Detection And Identification Of Bursaphelenchus Xylophilus With Real-time Fluorogenic Polymerase Chain Reaction

Pine wood nematode (Bursaphelenchus xylophilus), one of the most important quarantine organisms, is the great threat to the pine forestry in Asia. Morphologial identification of this nematode is time-consuming and difficulty due to its morphological similarity with Bursaphelenchus mucronatus. Many attempts to develop detecting and diagnosing methods for Bursaphelenchus xylophilus have been conducted. Protein-based methods including enzyme characteristics and ELISA, DNA-based methods such as restriction fragment length profiles and random amplified polymorphic DNA of the internal transcribed spacer region of ribosomal genes (ITS), and primers targeting repeated DNA sequence element as DNA fingerprinting probe have been successfully used to detect B. xylophilus and distinguish it from B. mucronatus. However, those were very time consuming and instable. The faster and practicable method for detecting and diagnosis of B. xylophilus for the quadrantine purpose is to be developed. A real-time quantitative polymerase chain reaction method for diagnosis of Bursaphelenchus xylophilus was developed in present study. The probes and primers for B. xylophilus were designed on the basis of the align sequence of the amplified internal transcribed spacer region of ribosomal DNA of Bursaphelenchus spp. by using the CLUSTSAL 1.81 of the sequence software. The probe carried two fluorescent dyes, a reporter of 6-carboxy-fluorescein (FAM) at the 5'end and a quencher of 6-carboxy-tetramethhylrhodamine (TAMRA) at the 3'end. The concentrations of primers and Mg2+ were optimized and the highest reporter fluorescence (?Rn) value was obtained for forward / reverse primer at 400nM / 400nM, and the lowest threshold cycle (CT) was obtained for Mg2+ at 3 mM with extend temperature at 60 °C. This profile is sensitive and specific to detect a single nematode of B. xylophilus, but does not react with other related species of Bursaphelenchus and other nematode. Comparison with other diagnosis method for B. xylophilus, the real-time PCR method developed in present study is time-saving (only 30min to 1.5 hr), and more stable and precise.