Duplex PCR based identification of Bursaphelenchus xylophilus (Steiner Buhrer, 1934) Nickle, 1970.

A duplex PCR combining species-specific primers and universal primers was developed to identify Bursaphelenchus xylophilus. The design of the specific primers was based on differences in the Internal Transcribed Spacer sequences Of rRNA between Bursaphelenchus xylophilus and other Bursaphelenchus species. The method was validated Using 24 populations of different Bursaphelenchus spp. and four other nematode species. The duplex PCR generated a specific amplicon of 580 bp for all populations of B. xylophilus and a control amplicon, obtained after amplification of D2-D3 expansion fragments of the 28S rRNA gene, of about 770 bp. All non B. xylophilus samples generated only the 770 bp fragment. Compared to other molecular methods, duplex PCR is more rapid, reliable and cheaper. It allows the detection of single specimen of B. xylophilus in a sample.