Detection of Prions

朊病毒检测

• 批准号: 9354863
• 负责人: BYRON CAUGHEY
• 金额: $ 127.34万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9354863
• 关键词: Affect; Agriculture; Animals; Autopsy; Biological Assay; Biopsy; Bovine Spongiform Encephalopathy; Brain; Cattle; Cerebrospinal Fluid; Chronic Wasting Disease; Clinic; Clinical; Collaborations; Colorado; Conversion disorder; Creutzfeldt-Jakob Syndrome; Data; Detection; Development; Diagnosis; Diagnostic tests; Discrimination; Disease; Early Diagnosis; Endopeptidase K; Enzyme-Linked Immunosorbent Assay; Evaluation; False Positive Reactions; Farming environment; Fluorescence; Future; Genotype; Geological Survey; Goat; Hamsters; Head; High temperature of physical object; Human; Immunoblotting; Immunohistochemistry; Infection; Investigation; Kansas; Kinetics; Length; Lymphoid Tissue; Mus; N-terminal; Neurodegenerative Disorders; North America; Nose; Patients; Performance; Population; PrP; PrPSc Proteins; Prevalence Study; Prion Diseases; Prions; Protein Isoforms; Reaction; Reaction Time; Reader; Reading; Recombinants; Sampling; Scrapie; Services; Sheep; Sodium Chloride; Sodium Dodecyl Sulfate; Source; South Korea; Specificity; Speed; Techniques; Temperature; Testing; Time; Tissue Sample; Tissues; Universities; Variant; Western Blotting; Writing; Wyoming; base; brain tissue; cervid; diagnostic assay; disease diagnosis; human tissue; improved; polymerization; prion seeds; rectal; response

1) RT-QuIC is a rapid, specific, and sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect sub-infectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully used to detect PrPSc in various tissues from humans and animals, including sheep, tissues from goats infected with classical scrapie had not yet been tested. Therefore, in collaboration with Dr. Rohana Dassanayake and colleagues at the USDA, we aimed to (a) evaluate whether prion seeds in brain tissues of goats with scrapie could be detected using RT-QuIC, (b) further optimize RT-QuIC conditions to improve scrapie detection, and (c) compare the performance of the RT-QuIC for the detection of PrPSc to the more commonly used western blot and enzyme-linked immunosorbent assays (ELISA). We further optimized RT-QuIC conditions for sensitive and specific detection of goat scrapie seeding activity in brain tissue from clinical animals. When used with 200 mM sodium chloride, both full-length sheep rPrPSen substrates (PrP genotypes A136R154Q171 and V136R154Q171) provided good discrimination between scrapie infected and normal goat brain samples within 15 h. Our findings indicated that the RT-QuIC is at least 10,000-fold more sensitive than the ELISA and western blot assays for the detection of scrapie seeding activity in goat brain samples. Taken together, these findings suggested that RT-QuIC sensitively detects prion seeding activity in classical scrapie-infected goat brain samples. 2) Prion diseases of cattle include the classical (C) bovine spongiform encephalopathy (BSE), as well as the atypical H-BSE and L-BSE strains. Although the C- and L-BSE strains can be detected and discriminated by ultrasensitive real time quaking-induced conversion (RT-QuIC) assays, no such test had been described for the detection of H-BSE or the discrimination of each of the major bovine prion strains. We demonstrated an RT-QuIC assay for H-BSE that can detect as little as 10-9 dilutions of brain tissue and neat cerebrospinal fluid from clinically affected cattle. Moreover, comparisons of reactivities with different recombinant prion protein substrates and/or immunoblot band profiles of proteinase K-treated RT-QuIC reaction products indicated that H-, L- and C -BSE have distinctive prion seeding activities and can be discriminated by RT-QuIC on this basis. 