order to investigate the feasibility of a microarray-based method for diagnostics of plant-parasitic nematodes, we have developed a DNA oligonucleotide microarray to detect the nematode species Meloidogyne chitwoodi, which is listed as a quarantine organism in Europe. Oligonucleotide capture probes were designed from nematode SCAR and satellite DNA sequences and spotted onto epoxy-coated glass slides. PCR products were generated using specific primers, labeled with Cyanine 3 or Cyanine 5 fluorescent dyes, and hybridized overnight to the microarray. This methodology allowed the specific detection of M. chitwoodi DNA in pure and mixed samples (i.e. when M. chitwoodi DNA was mixed with DNA from a congeneric nematode species). Simultaneous hybridization of the microarray with two amplified targets labeled with different dyes proved to be efficient, without any competition between the targets. These results illustrate a significant step forward in the development of the DNA chip technology for nematode detection, and constitute to our knowledge the first report of production and use of oligonucleotide microarrays for the detection of plant-parasitic nematodes, using the quarantine species M. chitwoodi as a test organism.