α变形菌纲磁螺菌属TaqMan探针实时荧光定量PCR快速检测方法的建立

目的建立一种快速有效检测环境中q变形菌纲磁螺菌属细菌的方法。方法本研究以α变形菌纲磁螺菌属保守且特异的16SrRNA片段为靶序列，化学合成基因序列，制备重组质粒作为磁螺菌属检测的标准品；用实时荧光定量聚合酶链反应（FQ—PCR）技术，针对目的片段基因设计特异性引物和TaqMan荧光探针，进行实时荧光定量PCR检测，运用统计学方法评价该方法的特异性、敏感性及重复性。结果本实验成功构建了质粒PUC19-QC，引物、探针特异性良好，标准曲线在5．10×10^1～5．10×10^7拷贝数之间具有较好的线性关系，相关系数R2=0．999。该法最低可检测到5．10x10个DNA拷贝数，灵敏度明显高于普通PCR；重复性试验表明．荧光定量PCR检测结果Ct值波动范围较小，α值的标准差均小于0．23，表明该法重复性好，可靠性高。析因方差分析表明不同稀释度之间的ct值差异有统计学意义（F=3125．305，P〈0．001），三个批次的再现性良好，差异无统计学意义（F=0．057,P=0．945），而浓度分组与时间之间也无交叉效应（F=0．533，P=0．873）。结论本实验所建立的TaqMan探针实时荧光定量PCR检测技术，能够快速有效地检测仅变形菌纲磁螺菌属细菌，且检测结果稳定，受外界条件如时间、气温等影响小。

Rapid detection of Magnetospirillum with TaqMan probe by real time fluorescent quantitative PCR

Objective To effectively investigate the diversity and distribution of Magnetospirillum in environment. Methods The recombinant plasmid PUC19-QC with special 16S rRNA gene sequences of A Magnetospirillum was chemical synthesized. Specific primers and TaqMan probe were developed for target sequences. Standard curve was generated by amplifying 10-fold dilutions of the PUC19-QC by real-time fluorescence quantitative polymerase chain reaction （FQ-PCR） in triplicate. Farther more,FQ-qPCR was performed to evaluate the specificity, sensitivity, applicability of primers and probe. Statistical analysis was performed to evaluate the repeatability and stability. Results We successfully plot a linear relevant standard curve （Y=-3.207LOG（X）＋35.93） which could detect as low as 5.1×10^1 copies compare to 5.1×10^3 for ordinary PCR. Primers and TaqMan probe could specifically test Magnetospirillum with no cross reaction with other germs. Repeatability and stability tests under the same conditions and different time obtained a 0.23 standard deviation.Statistical analysis performed with ANOVA for the Repeated measure.There was significant difference （F=3 125.305,P〈0.001） when copy number change,no significant difference when time change （F=0.057,P=0.945）.There was no cross effect between copy number and time（F=0.533,P=0.873）. Conclusion These new primers and probe provide a viable method to detect Magnetospirillum, which will improve our understanding of the diversity, distribution, and ecological role of Magnetospirillum.