3) Chronic wasting disease (CWD), a transmissible spongiform encephalopathy of cervids, was first documented nearly fifty years ago in Colorado and Wyoming, and has since spread across North America and to the Republic of Korea. The expansion of this disease makes the development of sensitive diagnostic assays and antemortem sampling techniques crucial for the mitigation of spread; this is especially true in cases of relocation/reintroduction, or prevalence studies in large or protected herds where depopulation may be contraindicated. We collaborated with Dr. Nicholas Haley of the University of Kansas, and other colleagues at Colorado State University, the U.CS. Geological Survey and the National Park Service in their evaluation of the sensitivity of the real-time quaking-induced conversion (RT-QuIC) assay in rectal mucosal associated lymphoid tissue (RAMALT) biopsies and nasal brushings collected antemortem. Antemortem findings were then compared to results from ante- and postmortem samples evaluated using immunohistochemistry (IHC). RAMALT samples were collected from populations of both farmed and free-ranging Rocky Mountain elk (n=316), with nasal brushes collected from a subpopulation of these animals (n=205). We hypothesized the sensitivity of RT-QuIC would be comparable to IHC in RAMALT, and would correspond to postmortem results. We found a high sensitivity through RAMALT testing (77.3%), which was highly correlative between RT-QuIC and IHC. Sensitivity was lower in nasal brushings (34%), though both RAMALT and nasal brush sensitivities were highly dependent on both genotype and obex score. These data suggested that RT-QuIC, like IHC, is a fairly sensitive assay for detection of CWD prions in RAMALT biopsies and other antemortem samples, and with further investigation has potential for large scale and rapid automated testing for CWD diagnosis. 4) The real-time quaking-induced conversion (RT-QuIC) assay for prion seeding activity has been applied to many prion diseases and provides for specific antemortem diagnostic testing. We evaluated RT-QuIC's long-term consistency and varied multiple reaction parameters. Repeated assays of a single scrapie sample using multiple plate readers and recombinant prion protein (rPrP(Sen)) substrates gave comparable results. N-terminal truncated hamster rPrP(Sen) (residues 90-231) hastened both prion-seeded and prion-independent reactions but maintained a clear kinetic distinction between the two. Raising temperatures or shaking speeds accelerated RT-QuIC reactions without compromising specificity. When applied to nasal brushings from Creutzfeldt-Jakob disease patients, higher temperatures accelerated RT-QuIC kinetics, and the use of hamster rPrP(Sen) (90-231) strengthened RT-QuIC responses. Elongation of shaking periods reduced scrapie-seeded reaction times, but continuous shaking promoted false-positive reactions. Furthermore, pH 7.4 provided for more rapid RT-QuIC reactions than more acidic pHs. Additionally, we showed that small variations in the amount of sodium dodecyl sulfate (SDS) significantly impacted the assay. Finally, RT-QuIC performed in multiplate thermoshakers followed by fluorescence readings in separate plate readers enhanced assay throughput economically. Collectively, these results demonstrated improved speed, efficacy and practicality of RT-QuIC assays and highlight variables to be optimized for future applications. 