Detection of bacterial 16S rRNA and identification of four clinically important bacteria by real-time PCR.
Detection of bacterial 16S rRNA and identification of four clinically important bacteria by real-time PCR.
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DOI：
10.1371/journal.pone.0048558
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Mc Gann P
中科院分区：
文献类型：
--
作者：
Clifford RJ;Milillo M;Prestwood J;Quintero R;Zurawski DV;Kwak YI;Waterman PE;Lesho EP;Mc Gann P
在临床传染病研究的范式中，鲍曼不动杆菌、大肠杆菌、肺炎克雷伯菌和铜绿假单胞菌代表了四种与临床最相关、因此研究最广泛的细菌。目前用于识别这些生物体的基于培养的方法缓慢且繁琐，并且越来越需要更快速和准确的分子检测方法。使用生物信息学工具，对 962,279 个细菌 16S rRNA 基因序列进行了比对，并选择同源区域来生成一组实时 PCR 引物，该引物靶向迄今为止已发表的所有细菌 16S rRNA 序列中的 93.6%。还设计了一组四种物种特异性实时 PCR 引物对，能够检测鲍曼不动杆菌、大肠杆菌、肺炎克雷伯菌和铜绿假单胞菌的不到 100 个基因组拷贝。所有引物均在体外测试了针对 50 种革兰氏阳性和阴性细菌的特异性。此外，还针对每个物种的 200 个临床分离株对物种特异性引物进行了测试，这些临床分离株是从来自不同地区和来源的大型临床分离株中随机选择的。培养物和实时 PCR 的比较显示 100% 的一致性。将引物纳入快速测定中，能够在 90 分钟内从平板或肉汤培养物中进行阳性鉴定。此外，我们的数据表明，当前的靶标，例如大肠杆菌中的uidA基因，由于序列变异而不适合作为物种特异性基因。本文描述的测定快速、经济高效且准确，并且可以轻松并入任何能够进行实时 PCR 的研究实验室。
Within the paradigm of clinical infectious disease research, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa represent the four most clinically relevant, and hence most extensively studied bacteria. Current culture-based methods for identifying these organisms are slow and cumbersome, and there is increasing need for more rapid and accurate molecular detection methods. Using bioinformatic tools, 962,279 bacterial 16S rRNA gene sequences were aligned, and regions of homology were selected to generate a set of real-time PCR primers that target 93.6% of all bacterial 16S rRNA sequences published to date. A set of four species-specific real-time PCR primer pairs were also designed, capable of detecting less than 100 genome copies of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa. All primers were tested for specificity in vitro against 50 species of Gram-positive and –negative bacteria. Additionally, the species-specific primers were tested against a panel of 200 clinical isolates of each species, randomly selected from a large repository of clinical isolates from diverse areas and sources. A comparison of culture and real-time PCR demonstrated 100% concordance. The primers were incorporated into a rapid assay capable of positive identification from plate or broth cultures in less than 90 minutes. Furthermore, our data demonstrate that current targets, such as the uidA gene in E.coli, are not suitable as species-specific genes due to sequence variation. The assay described herein is rapid, cost-effective and accurate, and can be easily incorporated into any research laboratory capable of real-time PCR.
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影响因子：
14.9
作者：
Cole JR;Wang Q;Cardenas E;Fish J;Chai B;Farris RJ;Kulam-Syed-Mohideen AS;McGarrell DM;Marsh T;Garrity GM;Tiedje JM
通讯作者：
Tiedje JM
影响因子：
4.1
作者：
Hartman, Laurie J.;Selby, Edward B.;Kulesh, David A.
通讯作者：
Kulesh, David A.
影响因子：
9.4
作者：
O'Leary, James;Corcoran, Daniel;Lucey, Brigid
通讯作者：
Lucey, Brigid
影响因子：
2
作者：
Anklam, Kelly S.;Kanankege, Kaushi S. T.;Doepfer, Doerte
通讯作者：
Doepfer, Doerte
影响因子：
2.4
作者：
Chern, E. C.;Siefring, S.;Haugland, R. A.
通讯作者：
Haugland, R. A.