Paper-Based Nucleic Acid Amplification Test for Rapid Diagnosis of Hepatitis C Viral Infection

摘要 慢性丙型肝炎病毒（HCV）感染是肝硬化、肝细胞癌和肝硬化的主要原因。 移植随着2013年成功推出一种副作用少的直接作用抗病毒（DAA）药物， 效果，负担得起的价格和>90%的治愈率用于治疗HCV感染，世卫组织首次采用了 到2030年消除病毒性肝炎这一公共卫生问题的全球战略。这一宏伟目标需要 这不仅是为了预防新的感染，也是为了诊断那些还没有意识到被感染的人。 感染了据估计，在7100万丙型肝炎病毒感染者中，有一半人不知道自己的病情。 感染，因此不能治疗。为这些未经治疗的人群提供医疗护理的关键， 停止感染的传播，是在感染被诊断出来后立即在现场进行治疗， 病人定期去看医生这将需要一个快速、具有成本效益的诊断平台， 在护理点（POC）使用，并在30分钟内提供诊断结果。目前的诊断， HCV抗体（Ab）检测记录暴露，然后是更复杂和昂贵的HCV RNA检测， 确认病毒血症，不能满足POC需求。由于血清HCVRNA最早可在1-2周内检测到， 感染后，基于聚合酶链反应（PCR）的RNA分析是HCV的金标准方法 诊断.然而，在POC实施基于PCR的分析受到周转时间缓慢的限制 成本/效益比高，特别是在资源贫乏的环境中。这项建议旨在填补差距， 开发一种新的诊断平台，通过整合能够在20分钟内识别HCV感染， 重组酶聚合酶扩增（RPA）、CRISPR-Cas 12 a和带正电荷的金纳米颗粒 （+GNP）为基础的侧向流分析（LFA）为一个统一的单步分析核酸扩增（NAA）测试。 为了实现这一目标，并解决基于LFA的POC NAA测试所面临的技术挑战， 1）使用RNA引物进行RPA核酸扩增，以解决 在统一RPA和CRISPR-12 a反应中的不相容性问题，2）选择性运输出带电的 +由CRISPR扩增过程释放的GNP，以解决试剂/酶洗脱问题，3）设计 具有分级靶结合和固有反应“计时器”的新型靶测试线，以解决定量问题 与基于LFA核酸诊断技术相关的问题，以及4）基于微型智能手机的 检测，这将使HCV诊断更加POC和移动的健康兼容。我们期待成功 该项目的完成将为快速NAA诊断带来一种新颖、稳健、具有成本效益的POC技术 这将对HCV感染的早期诊断和治疗产生广泛的积极影响， 传染病

Abstract Chronic hepatitis C virus (HCV) infection is a leading cause of cirrhosis, hepatocellular carcinoma, and liver transplantation. With the successful introduction in 2013 of a direct-acting antiviral (DAA) drug with few side effects, an affordable price and a >90% cure rate for the treatment of HCV infection, WHO adopted the first-ever global strategy for eliminating viral hepatitis as a public health problem by 2030. This ambitious goal will require major efforts, not only to prevent new infections but also to diagnose those who are not yet aware of being infected. It is estimated that of the 71 million people who are living with HCV infection, half are unaware of their infection and are thus untreated. A key to providing medical care to this untreated population, and thus to stopping spread of the infection, is to initiate immediate treatment on site once the infection is diagnosed during a patient's regular visit to a doctor's office. This will require a rapid, cost-effective diagnostic platform that can be used at the point of care (POC) and provide diagnostic results in <30 minutes. The current diagnostics, an initial HCV antibody (Ab) test to document exposure followed by the more complex and expensive HCV RNA test to confirm viremia, cannot meet the POC need. Since serum HCV RNA can be detected as early as 1-2 weeks after infection, polymerase chain reaction (PCR) based RNA analysis is the gold standard method for HCV diagnosis. However, implementation of PCR-based analysis at the POC is limited by its slow turnaround time and high cost/efficacy ratio, especially in resource-poor settings. This proposal is aimed at filling the gap by developing a novel diagnostic platform capable of identifying HCV infection in <20 minutes by integrating recombinase polymerase amplification (RPA), CRISPR-Cas12a and a positively charged gold nanoparticle (+GNP) based lateral flow assay (LFA) into a unified single-step assay for nucleic acid amplification (NAA) test. To achieve the objective and address the technology challenges facing an LFA-based POC NAA test, this project will implement several principal innovations: 1) use of RNA primers for RPA nucleic acid amplification to address the incompatibility issue in unifying RPA and CRISPR-12a reactions, 2) selective transporting out of the charged +GNPs released by the CRISPR amplification process to address the reagent/enzyme washout issue, 3) design of novel target test lines with graded target-binding and an intrinsic reaction “timer” to address the quantification issue associated with LFA-based nucleic acid diagnostic technology, and 4) miniaturized smartphone-based detection, which will make HCV diagnosis more POC and mobile health compatible. We expect that successful completion of this project will lead to a novel, robust, cost-effective POC technology for the rapid NAA diagnosis of HCV infection, which will have broad positive impacts on the early diagnosis and treatment of HCV and other infectious diseases.