结核分枝杆菌自身淬灭探针荧光定量PCR检测方法的建立

Detection of Mycobacterium tuberculosis by fluorescent quantitative PCR using self-quenched probe

目的利用自身淬灭探针技术建立敏感、特异、快速、价廉且能广泛应用的结核分枝杆菌荧光定量PCR检测方法。方法构建重组质粒PET-32a(+)-16s rRNA作为标准品,自行设计自身淬灭探针,建立、优化定量PCR体系,并进行方法学评价及临床应用。结果所建立方法的线性检测范围为102～109copies/μL,灵敏度为1copy/μL,特异性为100%。高拷贝样品的天间CV为2.65%、批内CV为1.86%,低拷贝样品的天间CV为2.12%、批内CV为0.78%。该方法比培养方法更快速、灵敏。结论以自身淬灭探针技术为平台的结核分枝杆菌荧光定量PCR方法灵敏度高、特异性好,对结核病的早期快速诊断有较高价值。

Ahn To establish a novel real-time fluorescence PCR method to detect M.tuberculosis with high sensitivity, specificity,stability and lower price using the self-quenched primer. Methods The 16s rRNA gene of Mycobacterium tuberculosis was cloned into the vector PET-32a （＋）.The recombinant vector was used as standard template. The self quenched primer was designed according to the 16s rRNA gene sequence.The optimization of the PCR reaction system and method was evaluated. Results Restriction maps showed that the structure of recombinant plasmid PET-32a （＋）-16s rRNA was the same as expected. Methodology analysis suggested that self-quenched probe was successful established with high sensitivity,specificity and accuracy. Conclusion The new real time fluorescence PCR is sensitive and specific, and is valuable for genetic diagnosis of Mycobacterium tuberculosis infection.