Rapid point-of-care detection of Hepatitis C viral RNA using multiplexed CRISPR/Cas platforms

项目摘要/摘要 2017年，世卫组织估计有7100万人感染了慢性丙型肝炎病毒(HCV)，但有40%-50%的人活着 患者并不知道自己的感染状况。仅在2016年，估计有39.9万人死于丙型肝炎病毒 由世卫组织报告。疾控中心估计，2013-2016年间，约有240万人感染了丙型肝炎病毒 在美国，只有一小部分人得到了正确的诊断。一种快速廉价的丙型肝炎病毒检测方法 RNA将允许更快的干预，并可以显著降低死亡风险和感染率，特别是 在资源有限的情况下。通过以独特的方式设计和多路复用CRISPR/CAS系统， 可以在30分钟内对血液样本进行灵敏的低拷贝丙型肝炎病毒RNA检测。这个项目 提出两种基于CRISPR的高度创新的检测方法的开发和临床验证 侧向流动试验中的丙型肝炎病毒分型。与特定目标绑定时的V和VI型CRISPR/CAS系统 核酸序列，激活二级并行核酸酶活性，可以快速切割单链 核酸以非特异性的多次周转方式。这种侧枝核酸酶活性已被用于 快速检测核酸。然而，它们具有纳摩尔灵敏度，需要预先放大靶标 以实现临床所需的大气压检测。而前置放大可以通过以下方式实现 等温技术这需要额外的操作步骤和稳定的温度控制增加 化验的时间和成本。 为了消除对靶前扩增的需要，同时保持高灵敏度和特异性，这 高风险/高回报项目提出了两种实现快速检测丙型肝炎病毒RNA的创新方法 不需要任何靶标扩增。为了实现第一个目标，最近开发了一个来自 PI的实验室在30分钟内实现了对核酸的毫微摩尔检测，而不需要任何目标预扩增 (Nguyen et al.，NAT.将以组合的方式进行测试，以进一步增强对 用侧向流动试验检测临床水平的丙型肝炎病毒核糖核酸。第二个目标是发展和临床 验证基于CRISPR链式反应(CCR)的多重检测方法检测丙型肝炎病毒RNA和基因 使用一种简单的基于荧光的护理点设备进行横向流动分析和RNA定量。这两个 这些方法将在储存的急性/慢性感染样本以及纵向样本中进行临床验证 监测与丙型肝炎治疗注册中心合作进行抗病毒治疗的患者 研究网络(丙型肝炎病毒-TARGET)，由领先的丙型肝炎病毒研究人员组成的国际联盟。所有的 受保人定义的组件(负担得起的、敏感的、特定的、用户友好的、快速和可靠的、 无设备和可交付给最终用户)标准。一种快速诊断平台的研制 将允许更快的治疗，减少暴发和患者更快的反应。在未来，这种方法将 能够检测丙型肝炎病毒的其他基因类型和病原体。

PROJECT ABSTRACT/SUMMARY In 2017, WHO estimated 71 million people had chronic Hepatitis C Virus (HCV) but 40-50% of the living patients were unaware of their infection status. In 2016 alone, an estimated 399,000 HCV-related deaths were reported by WHO. CDC estimates that between 2013-2016, around 2.4 million people were infected with HCV within the US and only a fraction of them were diagnosed properly. A rapid and inexpensive detection of HCV RNA would allow quicker intervention and can significantly reduce the risk of death and infection rate, especially in resource-limited settings. By engineering and multiplexing CRISPR/Cas systems in unique ways ultra- sensitive detection of low copies of HCV RNA can be achieved for blood samples within 30 minutes. This project proposes development and clinical validation of two highly innovative CRISPR-based approaches for detecting HCV genotype in a lateral flow assay. Type V and VI CRISPR/Cas systems when bound with their specific target nucleic acid sequence, activate a secondary collateral nuclease activity that can rapidly cleave single-stranded nucleic acids in a non-specific multiple turnover manner. This collateral nuclease activity has been utilized for rapidly detecting nucleic acids. However, they have nanomolar sensitivity and require pre-amplification of a target to achieve attomolar detection that is desirable for clinical use. While pre-amplification can be achieved by isothermal techniques this requires additional manipulation steps and a stable temperature control increasing the time and cost of an assay. To eliminate the need for target pre-amplification while maintaining high sensitivity and specificity, this high-risk/high-reward project proposes two innovative approaches to achieve rapid detection of HCV RNA without any target amplification. For the first aim, a recently developed `CRISPR-ENHANCE' (CE) platform from the PI's lab that achieved femtomolar detection of nucleic acids in 30 minutes without any target pre-amplification (Nguyen et al., Nat. Comm., 2020) will be tested in a combinatorial fashion to further enhance the sensitivity for detecting clinical levels of HCV RNA using a lateral flow assay. The second aim is to develop and clinically validate a CRISPR Chain Reaction (CCR) based test for detecting HCV RNA and genotypes using multiplexed lateral flow assay and quantifying RNA using a simple fluorescence-based point-of-care device. Both the approaches will be clinically validated in banked samples with acute/chronic infections as well as longitudinally monitor patients undergoing anti-viral therapy in collaboration with Hepatitis C Therapeutic Registry and Research Network (HCV-TARGET), an international consortium of leading HCV investigators. All the components as defined by the ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users) criteria by WHO. The development of a rapid diagnostic platform would allow quicker treatment, reduce outbreak and faster response from patients. In future, this approach would enable detection of other genotypes of HCV and etiologic agents.