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An effective PCR-based diagnostic method for the detection of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) in wood samples from lodgepole pine

In: Nematology
Authors:
Isabel Leal
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Margaret Green
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Eric Allen
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Leland Humble
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Michael Rott
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Abstract

A molecular diagnostic method has been designed for the detection and identification of Bursaphelenchus xylophilus. Heat shock protein 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 PCR. As a control, primers Bm701F and Bm701R were designed which specifically amplify a 168 base pair fragment from B. mucronatus. After optimisation, B. xylophilus primers were shown to be highly sensitive and could easily detect 23 target copies, or less than one nematode. Species-specific detection of B. xylophilus was carried out directly from concentrated Baermann funnel extracts using wood samples from lodgepole pine (Pinus contorta var. latifolia) trees from British Columbia, Canada, containing an unknown nematode population, thus bypassing the need for culturing or recovering the nematode before analysis.

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