用于病原菌单步富集的固体相的开发与表征

M. Archer, D. Stenger, B. Lin
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引用次数: 3

摘要

在复杂基质中鉴定低丰度目标核酸可能具有挑战性,因为丰度背景材料。目前的方法采用两步法,耗时长,容易受到污染,而且通常仅限于一种病原体。在这项研究中,我们描述了一种单步目标捕获方法,使用磁性微珠和捕获探针通过磷树突连接物共价连接。这种方法也被成功地用于同时捕获两种低丰度的致病性核酸,存在于一个复杂的基质(800倍过量的背景核酸),通过使用多病原体固相。固相的热稳定性允许变性和捕获顺序进行,目标的恢复可以通过热变性来执行,而不会有探针脱落的风险。讨论了涉及固相发展的关键变量和进一步优化所需的步骤。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Development and Characterization of a Solid Phase for Single-Step Enrichment of Pathogenic Targets
The identification of low abundance target nucleic acids in a complex matrix can be challenging due to the abundance background material. Current methods use two-step processes which are time consuming, prone to contamina- tion and usually limited to one pathogen. In this study we describe a single-step target-capture approach using magnetic microbeads with capture probes covalently attached through a phosphorus dendrimer linker. This approach was also used successfully for simultaneous capturing of two low abundance pathogenic nucleic acids present in a complex matrix (800- fold excess of background nucleic acids) by using a multi-pathogen solid phase. The thermal stability of the solid phase allows denaturation and capture to proceed sequentially and the recovery of the targets to be performed by heat denatura- tion without the risk of probe shedding. The critical variables involved in the development of the solid phase and the steps required for further optimization are discussed.
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