聚合酶链反应-高分辨率熔解曲线技术在下呼吸道细菌鉴定中的应用

Application of polymerase chain reaction-high-resolution melt technology for bacterial identification in samples collected from lower respiratory tract

为探讨聚合酶链反应-高分辨率熔解曲线(PCR-HRM)技术在检测呼吸道菌群中的应用,本研究用支气管镜从慢性阻塞性肺病患者下呼吸道采集分泌液,提取细菌总DNA,以16S通用引物27F/1492R扩增16S rDNA全长,PCR产物经电泳、纯化后连接到pGEMT-Easy载体上,构建16SrDNA克隆文库。然后以16S rDNA V3区通用引物338F/518R扩增克隆文库,V3区PCR-HRM分析在罗氏LightCycler 480实时荧光定量PCR系统中进行,采用HRM基因扫描软件分析数据。根据不同HRM图型,挑选克隆株测序并鉴定菌株。结果显示,PCR-HRM技术可灵敏区分下呼吸道不同细菌16SrDNA V3区,根据HRM图谱可极大减少克隆菌株测序样本,提示PCR-HRM技术可作为一种快速、高通量、敏感、经济的菌群多样性检测方法。

To study the role of polymerase chain reaction-high-resolution melt （PCR-HRM） in the detection of mixed bacteria from respiratory tract, the fluid from the lower respiratory tract of patients with chronic obstructive pulmonary disease （COPD） was collected through bronchoscope. Total DNA was extracted and the full-length 16S rDNA was amplified by PCR with 16S primers 27F/1492R. The PCR products were purified and connected to pGEMT-Easy to build a 16S rDNA clone library. The cloned library was amplified by 338F/518R （16S rDNA-V3 region universal primer）. PCR-HRM in V3 region was performed on Roche LightCycler 480 real-time fluorescence quantitative PCR system. The data were analyzed by HRM GeneScan analysis software. Strains with different HRM curve were selected for sequencing and strain identification. The results showed that PCR-HRM could be used to distinguish the 16S rDNA-V3 regions from different bacteria of the lower respiratory tract. PCR-HRM technology can be used as a rapid, high-throughput, sensitive and economic detection method for bacterial diversity in clinical samples.