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)感染是导致肝硬变、肝细胞癌和肝脏的主要原因 移植。随着2013年成功推出一种几乎没有副作用的直接作用抗病毒(DAA)药物 效果，负担得起的价格和90%的治愈率治疗丙型肝炎病毒感染，世卫组织通过了有史以来第一次 到2030年消除病毒性肝炎这一公共卫生问题的全球战略。这一雄心勃勃的目标需要 重大努力，不仅是为了预防新的感染，而且是为了诊断那些还没有意识到 被感染了。据估计，在7100万感染丙型肝炎病毒的人中，有一半人没有意识到他们的 感染，因此得不到治疗。向这些未经治疗的人群提供医疗保健的关键，从而 阻止感染的传播，是在感染被诊断出后立即开始现场治疗 病人定期去医生办公室就诊。这将需要一个快速、经济高效的诊断平台，它可以 在护理点(POC)使用，并在&lt；30分钟内提供诊断结果。目前的诊断，初步的 丙型肝炎病毒抗体(Ab)检测以记录暴露情况，然后进行更复杂和更昂贵的丙型肝炎病毒RNA检测 确认病毒血症，不能满足POC需要。由于最早1-2周就可以检测到血清中的丙型肝炎病毒核糖核酸 感染后，基于聚合酶链式反应(Pcr)的rna分析是检测丙型肝炎病毒的金标准方法。 诊断。然而，基于PCR的分析在PoC的实施受到其缓慢周转时间的限制 以及高性价比，特别是在资源匮乏的环境中。这项提议旨在通过以下方式填补这一空白 通过集成开发一种新型诊断平台，可在20分钟内识别丙型肝炎病毒感染 重组酶聚合酶扩增(RPA)、CRISPR-Cas12a和带正电荷金纳米颗粒 将基于(+GNP)的侧向流动分析(LFA)纳入统一的一步法进行核酸扩增(NAA)检测。 为了实现这一目标并解决基于LFA的POC NAA测试面临的技术挑战，该项目 将实施几项主要创新：1)使用RNA引物进行RPA核酸扩增，以解决 RPA和CRISPR-12A反应统一中的不相容问题，2)带电选择性输运 +CRISPR扩增过程释放的GNPs，以解决试剂/酶洗出问题，3)设计 具有分级的靶标结合和用于解决量化问题的本征反应“定时器”的新型靶标测试系 与基于LFA的核酸诊断技术相关的问题，以及4)基于智能手机的小型化 检测，这将使丙型肝炎诊断更POC和移动健康兼容。我们期待这一成功 该项目的完成将为NAA的快速诊断带来一种新的、强大的、经济高效的POC技术 对丙型肝炎病毒感染的早期诊断和治疗将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.