Center for Neuropathology and Prion research, Ludwig-Maximilians-University, Munich, Germany.
Acta Neuropathol Commun. 2013 Aug 2;1:44. doi: 10.1186/2051-5960-1-44.
PrPSc, the only known constituent of prions, the infectious agents causing prion diseases, can be detected by real-time quaking-induced conversion (RT-QuIC). However, there is no efficient method to quantify the amount of PrPSc by RT-QuIC.
Here we introduce quantitative RT-QuIC (qRT-QuIC) to quantify with high accuracy minute amounts of PrPSc in the brain and various peripheral tissues at levels far below detection by in vivo transmission. PrPSc is relatively resistant to treatment with proteinase K (PK). However, as there can also be a fraction of pathological PrP that is digested by PK, we use the term PrP27-30 to denote to the amount of PrPSc that can be detected by immunoblot after PK treatment. qRT-QuIC is based upon the quantitative correlation between the seeded amount of PrP27-30 and the lag time to the start of the conversion reaction detected by RT-QuIC. By seeding known amounts of PrP27-30 quantified by immunoblot into qRT-QuIC a standard calibration curve can be obtained. Based on this calibration curve, seeded undetermined amounts of PrP27-30 can be directly calculated. qRT-QuIC allowed to quantify PrP27-30 concentrations at extremely low levels as low as 10-15.5 g PrP27-30, which corresponds to 0.001 LD50 units obtained by in vivo i.c. transmission studies. We find that PrP27-30 concentration increases steadily in the brain after inoculation and can be detected at various time points during the incubation period in peripheral organs (spleen, heart, muscle, liver, kidney) in two experimental scrapie strains (RML, ME7) in the mouse.
We suggest that an automatic quantitative system to measure disease progression as well as prion contamination of organs, blood and food product is feasible. Moreover, the concept of qRT-QuIC should be applicable to measure other disease-associated proteins rich in β-pleated structures (amyloid) that bind ThT and that show seeded aggregation.
朊病毒，即传染性朊病毒疾病的病原体，仅由 PrPSc 构成，可通过实时震颤诱导转换（RT-QuIC）检测。然而，目前尚无通过 RT-QuIC 定量检测 PrPSc 含量的有效方法。
本研究引入了定量 RT-QuIC（qRT-QuIC），以高精度定量检测脑和各种外周组织中极少量的 PrPSc，检测水平远低于体内传播。PrPSc 相对耐受蛋白酶 K（PK）处理。然而，由于存在被 PK 消化的病理性 PrP 片段，我们使用 PrP27-30 来表示经 PK 处理后通过免疫印迹检测到的 PrPSc 量。qRT-QuIC 基于种子 PrP27-30 量与 RT-QuIC 检测到的转换反应起始时滞之间的定量相关性。通过将经免疫印迹定量的已知量 PrP27-30 种子到 qRT-QuIC 中，可以获得标准校准曲线。基于该校准曲线，可以直接计算未知量的 PrP27-30 种子。qRT-QuIC 可定量检测低至 10-15.5 g PrP27-30 的极微量 PrP27-30，相当于体内 i.c. 接种研究获得的 0.001 LD50 单位。我们发现接种后大脑中的 PrP27-30 浓度稳步增加，在两种实验性瘙痒病株（RML、ME7）的潜伏期内，可在各种外周器官（脾、心、肌肉、肝、肾）中检测到。
我们建议建立一种自动定量系统，以测量疾病进展以及器官、血液和食品的朊病毒污染情况是可行的。此外，qRT-QuIC 的概念应适用于测量其他富含β-折叠结构（淀粉样）的疾病相关蛋白，这些蛋白结合 ThT 并表现出种子聚集。
