Rapid detection of Hepatitis C virus using CRISPR/Cas

使用 CRISPR/Cas 快速检测丙型肝炎病毒

基本信息

批准号：
10477938
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：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10477938
关键词：
Antibodies Base Sequence Blood Blood specimen Cause of Death Centers for Disease Control and Prevention (U.S.)Cessation of life Chronic Hepatitis C Clinic Clinical Clustered Regularly Interspaced Short Palindromic Repeats Color Complement Coupled DNA DNA amplification Data Death Rate Detection Development Devices Diagnosis Disease Outbreaks Early Diagnosis Early Intervention Early treatment Engineering Enzyme Immunoassay Equipment Eye Fluorescence Resonance Energy Transfer Future Goals Guide RNA HCV screening HIV Hepatitis B Virus Hepatitis C virus Hour Human Papillomavirus Infection Intervention Location Methods Modification Nucleic Acids Orthologous Gene Paper Patients Persons Pilot Projects Polymerase Population RNA RNA-Directed DNA Polymerase Rapid diagnostics Reagent Reporter Reporting Resource-limited setting Resources Sampling Sensitivity and Specificity Serum Single Stranded DNA Virus Specificity System Technology Telephone Testing Time Viral Load result World Health Organization acute infection base co-infection cohort cost design detection limit detection platform detection sensitivity diagnostic platform diagnostic strategy ds-DNA high reward high risk improved infection rate innovation instrumentation lateral flow assay mortality risk mutant nanoGold novel novel strategies nuclease patient response point of care point-of-care diagnostics prevent rapid detection recombinase screening self testing seroconversion single molecule unpublished works user-friendly viral RNA viral detection

项目摘要

PROJECT ABSTRACT/SUMMARY In 2017, WHO estimated 71 million people had chronic Hepatitis C Virus (HCV) but 81% 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 2.1 million people were infected with HCV within the US and only a fraction of them were diagnosed properly. A rapid and reliable detection of an early-stage HCV would allow quicker intervention and can significantly reduce the risk of death and infection rate. An innovative self-testing diagnostic platform for early detection of HCV RNA using engineered type V and VI CRISPR/Cas systems can be created. 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. By detecting the collateral activity of CRISPR/Cas systems using a FRET-based reporter, up to 10 nM of target sequence has been fluorescently detected by Doudna and Zhang labs. By pre-amplifying a target using a reverse transcriptase and/or isothermal DNA amplification, single molecule detection of RNA or DNA has been achieved with concentrations as low as 2 aM. In preliminary unpublished work, engineered CRISPR RNA (crRNA) for CRISPR/Cas12a were discovered to amplify this further and achieved up to > 400,000-fold improved sensitivity with the limit of detection of 25 fM of PCA3 dsDNA in 6 hours, 700 fM of HIV ssDNA in 30 minutes, and 290 fM HCV ssDNA in 30 minutes without requiring target pre- amplification. Additional modifications on the CRISPR RNA improved the specificity of detection discriminating single point mutants. Based on the preliminary data, there are following specific goals. 1) To enhance sensitivity and specificity of CRISPR/Cas systems by applying novel engineering rules to different orthologs of CRISPR/Cas12a, CRISPR/Cas13a, and CRISPR/Cas14a systems that can identify known viral RNA copies with a sensitivity of 100 copies of RNA in 1 mL of blood. 2) To develop a paper-based device for colorimetric detection of a HCV target by naked eye at 100 target copies/mL concentration. 3) To perform a pilot study with the optimized device for validating HCV detection in clinical blood samples from healthy, high-risk, infected and treated patients with 95% accuracy. This integrated approach will have 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 this rapid diagnostic platform would allow quicker treatment, reduce outbreak and faster response from patients. In future, this approach would enable detection of co-infections including TB, HPV, and HIV, and HBV that are the major causes of death in HCV-infected populations.

