新型荧光定量PCR检测孕妇生殖道解脲脲原体方法的建立

目的 探讨建立自身淬灭探针荧光定量PCR方法检测解脲脲屏体（Uu）的可行性及价值。方法 根据Uu UreA基因序列设计合成引物和自身淬灭引物，采用基因克隆技术，将UreA基因片段插入到载体pMD18-T，以此作为标准品。优化PCR条件，建立以自身淬灭引物技术为平台的实时荧光定量聚合酶链反应（FQ-PCR）方法，并对所建立的方法进行初步方法学评价。结果 成功构建了重组质粒pMD18-T-UreA；建立了以自身淬灭探针技术为基础的实时荧光定量PCR方法，其线性检测范围为10^1～10^9copies／μl；灵敏度为10copies／μl；重复性实验批内CV为2．35％，批间CV为3．68％，天间CV为4．92％；特异性好；通过初步临床应用发现，所建立的FQ-PCR方法与经典的培养方法相比，不仅能实时监测反应进程、绝对定量和快速检测，且检出率显著高于培养方法。结论自身淬灭探针荧光定量PCR方法检测Uu具有快捷、敏感、廉价等优点，可在临床诊断中应用。

Development of a new fluorescent quantitative PCR detective method of ureaplasma urealyticum

Objective To establish a quickly real-time fluorescence PCR method to detect ureaplasma urealyticum with high sensitivity and lower price by the self-quenched primer. Methods The self-quenched probe and primers were dew signed according to the Urea gene sequence of Ureaplasma Urealyticum. The targeted sequence was cloned into the vector pMD-18-T. The recombinant vector was used as standard template. After PCR reaction system optimization and method evaluation, the new real time fluorescence PCR method was used to detect clinic examples. Results Recombinant plasmid pMD-18-T-UreA was cloned as expected. Methodology analysis showed that self-quenched probe was successfully established. After applied in clinical experiments, this new fluorescent quantitative method was found having more advantages than classic cultural method. Conclusion The new method is better for positive rate than the culture method and can be used in clinical diagnosis.