5) We also provided assistance in a project spear-headed by Bruce Chesebro and coworkers to assess early accumulation of PrPSc and prion seeding activity after local stereotactic inoculation of scrapie into the brains of mice. We performed RT-QuIC assays to help them assess the loss and replication, respectively in mice that either lacked or expressed PrPC. 6) We also participated in the writing of two major reviews of the applications of RT-QuIC to the diagnosis of human prions diseases.

1）RT-Quic是一种快速，特异和敏感的prion播种活性检测测定法，该测定法使用重组prion蛋白（RPRPSEN）检测prion蛋白（PRPSC）异常同工型的亚感染水平。尽管RT Quic已成功地用于检测来自人类和动物（包括绵羊）的各种组织中的PRPSC，但尚未对感染经典crap的山羊的组织进行测试。 Therefore, in collaboration with Dr. Rohana Dassanayake and colleagues at the USDA, we aimed to (a) evaluate whether prion seeds in brain tissues of goats with scrapie could be detected using RT-QuIC, (b) further optimize RT-QuIC conditions to improve scrapie detection, and (c) compare the performance of the RT-QuIC for the detection of PrPSc to the more commonly used western blot and酶联免疫吸附测定法（ELISA）。我们进一步优化了RT Quic条件，用于从临床动物中对脑组织中山羊草皮播种活性的敏感和特异性检测。当与200 mM氯化钠一起使用时，全长绵羊rprpsen底物（PRP基因型A136R154Q171和V136R154Q171）在15小时内均提供了良好的歧视。我们的发现表明，RT Quic至少比ELISA和Western印迹分析高10,000倍，以检测山羊脑样品中的crape籽活性。综上所述，这些发现表明，RT Quic敏感检测经典感染的山羊脑样品中的prion播种活性。 2）牛的prion病包括经典的（C）牛海绵状脑病（BSE），以及非典型H-BSE和L-BSE菌株。尽管可以通过超灵敏的实时Quaking诱导的转化率（RT-QUIC）测定来检测和区分C-和L-BSE菌株，但尚未描述过检测H-BSE或歧视每个主要牛prion菌株的测试。我们证明了用于H-BSE的RT Quic分析，该测定方法可以检测到临床受影响的牛的脑组织和整齐的脑脊髓液的10-9稀释液。此外，对蛋白酶K处理的RT Quic反应产物的反应活力与不同的重组prion蛋白底物的比较和/或免疫印迹带轮廓表明H-，L-和C-BSE具有独特的prion播种活性，并且可以在此基础上通过RT-quic区分。 3）慢性浪费疾病（CWD）是一种可传播的子宫颈的海绵状脑病，最早在五十年前在科罗拉多州和怀俄明州记录，此后一直遍布北美和大韩民国。 这种疾病的扩展使得敏感诊断测定法的发展和反典型抽样技术对于缓解扩散至关重要。在搬迁/重新引入的情况下，或在可能会禁忌人口减少的大型或受保护的牧群中，尤其如此。 我们与堪萨斯大学的尼古拉斯·海莉（Nicholas Haley）以及科罗拉多州立大学的其他同事（U.C.）合作。地质调查和国家公园管理局在评估直肠粘膜相关淋巴组织（Ramalt）活检（Ramalt）活检和收集的鼻刷中的实时Quaking诱导转化率（RT-QUIC）测定的敏感性时。 然后，将生物质地发现与使用免疫组织化学（IHC）评估的前后样品的结果进行了比较。 从耕种和自由放养的落基山麋鹿种群中收集了拉马特样品（n = 316），并从这些动物的亚群中收集了鼻刷（n = 205）。 我们假设RT Quic的灵敏度与Ramalt中的IHC相当，并且与验尸结果相对应。 我们通过拉马特测试（77.3％）发现了高灵敏度，这在RT-Quic和IHC之间高度相关。 鼻刷中的灵敏度较低（34％），尽管拉马特和鼻刷敏感性都高度依赖于基因型和OBEX评分。 这些数据表明，像IHC一样，RT-Quic是一种相当敏感的测定法，用于检测Ramalt活检中的CWD Prions和其他动物质量样品，并且随着进一步的研究，可能会进行大规模和快速自动化测试以进行CWD诊断。 4）实时Quaking诱导的转化率（RT-QUIC）用于prion植物活性，已应用于许多prion疾病，并提供了特定的激进症诊断测试。我们评估了RT Quic的长期一致性和多个多个反应参数。使用多个读板和重组prion蛋白（RPRP（SEN））底物对单个crapie样品进行重复测定，得出了可比的结果。 N末端截短的仓鼠RPRP（SEN）（残基90-231）加快了prion种子和非prion的反应，但在两者之间保持了明显的动力学区别。提高温度或摇动速度会加速RT Quic反应，而不会损害特异性。当应用于Creutzfeldt-Jakob疾病患者的鼻刷时，较高的温度加速了RT-Quic动力学，并使用仓鼠RPRP（SEN）（90-231）增强了RT-QUIC反应。摇动时期的伸长减少了刮屑种子的反应时间，但连续摇动促进了假阳性反应。此外，与更多酸性pH相比，pH 7.4提供了更快的RT Quic反应。此外，我们表明，十二烷基硫酸钠（SDS）的量的较小变化显着影响了该测定法。最后，在乘数热射手中进行的RT Quic，然后在单独的板块读取器中进行荧光读数，从而在经济上增强了测定吞吐量。总的来说，这些结果表明RT Quic分析的速度，功效和实用性提高了，并突出了将用于未来应用的变量。 5）我们还为布鲁斯·切斯布罗（Bruce Chesebro）和同事的矛头的一个项目提供了帮助，以评估局部立体定位接种小crapie的早期积累，并在小鼠的大脑中接种crapie的早期积累。我们进行了RT Quic测定法，以帮助他们评估缺乏或表达PRPC的小鼠的损失和复制。 6）我们还参与了对RT-Quic在诊断人类prion病疾病诊断的两项主要评论的撰写。