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/摘要 2017年，估计有7100万人患有慢性丙型肝炎病毒（HCV），但有40％至50％的生活患者不知道自己的感染状况。仅在2016年，估计有399,000人与HCV相关的死亡是由谁报告。 CDC估计，在2013 - 2016年之间，大约有240万人感染了HCV 在美国，只有一小部分被正确诊断出来。快速且廉价的HCV检测 RNA将允许更快的干预措施，并可以大大降低死亡和感染率的风险，尤其是在资源有限的设置中。通过以独特的方式工程和多重CRISPR/CAS系统超级可以在30分钟内实现对血液样本的低拷贝HCV RNA的敏感检测。这个项目提出开发和临床验证，以检测两种高度创新的CRISPR方法横向流量测定中的HCV基因型。 V型和VI CRISPR/CAS系统与其特定目标绑定核酸序列激活二级副核酸酶活性，该活性可以快速切割单链核酸以非特异性的多个周转方式。这种副核酸酶活性已用于快速检测核酸。但是，它们具有纳摩尔敏感性，需要预先放大目标为了实现临床使用期望的attomolor检测。虽然可以通过等温技术需要额外的操纵步骤，并且稳定的温度控制增加测定的时间和成本。为了消除目标预扩增的需求，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创新的方法来快速检测HCV RNA 没有任何目标扩增。为了第一个目标，最近开发的“ CRISPR-ENHANCE”（CE）平台 PI的实验室在30分钟内实现了核酸检测到没有任何目标预扩增的核酸检测（Nguyen等，Nat。Comm。，2020）将以组合方式进行测试，以进一步增强对使用横向流量测定法检测HCV RNA的临床水平。第二个目的是开发和临床验证基于CRISPR链反应（CCR）测试使用多路复用横向流量测定和使用简单的基于荧光的护理设备来量化RNA。两者方法将在具有急性/慢性感染的银行样本中对临床验证以及纵向验证监测接受抗病毒治疗的患者与丙型肝炎治疗注册中心合作研究网络（HCV-TARGEG），一个领先的HCV研究者的国际财团。全部由保证的组件（负担得起，敏感，特定，用户友好，快速和健壮， WHO。快速诊断平台的开发可以更快地治疗，减少患者的爆发和更快的反应。将来，这种方法将能够检测HCV和病因的其他基因型。

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：
10.21203/rs.3.rs-2549171/v1
发表时间：
2023
期刊：
Research square
影响因子：
0
作者：
Rananaware,SantoshR;Vesco,EmmaK;Shoemaker,GraceM;Anekar,SwapnilS;Sandoval,LukeSamuelW;Meister,KatelynS;Macaluso,NicolasC;Nguyen,LongT;Jain,PiyushK
通讯作者：
Jain,PiyushK

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

DOI：
10.1016/j.xcrm.2023.101037
发表时间：
2023-05-16
期刊：
CELL REPORTS MEDICINE
影响因子：
14.3
作者：
Nguyen, Long T.;Rananaware, Santosh R.;Yang, Lilia G.;Macaluso, Nicolas C.;Ocana-Ortiz, Julio E.;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.;Chang, Liwei;Rakestraw, Noah R.;Zachary, Jon F.;Guerra, Sebastian;Perez, Alberto;Jain, Piyush K.
通讯作者：
Jain, Piyush K.