Microbial DNA typing by automated repetitive-sequence-based PCR
Microbial DNA typing by automated repetitive-sequence-based PCR
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DOI：
10.1128/jcm.43.1.199-207.2005
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发表时间：
2005-01-01
影响因子：
9.4
通讯作者：
Lupski, JR
中科院分区：
文献类型：
--
作者：
Healy, M;Huong, J;Lupski, JR
基于重复序列的PCR（rep-PCR）已被认为是细菌菌株分型的有效方法。最近，rep-PCR已在商业上适用于称为DiversiLab系统的自动化格式，为临床实验室提供可靠的基于PCR的分型系统。我们描述了自动化rep-PCR的适应性，并探讨了该系统作为细菌菌株分型的分子基因分型工具的性能和重现性。自动化的修改包括rep-PCR化学和热循环参数的变化，基于微流体的DNA扩增子分级和检测的合并，以及基于互联网的计算机辅助分析，报告和数据存储。在DNA分离前，通过多个实验室、人员、仪器、DNA模板浓度和培养条件进行DNA分型和重复检测，检查自动rep-PCR的性能和重现性。最后，我们通过使用脑膜炎奈瑟菌感染爆发的分离株，证明了自动rep-PCR在临床实验室应用中的用途。N.脑膜炎暴发相关菌株与其他分离株不同。DiversiLab系统是一个高度集成、方便和快速的测试平台，可以让临床实验室实现微生物DNA分型的潜力。
Repetitive sequence-based PCR (rep-PCR) has been recognized as an effective method for bacterial strain typing. Recently, rep-PCR has been commercially adapted to an automated format known as the DiversiLab system to provide a reliable PCR-based typing system for clinical laboratories. We describe the adaptations made to automate rep-PCR and explore the performance and reproducibility of the system as a molecular genotyping tool for bacterial strain typing. The modifications for automation included changes in rep-PCR chemistry and thermal cycling parameters, incorporation of microfluidics-based DNA amplicon fractionation and detection, and Internet-based computer-assisted analysis, reporting, and data storage. The performance and reproducibility of the automated rep-PCR were examined by performing DNA typing and replicate testing with multiple laboratories, personnel, instruments, DNA template concentrations, and culture conditions prior to DNA isolation. Finally, we demonstrated the use of automated rep-PCR for clinical laboratory applications by using isolates from an outbreak of Neisseria meningitidis infections. N. meningitidis outbreak-related strains were distinguished from other isolates. The DiversiLab system is a highly integrated, convenient, and rapid testing platform that may allow clinical laboratories to realize the potential of microbial DNA typing.