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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
赵焕英1,薛冰1,方霄2,陈瑛3,郭伟4
1. 首都医科大学医学实验与测试中心,北京100069; 2. 济宁医学院药学院2013年级药物制剂技术,日照 276826; 3. 首都医科大学附属友谊医院妇产科,北京100050; 4. 首都医科大学附属北京天坛医院急诊科,北京 100050
Application of polymerase chain reaction-high-resolution melt technology for bacterial identification in samples collected from lower respiratory tract
ZHAO Huan-Ying1, XUE Bing1, FANG Xiao2, CHEN Ying3, GUO Wei4
1. Core Facilities Center, Capital Medical University, Beijing 100069 China; 2. Grade 2013 of Pharmaceutical Preparation Technology, Jining Medical University, Rizhao 276826, China; 3. Department of Obstetrics and Gynecology, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China;4. Emergency Department,Beijing Tian Tan Hospital, Capital Medical University,Beijing 100050, China
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摘要: 为探讨聚合酶链反应-高分辨率熔解曲线(PCR-HRM)技术在检测呼吸道混合细菌菌群中的应用,本研究以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LightCycler480实时荧光定量PCR系统中进行,采用HRM基因扫描分析软件分析数据。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。

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赵焕英1
薛冰1
方霄2
陈瑛3
郭伟4
关键词 聚合酶链反应-高分辨率熔解曲线技术微生物菌群16S rDNA文库慢性阻塞性肺病哮喘    
Abstract: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 bronchoscopy. Total DNA of bacteria was extracted and the full-length 16S rDNA was amplified by PCR with 16S primers 27F/1492R. PCR products were electrophoresed and purified, and then connected to pGEMT-Easy carrier to build 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 by Roche LightCycler 480 real-time fluorescence quantitative PCR system. The data were analyzed by HRM GeneScan analysis software. According to the different HRM patterns, clonal strains were selected for sequencing to identify bacterial strain. The results showed that PCR-HRM could be responsive to distinguish the 16S rDNA-V3 regions from different bacteria of the lower respiratory tract and cloned strain sequencing sample size was significantly reduced according to HRM patterns. PCR-HRM technology can be used as a rapid, high-throughput, sensitive and economic detection method of bacterial diversity.

Key wordsPolymerase chain reaction-high-resolution melt technology    Microbial flora    16S rDNA library    Chronic obstructive pulmonary disease    Asthma
基金资助:国家自然科学基金资助项目(30901598),首都医科大学基础临床科研合作基金(13JL28、14JL29),首都医科大学校基金技术类项目(2014JS08)

通讯作者: 郭伟   
引用本文:   
赵焕英1,薛冰1,方霄2,陈瑛3,郭伟4. 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[J]. 微生物与感染, 2015, 10(5): 308-314.
ZHAO Huan-ying1, XUE Bing1, FANG Xiao2, CHEN Ying3, GUO Wei4. Application of polymerase chain reaction-high-resolution melt technology for bacterial identification in samples collected from lower respiratory tract. JOURNAL OF MICROBES AND INFECTIONS, 2015, 10(5): 308-314.
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