巢式-荧光定量PCR技术快速检测生殖支原体

Nested-qPCR for Rapid and Quantitative Identification of Mycoplasma Genitalium

目的建立生殖支原体巢式-荧光定量PCR快速检测技术。方法针对生殖支原体MgPa基因的保守片段(1414~1561bp),利用Primer express3.0设计引物及探针,巢式PCR扩增临床样本,纯化后克隆到载体,制备阳性标准品。优化PCR反应条件,研究其特异性、灵敏性和重现性。结果构建了含MgPa基因的重组质粒,以不同浓度的重组质粒制作标准曲线,在101~109拷贝数之间有较好的线性关系。该方法检测阳性率高达15.4%,能特异性检测生殖支原体,而人型支原体、解脲脲原体、沙眼衣原体等检测为阴性;灵敏度高,可检测到10个拷贝数;3组重复试验的变异系数均小于3%。结论本研究建立了生殖支原体的快速检测技术,具有检测阳性率高、特异性强、灵敏度高、重现性好等特点。

Objective To develop a nested-qPCR method for rapid and quantitative identification of Mycoplasma genitalium (Mg).Methods The primers and probe were designed using Primer Express 3.0 software.Conserved fragment of MgPa gene(1 414~1 561bp)was amplified by nested PCR and cloned into a vector as the positive standard.The conditions of PCR reaction were optimized to study its specificity,sensitivity and reproducibility.Three replicates were tested at each concentration with the same batch of reagents to assess assay reproducibility.Results The positive rate of the method was up to 15.4%,and Mycoplasma genitalium could be detected specifically.The sensitivity was high,10 copies could be detected,and the variation coefficient of three groups in repetitive tests was less than 3%.Conclusion QPCR method of Mycoplasma genitalium has high positive detection rate,is sensitive,specific and reproducible.