枣疯病植原体 TaqMan 探针实时荧光定量 PCR 检测方法的建立

摘要

A set of primers and TaqMan probe specific for Jujube witches’broom phytoplasma were designed ac-cording to the conserved region of 16S rDNA gene,and the recombinant plasmid was constructed as a standard control.A TaqMan real-time fluorescent PCR assay for quantitative detection of the pathogen was established and optimized.The evaluation assay indicated that a good linear correlation was demonstrated in the standard curve for the real-time fluorescent PCR assay,with the correlation coefficient (r 2 )of 0.998.The method was specific to jujube witches’broom phytoplasma,and it can detect 60 copies/μL plasmid DNA.It not only provides a sensi-tive,specific and reproducible method for detection of jujube witches’broom phytoplasma,but also lays the foun-dation for the grading system of the pathogen at quantitative level.%根据枣疯病植原体16S rDNA 基因保守区域设计、合成特异性引物和 TaqMan 探针，以构建的重组质粒作为阳性标准品，建立并优化了对枣疯病植原体的 TaqMan 实时荧光定量 PCR 检测方法。对优化后的方法进行灵敏度、特异性及稳定性评价，制作了标准曲线。结果显示，制作的标准曲线有极好的线性关系，相关系数（r2）达到0．998，建立的实时荧光定量 PCR 检测方法能够特异性地检测枣疯病植原体，能检测到60拷贝的质粒 DNA。本研究建立的实时荧光定量 PCR 检测方法灵敏度、特异性、重复性好，不仅能够实现对枣疯病植原体的快速检测，而且为实现从病原定量水平上对枣疯病病情分级奠定了基础。