O'Connor Matthew J, Bishop Keith, Workman Robert G, Maddison Ben C, Gough Kevin C
a School of Veterinary Medicine and Science, The University of Nottingham, Sutton Bonington Campus , College Road, Sutton Bonington , Leicestershire , UK.
b ADAS UK, School of Veterinary Medicine and Science, The University of Nottingham, Sutton Bonington Campus , College Road, Sutton Bonington , Leicestershire , UK.
Prion. 2017 Jan 2;11(1):54-64. doi: 10.1080/19336896.2016.1259051. Epub 2017 Feb 8.
The in vitro amplification of prions by serial protein misfolding cyclic amplification has been shown to detect PrP to levels at least as sensitive as rodent bioassay but in a fraction of the time. Bovine spongiform encephalopathy is a zoonotic prion disease in cattle and has been shown to occur in 3 distinct forms, classical BSE (C-BSE) and 2 atypical BSE forms (L-BSE and H-BSE). Atypical forms are usually detected in asymptomatic, older cattle and are suggested to be spontaneous forms of the disease. Here, we show the development of a serial protein misfolding cyclic amplification method for the detection of H-BSE. The assay could detect PrP from 3 distinct experimental isolates of H-BSE, could detect PrP in as little as 1×10 g of brain material and was highly specific. Additionally, the product of serial protein misfolding cyclic amplification at all dilutions of seed analyzed could be readily distinguished from L-BSE, which did not amplify, and C-BSE, which had PrP with distinct protease K-resistance and protease K-resistant PrP molecular weights.
通过连续蛋白质错误折叠循环扩增进行的朊病毒体外扩增已被证明能够检测到朊蛋白，其灵敏度至少与啮齿动物生物测定法相当，但所需时间仅为后者的一小部分。牛海绵状脑病是牛的一种人畜共患朊病毒疾病，已被证明有三种不同形式，即经典型牛海绵状脑病（C-BSE）和两种非典型牛海绵状脑病形式（L-BSE和H-BSE）。非典型形式通常在无症状的老龄牛中检测到，被认为是该疾病的自发形式。在此，我们展示了一种用于检测H-BSE的连续蛋白质错误折叠循环扩增方法的开发。该检测方法能够从三种不同的H-BSE实验分离株中检测到朊蛋白，能够在低至1×10克脑材料中检测到朊蛋白，并且具有高度特异性。此外，在分析的所有种子稀释度下，连续蛋白质错误折叠循环扩增的产物都能够很容易地与未扩增的L-BSE以及具有不同蛋白酶K抗性和蛋白酶K抗性朊蛋白分子量的C-BSE区分开来。
