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

使用多重 CRISPR/Cas 平台快速即时检测丙型肝炎病毒 RNA

基本信息

批准号：
10613983
负责人：
Piyush K Jain
金额：
$ 22.88万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10613983
关键词：
Acute Acute Hepatitis Acute Hepatitis C Adult Antiviral Therapy Base Sequence Binding Biological Assay Blood Blood specimen Buffers CRISPR/Cas technology Capital Cessation of life Chronic Chronic Hepatitis C Clinical Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Coupled DNA Death Rate Detection Development Devices Diagnosis Diagnostic Disease Outbreaks Early Intervention Engineering Equipment Etiology Fluorescence Future Genotype HCV screening Hepatitis C Hepatitis C Antibodies Hepatitis C Therapy Hepatitis C virus Immunoassay Individual Infection International Intervention Kinetics Laboratories Lateral Life Liver Cirrhosis Liver diseases Methods Modality Nucleic Acids Patient Monitoring Patients Persons Plasma Pregnant Women Preparation Primary carcinoma of the liver cells RNA ROC Curve Rapid diagnostics Reaction Recommendation Registries Relapse Reporting Research Research Personnel Resource-limited setting Reverse Transcriptase Polymerase Chain Reaction Reverse Transcription Risk Reduction Sampling Sensitivity and Specificity Serum Single Stranded DNA Virus Specificity Specimen Speed System Techniques Temperature Testing Therapeutic Time Validation Viral Genome Virus World Health Organization anti-hepatitis C chronic infection cohort combinatorial cost design detection platform detection sensitivity diagnostic platform diagnostic strategy effective therapy high reward high risk improved infection rate innovation internal control isothermal amplification lateral flow assay nanomolar novel strategies nuclease nucleic acid detection patient response point of care point of care testing point-of-care detection point-of-care diagnostics prevent rapid detection rapid test screening seroconversion transmission process user-friendly viral RNA

项目摘要

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.

项目摘要/总结 2017 年，世界卫生组织估计有 7100 万人患有慢性丙型肝炎病毒 (HCV)，但 40-50% 的人患有慢性丙型肝炎病毒 (HCV) 患者不知道自己的感染状况。仅 2016 年，估计就有 399,000 例 HCV 相关死亡世界卫生组织报告。 CDC估计，2013年至2016年间，大约有240万人感染了HCV 在美国，只有一小部分人得到了正确诊断。快速且廉价的 HCV 检测 RNA将允许更快的干预，并可以显着降低死亡风险和感染率，尤其是在资源有限的环境中。通过以独特的方式设计和多重化 CRISPR/Cas 系统，可在30分钟内实现血样低拷贝HCV RNA的灵敏检测。这个项目提出开发和临床验证两种高度创新的基于 CRISPR 的检测方法侧流检测中的 HCV 基因型。 V 型和 VI 型 CRISPR/Cas 系统与特定靶标结合时核酸序列，激活可快速切割单链的次级附属核酸酶活性核酸以非特异性多次周转方式。这种附带核酸酶活性已被用于快速检测核酸。然而，它们具有纳摩尔级灵敏度并且需要对目标进行预放大实现临床应用所需的阿托摩尔检测。虽然预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测定的时间和成本。为了消除对目标预放大的需要，同时保持高灵敏度和特异性，这高风险/高回报项目提出两种创新方法来实现HCV RNA的快速检测没有任何目标放大。为了实现第一个目标，最近开发了“CRISPR-ENHANCE”（CE）平台 PI实验室在30分钟内实现飞摩尔级核酸检测，无需任何目标预扩增（Nguyen 等人，Nat. Comm.，2020）将以组合方式进行测试，以进一步增强对使用侧流检测检测 HCV RNA 的临床水平。第二个目标是开发并临床验证基于 CRISPR 链反应 (CCR) 的测试，用于使用多重检测 HCV RNA 和基因型使用简单的基于荧光的护理点设备进行侧流测定和定量 RNA。两者都这些方法将在急性/慢性感染的库样本中进行临床验证，并进行纵向验证与丙型肝炎治疗登记处合作监测接受抗病毒治疗的患者研究网络 (HCV-TARGET)，一个由领先的 HCV 研究人员组成的国际联盟。所有的 ASSURED 定义的组件（经济实惠、灵敏、具体、用户友好、快速且稳健、世卫组织的标准（无需设备且可交付给最终用户）。快速诊断平台的开发将允许更快的治疗、减少疫情爆发和患者更快的反应。未来，这种方法将能够检测 HCV 的其他基因型和病原体。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Programmable RNA detection with CRISPR-Cas12a.

使用 CRISPR-Cas12a 进行可编程 RNA 检测。

DOI：
发表时间：
2023-02-17
期刊：
影响因子：
0
作者：
Rananaware, Santosh R;Vesco, Emma K;Shoemaker, Grace M;Anekar, Swapnil S;Sandoval, Luke Samuel W;Meister, Katelyn S;Macaluso, Nicolas C;Nguyen, Long T;Jain, Piyush K
通讯作者：
Jain, Piyush K

Programmable RNA detection with CRISPR-Cas12a.

使用 CRISPR-Cas12a 进行可编程 RNA 检测。

DOI：
发表时间：
2023-01-30
期刊：
影响因子：
0
作者：
Rananaware, Santosh R;Vesco, Emma K;Shoemaker, Grace M;Anekar, Swapnil S;Sandoval, Luke Samuel W;Meister, Katelyn S;Macaluso, Nicolas C;Nguyen, Long T;Jain, Piyush K
通讯作者：
Jain, Piyush K

Programmable RNA detection with CRISPR-Cas12a.

使用 CRISPR-Cas12a 进行可编程 RNA 检测。

DOI：
发表时间：
2023-09-05
期刊：
影响因子：
16.6
作者：
Rananaware, Santosh R;Vesco, Emma K;Shoemaker, Grace M;Anekar, Swapnil S;Sandoval, Luke Samuel W;Meister, Katelyn S;Macaluso, Nicolas C;Nguyen, Long T;Jain, Piyush K
通讯作者：
Jain, Piyush K

Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids.

通过从头结构分析设计高度热稳定的 Cas12b，用于核酸的一锅检测。

DOI：
发表时间：
2023-05-16
期刊：
影响因子：
0
作者：
Nguyen, Long T;Rananaware, Santosh R;Yang, Lilia G;Macaluso, Nicolas C;Ocana;Meister, Katelyn S;Pizzano, Brianna L M;Sandoval, Luke Samuel W;Hautamaki, Raymond C;Fang, Zoe R;Joseph, Sara M;Shoemaker, Grace M;Carman, Dylan R;Cha
通讯作者：
Cha

Harnessing noncanonical crRNAs to improve functionality of Cas12a orthologs.

利用非规范 crRNA 来改善 Cas12a 直系同源物的功能。

DOI：
发表时间：
2024-02-27
期刊：
影响因子：
8.8
作者：
Nguyen, Long T;Macaluso, Nicolas C;Rakestraw, Noah R;Carman, Dylan R;Pizzano, Brianna L M;Hautamaki, Raymond C;Rananaware, Santosh R;Roberts, Isabel E;Jain, Piyush K
通讯作者：
Jain, Piyush K