Study On The Molecular Detection For Flavescence Dorée Phytoplasma

Investigated and sampled the Grape Yellow Phytoplasma in the main grape growing areas of Xinjiang during 2007～2008.And did not found Grape Yellow Phytoplasma in 191 samples have been collected by PCR.Two pairs of primers(R16mF2/ R16mR2,R16F2/ R16R2)which reported in the literatures have been used to detecte Flavescence dorée phytoplasma by regular PCR and Nested-PCR;Compared with the detection sensitivity of tow methods,the results showed that:The minimal amount of DNA was 20ng/μL when used R16mF2/R16mR2 primers.Useing the production of regular PCR as a template for the Nested-PCR,the reliability of detection has greatly increased,meanwhile sensitivity improved 10000 times higher than regular PCR;At the same time,PCR reaction conditions have been optimized,and created the optimal conditions for PCR reaction.The time of Nested-PCR detection about 5 hours.Genomic DNA of Flavescence dorée phytoplasma were amplified by the polymerase chain reaction (PCR)with the universal primers P1/P7.The production were cloned and sequenced.Sequences were aligned with 16S rDNA sequence in other Phytoplasmas in GeneBank.Phylogenetic dendrogram was constructed by DNASTAR software.The tested Strains were isolated and classified.Designed specific primers FDF09 and FDR09 and TaqMan probe FDX-Probe for Flavescence dorée phytoplasma based on differences of 16SrDNA sequences between Flavescence dorée phytoplasma and other Phytoplasmas;Established the Real-time fluorescence PCR method by optimizing reaction system and condition;Results of specific detection showed that:The probe was highly specific,with the exception of Flavescence dorée phytoplasma fluorescence signal enhancement,the other phytoplasma and bacteria were not fluorescent signal enhancement;The method sensitivity which was about 0.56fg/reaction;Reproducibility experiment show that this detection method haved highly duplication and stability;The time of detection about 1 hours;And did not found Flavescence dorée Phytoplasma in 191 samples have been collected by Real-time fluorescence PCR.Those primers and probe haved applicated the patent of invention in China,Application numbers were 200910113240.1.