CAJ | 학술논문

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16S rDNA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16S rDNA 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。
위탐토취합매련반응‐고분변솔용해곡선（PCR‐HRM）기술재검측호흡도균군중적응용，본연구용지기관경종만성조새성폐병환자하호흡도채집분비액，제취세균총DNA ，이16S통용인물27F／1492R확증16S rDNA전장，PCR산물경전영、순화후련접도pGEMT‐Easy재체상，구건16S rDNA극륭문고。연후이16S rDNA V3구통용인물338F／518R확증극륭문고，V3구PCR‐HRM분석재라씨LightCycler 480실시형광정량PCR계통중진행，채용HRM기인소묘연건분석수거。근거불동 HRM도형，도선극륭주측서병감정균주。결과현시，PCR‐HRM기술가령민구분하호흡도불동세균16S rDNA V3구，근거 HRM도보가겁대감소극륭균주측서양본，제시PCR‐HRM기술가작위일충쾌속、고통량、민감、경제적균군다양성검측방법。
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 .