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point-of-care diagnostics prevent rapid detection recombinase screening self testing seroconversion single molecule unpublished works user-friendly viral RNA viral detection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/摘要 2017年，估计有7100万人患有慢性丙型肝炎病毒（HCV），但有81％的活着患者不知道他们的感染状况。仅在2016年，WHO估计有399,000例HCV相关的死亡。疾病预防控制中心估计，在2013 - 2016年之间，美国大约有210万人感染了HCV，只有一个其中的一部分被正确诊断。快速可靠的对早期HCV的检测将允许更快干预，可以大大降低死亡和感染率的风险。创新的自我测试诊断平台为了尽早使用工程化的VI类和VI CRISPR/CAS系统来早日检测HCV RNA。 V类和VI CRISPR/CAS系统与其特定靶核酸序列结合时，激活二次副核酸酶可以以非特异性的多重周转方式快速裂解单链核酸的活性。通过检测使用基于FRET的记者使用CRISPR/CAS系统的抵押活动，最多10 nm的目标序列一直是 Doudna和Zhang Labs检测到荧光。通过使用逆转录酶和/或等温度预省点目标 DNA扩增，浓度低至2 AM的RNA或DNA的单分子检测。在发现了crispr/cas12a的初步未发表的工作，工程化的CRRNA RNA（CRRNA）来扩大这一点进一步并实现了> 400,000倍的提高灵敏度，在6中检测25 fm的PCA3 dsDNA的限制小时，在30分钟内，在30分钟内，HIV ssDNA的700 FM，在30分钟内290 FM HCV ssDNA，而无需目标pre- 放大。对CRISPR RNA的其他修改提高了检测单一的特异性点突变体。根据初步数据，有以下特定目标。 1）提高灵敏度和特异性 CRISPR/CAS系统通过将新颖的工程规则应用于CRISPR/CAS12A，CRISPR/CAS13A和 CRISPR/CAS14A系统可以鉴定已知的病毒RNA副本，其灵敏度为100份RNA，1 mL 1 ml 血。 2）开发一种基于纸张的设备，用于通过肉眼100份/ml的肉眼来比色检测HCV目标专注。 3）用优化的设备进行试验研究，以验证临床血液样本中的HCV检测从健康，高危，感染和治疗的患者中，精度为95％。这种综合方法将拥有所有由保证的组件（负担得起，敏感，特定，用户友好，快速且健壮，无设备以及最终用户的交付）标准。这个快速诊断平台的开发将允许更快治疗，减少患者的爆发和更快的反应。将来，这种方法将能够检测共同感染包括TB，HPV和HIV和HBV，是HCV感染人群中死亡的主要原因。

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）

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

DOI：
10.1038/s43856-021-00066-4
发表时间：
2022
期刊：
COMMUNICATIONS MEDICINE
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.

DOI：
10.1016/j.ebiom.2022.103926
发表时间：
2022-03
期刊：
EBioMedicine
影响因子：
11.1
作者：
Nguyen LT;Macaluso NC;Pizzano BLM;Cash MN;Spacek J;Karasek J;Miller MR;Lednicky JA;Dinglasan RR;Salemi M;Jain PK
通讯作者：
Jain PK