Abstract:

OPM₀₅-M₂₁₀₀, the specific RAPD fragment of B. xylophilus , was collected from agarose gels and purified. Then, the purified fragment was inserted into the pGEM^®-T Vector that was transformed into E. coli and cloned and sequenced. Based on the sequence of RAPD marker, the sequences characterized amplified region (SCAR) primers were designed by the aid of the software Oligo5.0. The forward primer is M₀₅F₂(5'-CGGGT CATGG CTGGA GGTAT CGT-3'),and the backward primer is M₀₅R₁(5'-TGGCT CAATG GCAAA TCCTT CGTA-3'). The specific fragment (OPM₀₅-M₂₁₀₀) was successfully converted to SCAR marker (SCAR-M₀₅-X₆₀₀) by using M₀₅F₂/R₁,which was the specific markers of B. xylophilus. Then, the DNA of 92 isolates of Bursaphelenchus, B. mucronatus, B. hofmanni, Aphelenchoides macronucleatus and Seinura sp. which were isolated from dead pines, were marked, and the DNA of a single nematode extracted with a simple method was detected using this set of specific primers. The results indicated that the PCR product of all 81 isolates of B. xylophilus was a clear and bright fragment about 600 bp with M₀₅F₂/R₁. But eight isolates of B. mucronatus, one B. hofmanni, one A. macronucleatus and one Seinura sp. had no any fragments. Assay M₀₅F₂/R₁ also successfully detected single pinewood nematode. Therefore, the specific pairwises would be used for constructing identification kits of B. xylophilus, implementing the aim of quick detection, and achieving the purpose of identify juvenile successfully.

Key words: Bursaphelenchus xylophilus, SCAR marker, specific fragment, detection technique