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Deployable CRISPR-Cas13a diagnostic tools to detect and report Ebola and Lassa virus cases in real-time
bioRxiv - Molecular Biology Pub Date : 2020-05-27 , DOI: 10.1101/2020.05.26.116442
Kayla G. Barnes , Anna E. Lachenauer , Adam Nitido , Sameed Siddiqui , Robin Gross , Brett Beitzel , Katherine J. Siddle , Catherine A. Freije , Bonnie Dighero-Kemp , Samar Mehta , Amber Carter , Jessica Uwanibe , Fehintola Ajogbasile , Testimony J. Olumade , Ikponmwosa Odia , John Demby Sandi , Mambu Momoh , Hayden C. Metsky , Chloe K. Boehm , Aaron E. Lin , Molly Kemball , Daniel J. Park , Donald S. Grant , Christian T. Happi , Luis Branco , Matt Boisen , Brian M. Sullivan , Mihret Amare , Abdulwasiu Tiamiyu , Zahra Parker , Michael Iroezindu , Kayvon Modjarrad , Cameron Myhrvold , Robert F. Garry , Gustavo Palacios , Lisa E. Hensley , Stephen F. Schaffner , Andres Colubri , Pardis C. Sabeti

Viral hemorrhagic fevers (VHFs) remain some of the most devastating human diseases, and recent outbreaks of Ebola virus disease (EVD) 1,2 and Lassa fever (LF) 3,4 highlight the urgent need for sensitive, field-deployable tests to diagnose them 5,6. Here we develop CRISPR-Cas13a-based (SHERLOCK) diagnostics targeting Ebola virus (EBOV) and Lassa virus (LASV), with both fluorescent and lateral flow readouts. We demonstrate on laboratory and clinical samples the sensitivity of these assays and the capacity of the SHERLOCK platform to handle virus-specific diagnostic challenges. Our EBOV diagnostic detects both the L and NP genes, thereby eliminating the potential for false positive results caused by the rVSVΔG-ZEBOV-GP live attenuated vaccine. Our two LASV diagnostics together capture 90% of known viral diversity and demonstrate that CRISPR-RNAs (crRNAs) can be effectively multiplexed to provide greater coverage of known viral diversity. We performed safety testing to demonstrate the efficacy of our HUDSON protocol in heat-inactivating and chemically treating VHF viruses before SHERLOCK testing, eliminating the need for an extraction. We developed a user-friendly field protocol and mobile application (HandLens) to report results, facilitating SHERLOCK's use in endemic regions. Finally, we successfully deployed our tests in Sierra Leone and Nigeria in response to recent outbreaks.

中文翻译:

可部署的CRISPR-Cas13a诊断工具可实时检测和报告埃博拉和拉萨病毒病例

病毒性出血热(VHF)仍然是最致命的人类疾病,最近爆发的埃博拉病毒病(EVD)1,2和拉沙热(LF)3,4突显了对进行现场诊断的敏感,紧急测试的迫切需要5,6。在这里,我们开发针对埃博拉病毒(EBOV)和拉萨病毒(LASV)的基于CRISPR-Cas13a(SHERLOCK)的诊断方法,同时具有荧光和横向流读数。我们在实验室和临床样本上证明了这些检测方法的敏感性以及SHERLOCK平台应对病毒特异性诊断挑战的能力。我们的EBOV诊断程序同时检测L和NP基因,从而消除了由rVSVΔG-ZEBOV-GP减毒活疫苗引起的假阳性结果的可能性。我们的两个LASV诊断程序共同捕获了90%的已知病毒多样性,并证明CRISPR-RNA(crRNA)可以有效地多路复用以提供更大的已知病毒多样性覆盖率。在进行SHERLOCK测试之前,我们进行了安全性测试,以证明我们的HUDSON协议在热灭活和化学处理VHF病毒方面的功效,从而无需提取。我们开发了用户友好的现场协议和移动应用程序(HandLens)报告结果,从而促进了SHERLOCK在流行地区的使用。最后,我们针对最近的疫情在塞拉利昂和尼日利亚成功部署了测试。在进行SHERLOCK测试之前,我们进行了安全性测试,以证明我们的HUDSON协议在热灭活和化学处理VHF病毒方面的功效,从而无需提取。我们开发了一种用户友好的现场协议和移动应用程序(HandLens)报告结果,从而促进了SHERLOCK在流行地区的使用。最后,我们针对最近的疫情在塞拉利昂和尼日利亚成功部署了测试。在进行SHERLOCK测试之前,我们进行了安全性测试,以证明我们的HUDSON协议在热灭活和化学处理VHF病毒方面的功效,从而无需提取。我们开发了一种用户友好的现场协议和移动应用程序(HandLens)报告结果,从而促进了SHERLOCK在流行地区的使用。最后,我们针对最近的疫情在塞拉利昂和尼日利亚成功部署了测试。
更新日期:2020-05-27
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