Application of conventional PCR and real-time PCR diagnostic methods for detection of the pinewood nematode, bursaphelenchus xylophilus, in wood samples from lodgepole pine

Abstract

Molecular diagnostic methods have been designed for the detection and identification of the pinewood nematode, Bursaphelenchus xylophilus. Heat shock protein 70 (Hsp 70) gene sequences from B. xylophilus and the closely related B. mucronatus, were compared and used to design primers Bx701F and Bx701R which amplify a 171 base pair fragment from B. xylophilus by polymerase chain reaction (PCR). As a control, primers Bm701F and Bm701R were designed which specifically amplify a 168 base pair fragment from B. mucronatus. After optimization, B. xylophilus primers were shown to be highly sensitive and could easily detect 23 target copies, or less than 1 nematode. In addition, a real-time PCR method was developed to detect and differentiate B. xylophilus from other wood-inhabiting nematode species. A primer set and a specific Taqman® fluorescent probe were designed to amplify target B. xylophilus Hsp 70 sequences. After optimization, this real-time PCR assay was shown to be highly specific and sensitive, detecting at least 5 pg of B. xylophilus genomic DNA, as well as DNA extracted from individual nematodes. The species-specific detection of B. xylophilus was carried out directly from concentrated Baermann funnel extracts using wood samples from lodgepole pine (Pinus contorta, Dougl. var. latifolia) trees. © 2008 Springer Science+Business Media B.V.