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Multiple displacement amplification of DNA for ITS-RFLP analysis of individual juveniles of Bursaphelenchus

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image of Nematology
For more content, see Nematologica.

Differentiation of the plant-pathogenic pinewood nematode, Bursaphelenchus xylophilus, from non-pathogenic Bursaphelenchus species is difficult because of high morphological similarities among closely related species. In recent years, ITS-RFLP analysis has become a useful tool for Bursaphelenchus species identification. Analysis of individual nematodes is hampered by the fact that sufficient template DNA for ITS-PCR cannot be extracted reliably. We have employed a whole genome amplification method, termed multiple displacement amplification (MDA), to 26 DNA extracts from individual juveniles to increase the amount of template DNA. Preamplification of the whole genomic DNA by MDA provided sufficient amounts of PCR product for ITS-RFLP analysis with 12 out of 20 B. xylophilus, two B. mucronatus, two B. fraudulentus and two B. eggersi samples tested. The introduction of MDA to ITS-RFLP analysis of nematodes may improve the reliability of diagnostic testing for limited samples and permit verification of analytical results.


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