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），但其中81%的活着的患者不知道自己的感染状况。仅 2016 年，世界卫生组织就估计有 399,000 例 HCV 相关死亡。 CDC估计，2013年至2016年间，美国境内约有210万人感染HCV，而只有其中一部分得到了正确诊断。快速可靠地检测早期丙型肝炎病毒将有助于更快地干预并可以显着降低死亡风险和感染率。创新的自测诊断平台可以创建使用工程化的 V 型和 VI 型 CRISPR/Cas 系统来早期检测 HCV RNA。 V 型和 VI 型 CRISPR/Cas 系统与特定靶核酸序列结合时，激活次级附属核酸酶能够以非特异性多次周转方式快速切割单链核酸的活性。通过检测使用基于 FRET 的报告基因的 CRISPR/Cas 系统的附带活性，高达 10 nM 的目标序列已被由Doudna 和Zhang 实验室进行荧光检测。通过使用逆转录酶和/或等温酶预扩增靶标 DNA 扩增、RNA 或 DNA 的单分子检测已经实现，浓度低至 2 aM。在初步未发表的工作，发现用于 CRISPR/Cas12a 的工程 CRISPR RNA (crRNA) 可以放大这一点进一步，灵敏度提高了 400,000 倍以上，PCA3 dsDNA 的检测限为 6 中的 25 fM 小时，30 分钟内 700 fM HIV ssDNA，30 分钟内 290 fM HCV ssDNA，无需目标预放大。对 CRISPR RNA 的额外修饰提高了检测区分单基因的特异性点突变体。根据初步数据，有以下具体目标。 1) 提高敏感性和特异性 CRISPR/Cas 系统，通过将新颖的工程规则应用于 CRISPR/Cas12a、CRISPR/Cas13a 和 CRISPR/Cas13a 的不同直系同源物 CRISPR/Cas14a 系统可以识别已知的病毒 RNA 拷贝，灵敏度为 1 mL 液体中 100 个 RNA 拷贝血。 2) 开发一种纸基设备，用于肉眼以 100 个目标拷贝/mL 比色检测 HCV 目标专注。 3) 使用优化的装置进行试点研究，以验证临床血液样本中的 HCV 检测来自健康、高危、感染和治疗的患者，准确率达 95%。这种综合方法将具有所有 ASSURED 定义的组件（经济实惠、灵敏、特定、用户友好、快速且稳健、无需设备）和可交付给最终用户）世界卫生组织的标准。这种快速诊断平台的开发将允许更快治疗、减少疫情爆发并加快患者反应。将来，这种方法将能够检测混合感染包括 TB、HPV、HIV 和 HBV，它们是 HCV 感染人群死亡的主要原因。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A Thermostable Cas12b from Brevibacillus Leverages One-pot Detection of SARS-CoV-2 Variants of Concern.

来自短芽孢杆菌的热稳定 Cas12b 利用一锅法检测值得关注的 SARS-CoV-2 变体。

DOI：
发表时间：
2021-10-18
期刊：
影响因子：
0
作者：
Nguyen, Long T;Macaluso, Nicolas C;Pizzano, Brianna L M;Cash, Melanie N;Spacek, Jan;Karasek, Jan;Dinglasan, Rhoel R;Salemi, Marco;Jain, Piyush K
通讯作者：
Jain, Piyush K

Clinical validation of engineered CRISPR/Cas12a for rapid SARS-CoV-2 detection.

用于快速 SARS-CoV-2 检测的工程化 CRISPR/Cas12a 的临床验证。

DOI：
发表时间：
2022
期刊：
影响因子：
0
作者：
Nguyen, Long T;Rananaware, Santosh R;Pizzano, Brianna L M;Stone, Brandon T;Jain, Piyush K
通讯作者：
Jain, Piyush K

A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern.

来自短芽孢杆菌的热稳定 Cas12b 利用一锅法区分所关注的 SARS-CoV-2 变体。

DOI：
发表时间：
2022-03
期刊：
影响因子：
11.1
作者：
Nguyen, Long T;Macaluso, Nicolas C;Pizzano, Brianna L M;Cash, Melanie N;Spacek, Jan;Karasek, Jan;Miller, Megan R;Lednicky, John A;Dinglasan, Rhoel R;Salemi, Marco;Jain, Piyush K
通讯作者：
Jain, Piyush K