Molecular diagnostics of the hop cyst nematode, Heterodera humuli Filipjev, 1934, using real-time PCR

Summary The hop cyst nematode, Heterodera humuli , is a pest of hop with the potential to substantially limit yields worldwide. To enable molecular-based diagnostics, a real-time PCR assay for the identification of H. humuli was designed. Polymorphism in the COIII gene fragments was used to design species-specific primers and a TaqMan probe. The specificity of the primer and probe set was tested against different populations of H. humuli and non-target cyst nematodes in multiplex reactions. In multiplex real-time PCR experiments with specific and universal primer and probe sets, fluorescent signals were simultaneously monitored for COIII and D3 of 28S rRNA target genes. Assays with species-specific primers and probe were able to detect DNA extracted from 0.0016 of a H. humuli second-stage juvenile per reaction while showing no reaction with non-target species.