Research On A Rapid PCR Detection Method For Pinewood Nematode (Bursaphelenchus Xylophilus)

Pinewilt disease, which is caused by pine wood nematode (Bursaphelenchus xylophilus), is a destructive quarantine disease for Pinus spp. worldwide because of its high mortality rate, rapid spreading and difficulty to control. Due to lack of high effective insecticides and eradication method, many countries taking action mainly rely on enhancement of quarantine regulations in non-quarantine area to prevent the introduction and establishment of the diseases, therefore, it is necessary to find an accurate, rapid and robust detection method which can be carried through the detection and early diagnosis for a regulatory response and effective management of pinewilt through the detection and early diagnosis. The traditional detection methods such as morphological diagnosis and biochemical detection for pinewilt, are limited due to its relatively lower sensitivity and specificity which impede them from early diagnosis, while molecular detection technology based on nucleic acid, especially the polymerase chain reaction (PCR) technology, is characteristic for its rapidness, sensitivity and specificity which can fulfill the requirements of plant quarantine in modern society.Research Purpose: A systematic research was carried out via molecular detection method for B. xylophilus in order to establish an accurate, rapid and robust detection system and serve for the non-quarantine area and disease-monitoring system construction.Research Content: Compared the modified CTAB method, Kit method and Soaking-filtration method for extracting DNA from B. xylophilus and B.mucroratus; designed primers with professional designing software Primer Premier 5.0; determined the specificity of the detection method with the templates including B.mucroratus, Caenorhabd itis spp, B. fraudulentus and several other saprophytic nematodes; determine the detection sensitivity with serial dilution samples from B. xylophilus genome DNA; determined the robustness of optimum protocol with different thermocyclers and different temperature controlling module; determined the efficiency through sequencing the PCR products after connected with vector pMD18-T and transformed into Escherichia coli JM109 with CaCl2 method; established the harmless positive control system with recombinant plasmid and transformed E. coli; developed he detection dry kit which can stored at room temperature using a freeze-dry-solidifying method dealing with a premix of all the reagents in a PCR reaction and an extra reagent for stabilization (MS); detected the field samples collected from Tianjin, Guangxi, Hunan, Chongqing and Sichuan with optimum PCR system; performed the primary study on the real-time fluorescent PCR (RTi-PCR) detection method for B. xylophilus with BIO-RAD iCycler iQ.Research Results:①For the DNA extraction method, the soaking-filtration method is the best for B. xylophilus DNA extraction which can exclude the interference from the plant pigments, protein and polysaccharide material. And the method could extract nematode DNA as templet for PCR reaction. The minimum test limit was one nematode in 1.5 gramme wood scraps. A rapid DNA extraction method from pine wood scraps was established.②For PCR detection method of B. xylophilus, an accurate, rapid, sensitive, robust and secure PCR detection system was established based on the primer pair cqubs01/cquba01 selected from the specific region of B. xylophilus DNA sequence. The detection limitation was 100pg/25uL.③For B.mucroratus, a selected primer pair cqubms01/cqubma01 was integrated with the B. xylophilus primer pair cqubs01/cquba01 and a duplex PCR system was established for simultaneous detection of both insects.④A SYBR Green I RTi-PCR detection system for B. xylophilus was established with the primer pair cqubs01/cquba01which had a better performance in sensitivity and rapidness. The detection limitation was 100fg/25uL using PCR detection method.⑤The total DNA extracted with soaking-filtration method was not suitable to serve as positive control due to its unstable DNA content and short storage period, while the recombinant plasmid used as harmless positive control in the detection system was quite robust in repeated experiments and could serve as standard samples in RTi-PCR system.⑥After MS added in the premix, it could stabilize the biological molecules e.g. enzyme and dNTPs and the whole mixture could be stored at room temperature for at least 0.5 year after vacant-freeze-drying treatment.Conclusion: Two qualitative and one quantitative PCR method for rapid, robust and reliable detection of B. xylophilus were established with improved DNA extraction method and recombinant positive control was constructed. From sample treatment to the final results, the whole detection procedure could be finished within 6h, which fulfilled the requirements in practical detection. This method system could be useful for custom and quarantine department and early diagnosis.