【摘要】 克隆并测定了中国柑桔黄龙病病原16SrDNA基因序列,经同源性比较,表明属于柑桔黄龙病菌(Liberobacterasiaticum)亚洲种中一个新株系(中国厦门株系)。依据获得的16SrDNA特异序列,建立了柑桔黄龙病病菌检测的实时荧光PCR方法,并应用此方法对柑桔样品进行了检测。结果显示,该检测方法具有快速、特异、敏感、重复性好等优点。该方法为柑桔黄龙病的检测,特别是早期诊断、检疫和病害的综合治理奠定了基础。更多还原

【Abstract】 The 16S rDNA gene of citrus Hunanglongbing pathogen(Liberobacter asiat icum ) in China was cloned and sequenced. The 16S rDNA of the Chinese pathogen w as 99.8% and 98.7% homogous to those of L.asiaticum and L.africanu. This means t hat the pathogen should be a new strain of L.asiaticum designated as China Xiame n strain. A real-time fluorescent PCR(RTF-PCR) method based on the 16S rDNA sequ ence was established to detect L.asiaticum. Its principle is that Taq Man probes relying on fluorescence resonance energy transfer (FRET) for quantitation are o ligonucleotides that contain a fluorescent dye, typically on the 5’ end, and a q uenching dye, typically located on the 3’ end. When irradiated, the excited fluo rescent dye transfers energy to the nearby quenching dye molecule without fluore scing. Taq Man probes are designed to hybridize to an internal region of a PCR p roduct. During PCR, when the polymerase replicates a template on which a Taq Ma n probe is bound, the 5’ exonuclease activity of the polymerase cleaves the prob e. This separates the fluorescent and quenching dyes and FRET no longer occurs. Fluorescence increases in each cycle, proportional to the rate of probe cleavage . Using RTF-PCR, 11 plant samples were detected . The described method has the a dvantage of high speed ,sensitivity, specifity and stable reproductivity. The me thod could identify the pathogen at early stage of infection in the diseased tre es without any symptom, it should be in favor of quarantine and control of citru s Hunanglongbing disease.更多还原