朊蛋白体外扩增技术对不同种属朊病毒检测敏感性分析  

Sensitivity Analysis of Prion Protein Amplification in Vitro for Detection of Different Prion Species

作　　者：刘楚眸 杨微 肖康 武月章 陈冬冬 董小平[1] 石琦[1] LIU Chumou;YANG Weit;XIAO Kang;WU Yuezhang;CHEN Dongdong;DONG Xiaoping;SHI Qi(State Key Laboratory of Infectious Disease Prevention and Control,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]中国疾病预防控制中心、病毒病预防控制所、传染病预防控制国家重点实验室,北京102206

出　　处：《病毒学报》2023年第2期372-383,共12页Chinese Journal of Virology

基　　金：国家重点研发计划(项目号:2020YFE0205700),题目:中国塞拉利昂“一带一路”联合实验室建设与重要传染病病原学研究;传染病预防控制国家重点实验室课题(项目号:2021SKLID101,2019SKLID501)。

摘　　要：朊蛋白体外扩增技术包括实时震动诱导蛋白扩增(Real-time quaking-induced conversion,RT-QuIC)技术及蛋白错误折叠循环扩增(Protein misfolding cyclic amplification,PMCA)技术。为了分析朊蛋白体外扩增技术与传统的蛋白免疫印迹技术对于朊病毒毒株检测敏感性的差异,应用此三种方法对朊病毒小鼠适应株139A、ME7和S15以及仓鼠适应株263K毒株进行了检测及对比分析。结果显示,应用RT-QuIC方法以及三轮PMCA扩增方法,均可检测到10-9稀释度139A小鼠脑匀浆中的朊病毒,比Western blot方法敏感度提高了106倍;应用RT-QuIC方法以及三轮PMCA扩增方法,分别可检测到10-7稀释度及10-8稀释度ME7小鼠脑匀浆中的朊病毒,分别比Western blot方法敏感度提高了104倍及105倍;应用RT-QuIC方法以及三轮PMCA扩增方法,分别可检测到10-8稀释度及10-9稀释度S15小鼠脑匀浆中的朊病毒,分别比Western blot方法敏感度提高了105倍及106倍;应用RT-QuIC方法可检测到10-9稀释度263K仓鼠脑匀浆中的朊病毒,比Western Blot方法敏感度提高了106倍,但三轮PMCA扩增方法可检测到10-3稀释度的263K小鼠脑匀浆中的朊病毒,与Western Blot方法比较敏感度没有明显提高。本实验为优化朊蛋白体外扩增技术的检测条件、检测敏感性及该技术的临床应用提供了实验数据参考。Prion protein amplification techniques in vitro include real-time quack-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA).The traditional detection method is Western blotting.Here,the sensitivity of detection of mouse prion strains 139A,ME7,and S15,and hamster Prion strain 263K was determined using these three methods.The results showed that both RT-QuIC and three rounds of PMCA amplification methods could detect Prions in 139A infected mice brain homogenate at 10-9dilution,which was 10~6 times more sensitive than Western blotting.The RT-QuIC and three rounds of PMCA amplification methods could detect prions in ME7 infected mice brain homogenates at 10-7dilution and 10-8dilution respectively,and the sensitivity was 10~4 and 10~5 times higher than that of Western blotting.The RT-QuIC method and three rounds of PMCA amplification methods could detect Prions in S15 infected mice brain homogenates at 10-8dilution and 10-9dilution respectively,which were 10~5 times and 10~6 times more sensitive than Western blotting.The RT-QuIC method could detect Prions in the 10-9dilution of 263K infected hamster brain homogenate,which was 10~6 times more sensitive than Western blotting,but the three-round PMCA amplification method could only detect Prions in 10-3dilution of 263K infected hamster brain homogenate.This study provides experimental data reference for optimizing the detection conditions,sensitivity and clinical application of Prion protein amplification in vitro.

关 键 词：朊病毒 实时震动诱导蛋白扩增 蛋白错误折叠循环扩增 蛋白免疫印迹 

分 类 号：R373.9[医药卫生—病原生物学]