Molecular fingerprinting of Clostridium difficile isolates:: Pulsed-field gel electrophoresis versus amplified fragment length polymorphism
Molecular fingerprinting of Clostridium difficile isolates:: Pulsed-field gel electrophoresis versus amplified fragment length polymorphism
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DOI：
10.1128/jcm.40.1.101-104.2002
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发表时间：
2002-01-01
影响因子：
9.4
通讯作者：
Horrevorts, AM
中科院分区：
文献类型：
--
作者：
Klaassen, CHW;van Haren, HA;Horrevorts, AM
采用脉冲场凝胶电泳（PFGE）和扩增片段长度多态性（AFLP）两种分子指纹技术，对3所综合性医院疑似暴发的艰难梭菌分离株进行流行病学关联性研究。PFGE分析由于广泛的DNA降解而产生不确定的数据。尽管在电泳缓冲液中加入硫脲可以在一定程度上防止这种降解，但以这种方式获得的弱DNA带型仍然远远不是最佳的。通过使用荧光标记的PCR引物和在ABI PRISM自动化DNA分析平台上分析获得AFLP数据。AFLP分析产生了高分辨率和高度可重复的DNA指纹图谱，从中可以很容易地确定菌株之间的流行病学相关性。AFLP结果可以在24小时内轻松获得，而完成冗长的PFGE方案通常需要3至4天。AFLP显然被证明是一种更可靠的C。艰难梭菌分离株，特别是对于那些标准PFGE程序由于DNA降解而产生不确定结果的分离株。
Two molecular fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP), were used to investigate the epidemiological relatedness among Clostridium difficile isolates from suspected outbreaks in three general hospitals. Analysis by PFGE yielded inconclusive data as a result of extensive DNA degradation. Although this degradation could be prevented to a certain extent by the inclusion of thiourea in the electrophoresis buffer, the weak DNA banding patterns obtained in this way were still far from optimal. AFLP data were obtained by using fluorescently labeled PCR primers and analysis on an ABI PRISM automated DNA analysis platform. AFLP analysis yielded high resolution and highly reproducible DNA fingerprinting patterns from which the epidemiological relatedness among the isolates could easily be determined. AFLP results could be readily obtained within 24 h, whereas 3 to 4 days were routinely required to complete the lengthy PFGE protocol. AFLP clearly proved to be a much more fail-safe fingerprinting method for C. difficile isolates, especially for those isolates for which a standard PFGE procedure yielded inconclusive results due to DNA degradation.