Real-time fluorescence loop mediated isothermal amplification for the detection of Acinetobacter baumannii.
Real-time fluorescence loop mediated isothermal amplification for the detection of Acinetobacter baumannii.
复制标题
DOI：
10.1371/journal.pone.0066406
复制
发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Liao K
中科院分区：
文献类型：
--
作者：
Wang Q;Zhou Y;Li S;Zhuo C;Xu S;Huang L;Yang L;Liao K
鲍曼不动杆菌的检测主要依赖于细菌培养，但往往不能及时返回有用的结果。虽然基于DNA的检测在检测病原体方面比细菌培养更敏感，但分子结果往往不一致，并受到假阳性的质疑，例如由于系统和环境来源的污染。此外，这些分子工具需要昂贵的实验室仪器。因此，建立现场使用的分子工具需要更简单的分子平台。环介导等温扩增方法相对简单，可以改进以更好地用于常规临床细菌学实验室。近年来，已经开发了一种能够在同一平台上进行LAMP的扩增和检测（通过荧光）的简单且便携的装置。该方法被称为实时环介导等温扩增。本研究尝试利用该方法对A.鲍曼不动杆菌。设计了种特异性引物，以检验该方法的实用性。A.与细菌培养和聚合酶链反应方法相比，使用鲍曼不动杆菌来确定该系统的灵敏度和特异性。所有痰标本经16S rRNA基因测序证实为不动杆菌属。实时荧光定量PCR方法简单，可实时检测DNA扩增，并能区分A.来自醋酸钙不动杆菌和不动杆菌基因组种的鲍曼不动杆菌3. DNA提取采用简易煮沸法。与细菌培养法相比，荧光素酶法检测A.鲍曼不动杆菌感染率分别为98.9%和75.0%。Realtase检测试剂盒仅需要一个单元，可通过目视检查验证检测试剂盒阳性。因此，该检测方法具有很大的应用潜力，可作为一种检测A.鲍曼不动杆菌。
Detection of Acinetobacter baumannii has been relying primarily on bacterial culture that often fails to return useful results in time. Although DNA-based assays are more sensitive than bacterial culture in detecting the pathogen, the molecular results are often inconsistent and challenged by doubts on false positives, such as those due to system- and environment-derived contaminations. In addition, these molecular tools require expensive laboratory instruments. Therefore, establishing molecular tools for field use require simpler molecular platforms. The loop-mediated isothermal amplification method is relatively simple and can be improved for better use in a routine clinical bacteriology laboratory. A simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in the same platform has been developed in recent years. This method is referred to as real-time loop-mediated isothermal amplification. In this study, we attempted to utilize this method for rapid detection of A. baumannii. Species-specific primers were designed to test the utility of this method. Clinical samples of A. baumannii were used to determine the sensitivity and specificity of this system compared to bacterial culture and a polymerase chain reaction method. All positive samples isolated from sputum were confirmed to be the species of Acinetobacter by 16S rRNA gene sequencing. The RealAmp method was found to be simpler and allowed real-time detection of DNA amplification, and could distinguish A. baumannii from Acinetobacter calcoaceticus and Acinetobacter genomic species 3. DNA was extracted by simple boiling method. Compared to bacterial culture, the sensitivity and specificity of RealAmp in detecting A. baumannii was 98.9% and 75.0%, respectively. The RealAmp assay only requires a single unit, and the assay positivity can be verified by visual inspection. Therefore, this assay has great potential of field use as a molecular tool for detection of A. baumannii.
登录
查看更多内容
影响因子：
3.7
作者：
Lucchi NW;Demas A;Narayanan J;Sumari D;Kabanywanyi A;Kachur SP;Barnwell JW;Udhayakumar V
通讯作者：
Udhayakumar V
影响因子：
9.4
作者：
Higgins, Paul G.;Lehmann, Marlene;Seifert, Harald
通讯作者：
Seifert, Harald
影响因子：
3.7
作者：
Clifford RJ;Milillo M;Prestwood J;Quintero R;Zurawski DV;Kwak YI;Waterman PE;Lesho EP;Mc Gann P
通讯作者：
Mc Gann P
影响因子：
9.4
作者：
Chang, HC;Wei, YF;Chang, TC
通讯作者：
Chang, TC
影响因子：
2.6
作者：
García-Arata, MI;Gerner-Smidt, P;Ibrahim, A
通讯作者：
Ibrahim, A