Molecular diagnostics of the Mediterranean olive cyst nematode, Heterodera mediterranea Vovlas, Inserra & Stone, 1981 using conventional and real-time PCR

The Mediterranean olive cyst nematode, Heterodera mediterranea, is a pest of olive trees that is found in Spain, Italy, and Tunisia. In this paper, conventional and real-time PCR assays for the identification of H. mediterranea was developed. The hsp90 gene fragment was used to design a species-specific primer and a TaqMan probe. The specificity of the primer/probe set was tested in singleplex or multiplex reactions against Mediterranean olive cyst nematode and non-target nematodes. In multiplex real-time PCR experiments with the specific and universal primer/probe sets, fluorescent signals were simultaneously monitored for hsp90 and D3 of 28S rRNA target genes. The results of the experiments showed that the assays with species-specific primer/probe were sensitive enough to detect the DNA from 0.03 of a second-stage juvenile (J2) H. mediterranea in a reaction tube.