The real-time quaking-induced conversion assay for detection of human prion disease and study of other protein misfolding diseases
The real-time quaking-induced conversion assay for detection of human prion disease and study of other protein misfolding diseases
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DOI：
10.1038/nprot.2016.120
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发表时间：
2016-11-01
期刊：
影响因子：
14.8
通讯作者：
Zerr, Inga
中科院分区：
文献类型：
--
作者：
Schmitz, Matthias;Cramm, Maria;Zerr, Inga
体外错误折叠蛋白扩增系统的发展和应用是检测人脑和脑脊液（CSF）样品中异常折叠朊病毒蛋白瘙痒症（PrPsc）的主要创新。在此，我们描述了一种快速有效的蛋白质扩增技术，实时振荡诱导转换（RT-QuIC），用于检测人脑和CSF中的PrPsc种子。与其他基于超声处理的体外错误折叠蛋白质扩增试验（如蛋白质错误折叠循环扩增（PMCA））相比，RT-QuIC技术基于朊病毒种子诱导的重组朊病毒蛋白质底物的错误折叠和聚集，通过在荧光板读数器中振荡和静止的交替循环加速。单个RT-QuIC检测通常使用96孔板格式一式三份分析多达32个样品。从样品制备到结果分析，该方案需要近似87小时才能完成。除了诊断之外，该技术还具有实质性的通用分析应用，包括药物筛选、朊病毒菌株鉴别、生物危害筛选（例如，以降低与朊病毒疾病相关的传播风险）和蛋白质错误折叠的研究;此外，它还可能用于研究其他蛋白质错误折叠疾病，如阿尔茨海默病和帕金森病。
The development and adaption of in vitro misfolded protein amplification systems has been a major innovation in the detection of abnormally folded prion protein scrapie (PrPsc) in human brain and cerebrospinal fluid (CSF) samples. Herein, we describe a fast and efficient protein amplification technique, real-time quaking-induced conversion (RT-QuIC), for the detection of a PrPsc seed in human brain and CSF. In contrast to other in vitro misfolded protein amplification assays-such as protein misfolding cyclic amplification (PMCA)-which are based on sonication, the RT-QuIC technique is based on prion seed-induced misfolding and aggregation of recombinant prion protein substrate, accelerated by alternating cycles of shaking and rest in fluorescence plate readers. A single RT-QuIC assay typically analyzes up to 32 samples in triplicate, using a 96-well-plate format. From sample preparation to analysis of results, the protocol takes similar to 87 h to complete. In addition to diagnostics, this technique has substantial generic analytical applications, including drug screening, prion strain discrimination, biohazard screening (e.g., to reduce transmission risk related to prion diseases) and the study of protein misfolding; in addition, it can potentially be used for the investigation of other protein misfolding diseases such as Alzheimer's and Parkinson's disease.