SCAR Marker and Detection Technique of Bursaphelenchus xylophilus

OPM05-M2100,the specific RAPD fragment of Bursaphelenchus xylophilus,was collected from agarose gels and purified.Then,the purified fragment was inserted into the pGEMR -T Vector that was transformed into Escherichia.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 M05 F2/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 M05 F2/R1.But eight isolates of B. mucronatus,one B.hofmanni,one A.macronucleatus and one Seinura sp.had no any fragments.Assay M05 F2/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.