Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Disease, Hamilton, Montana, United States of America.
PLoS Pathog. 2010 Dec 2;6(12):e1001217. doi: 10.1371/journal.ppat.1001217.
A major problem for the effective diagnosis and management of prion diseases is the lack of rapid high-throughput assays to measure low levels of prions. Such measurements have typically required prolonged bioassays in animals. Highly sensitive, but generally non-quantitative, prion detection methods have been developed based on prions' ability to seed the conversion of normally soluble protease-sensitive forms of prion protein to protease-resistant and/or amyloid fibrillar forms. Here we describe an approach for estimating the relative amount of prions using a new prion seeding assay called real-time quaking induced conversion assay (RT-QuIC). The underlying reaction blends aspects of the previously described quaking-induced conversion (QuIC) and amyloid seeding assay (ASA) methods and involves prion-seeded conversion of the alpha helix-rich form of bacterially expressed recombinant PrP(C) to a beta sheet-rich amyloid fibrillar form. The RT-QuIC is as sensitive as the animal bioassay, but can be accomplished in 2 days or less. Analogous to end-point dilution animal bioassays, this approach involves testing of serial dilutions of samples and statistically estimating the seeding dose (SD) giving positive responses in 50% of replicate reactions (SD(50)). Brain tissue from 263K scrapie-affected hamsters gave SD(50) values of 10(11)-10(12)/g, making the RT-QuIC similar in sensitivity to end-point dilution bioassays. Analysis of bioassay-positive nasal lavages from hamsters affected with transmissible mink encephalopathy gave SD(50) values of 10(3.5)-10(5.7)/ml, showing that nasal cavities release substantial prion infectivity that can be rapidly detected. Cerebral spinal fluid from 263K scrapie-affected hamsters contained prion SD(50) values of 10(2.0)-10(2.9)/ml. RT-QuIC assay also discriminated deer chronic wasting disease and sheep scrapie brain samples from normal control samples. In principle, end-point dilution quantitation can be applied to many types of prion and amyloid seeding assays. End point dilution RT-QuIC provides a sensitive, rapid, quantitative, and high throughput assay of prion seeding activity.
用于有效诊断和管理朊病毒疾病的一个主要问题是缺乏快速高通量的检测方法来测量低水平的朊病毒。此类测量通常需要在动物中进行长时间的生物测定。已经开发了高度敏感但通常是非定量的朊病毒检测方法，这些方法基于朊病毒将正常可溶性蛋白酶敏感形式的朊病毒蛋白转化为蛋白酶抗性和/或淀粉样纤维形式的能力。在这里，我们描述了一种使用称为实时震颤诱导转化测定（RT-QuIC）的新朊病毒接种测定来估计朊病毒相对量的方法。该反应结合了先前描述的震颤诱导转化（QuIC）和淀粉样纤维接种测定（ASA）方法的各个方面，涉及细菌表达的重组 PrP(C)的富含α螺旋形式的朊病毒接种转化为富含β片层的淀粉样纤维形式。RT-QuIC 与动物生物测定一样敏感，但可以在 2 天或更短的时间内完成。类似于终点稀释动物生物测定，该方法涉及对样品的连续稀释进行测试，并统计估计在 50%的重复反应中产生阳性反应的接种剂量（SD）（SD(50)）。来自 263K 瘙痒病感染仓鼠的脑组织给出了 SD(50)值为 10(11)-10(12)/g，使得 RT-QuIC 与终点稀释生物测定的灵敏度相似。对感染传染性水貂脑病的仓鼠的生物测定阳性鼻冲洗液进行分析，得到了 SD(50)值为 10(3.5)-10(5.7)/ml，表明鼻腔释放出大量可快速检测到的朊病毒感染性。来自 263K 瘙痒病感染仓鼠的脑脊髓液含有 SD(50)值为 10(2.0)-10(2.9)/ml。RT-QuIC 测定还区分了鹿慢性消耗病和绵羊瘙痒病脑组织与正常对照样本。原则上，终点稀释定量可以应用于许多类型的朊病毒和淀粉样纤维接种测定。终点稀释 RT-QuIC 提供了一种敏感、快速、定量和高通量的朊病毒接种活性检测方法。
