一种基于多点cas13的检测方法,可同时用于COVID-19诊断和变异监测。

IF 3.7 4区 生物学 Q2 GENETICS & HEREDITY CRISPR Journal Pub Date : 2023-04-01 DOI:10.1089/crispr.2022.0048
Maturada Patchsung, Aimorn Homchan, Kanokpol Aphicho, Surased Suraritdechachai, Thanyapat Wanitchanon, Archiraya Pattama, Khomkrit Sappakhaw, Piyachat Meesawat, Thanakrit Wongsatit, Artittaya Athipanyasilp, Krittapas Jantarug, Niracha Athipanyasilp, Juthamas Buahom, Supapat Visanpattanasin, Nootaree Niljianskul, Pimchai Chaiyen, Ruchanok Tinikul, Nuanjun Wichukchinda, Surakameth Mahasirimongkol, Rujipas Sirijatuphat, Nasikarn Angkasekwinai, Michael A Crone, Paul S Freemont, Julia Joung, Alim Ladha, Omar Abudayyeh, Jonathan Gootenberg, Feng Zhang, Claire Chewapreecha, Sittinan Chanarat, Navin Horthongkham, Danaya Pakotiprapha, Chayasith Uttamapinant
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引用次数: 0

摘要

即时护理(POC)核酸检测技术有望帮助金标准技术控制COVID-19大流行,但在执行急需的复杂检测(如病毒变异监测的多路检测)能力方面的缺陷可能会限制其广泛采用。在此,我们开发了一种基于LwaCas13a和PsmCas13b的强大的多路聚类定期间隔短重复序列(CRISPR)的检测方法,可以同时诊断严重急性呼吸综合征冠状病毒2 (SARS-CoV-2)感染,并确定引起SARS-CoV-2的致病变体(VOC),包括全球主要的VOCs Delta (B.1.617.2)和Omicron (B.1.1.529),同时在检测多个SARS-CoV-2基因靶点时保持高水平的准确性。该平台具有适合POC使用的几个属性:预混,冷冻干燥试剂,便于使用和储存;方便直接看眼或智能手机读数;以及多路检测的一锅变体。为了减少对专有试剂的依赖,并使这种技术能够在低收入和中等收入国家持续使用,我们在当地生产和配制了我们自己的重组酶聚合酶扩增反应,并证明了其与商业对应物的同等效率。我们的工具——基于CRISPR的同时诊断COVID-19和变异监测的检测,可以在当地生产——可以在POC环境中可持续地使用CRISPR诊断技术来诊断COVID-19和其他疾病。
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A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for Simultaneous COVID-19 Diagnosis and Variant Surveillance.

Point-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection-such as multiplexed detection for viral variant surveillance-may limit their widespread adoption. Herein, we developed a robust multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC)-including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529)-all the while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool-CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance that can be locally manufactured-may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.

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来源期刊
CRISPR Journal
CRISPR Journal Biochemistry, Genetics and Molecular Biology-Biotechnology
CiteScore
6.30
自引率
2.70%
发文量
76
期刊介绍: In recognition of this extraordinary scientific and technological era, Mary Ann Liebert, Inc., publishers recently announced the creation of The CRISPR Journal -- an international, multidisciplinary peer-reviewed journal publishing outstanding research on the myriad applications and underlying technology of CRISPR. Debuting in 2018, The CRISPR Journal will be published online and in print with flexible open access options, providing a high-profile venue for groundbreaking research, as well as lively and provocative commentary, analysis, and debate. The CRISPR Journal adds an exciting and dynamic component to the Mary Ann Liebert, Inc. portfolio, which includes GEN (Genetic Engineering & Biotechnology News) and more than 80 leading peer-reviewed journals.
期刊最新文献
Early Detection of Wildlife Disease Pathogens Using CRISPR-Cas System Methods. CRISPR-GRIT: Guide RNAs with Integrated Repair Templates Enable Precise Multiplexed Genome Editing in the Diploid Fungal Pathogen Candida albicans. Genome Editing in Apicomplexan Parasites: Current Status, Challenges, and Future Possibilities. Mutation-Specific CRISPR Targeting with SaCas9 and AsCas12a Restores Therapeutic Sensitivity in Treatment-Resistant Melanoma. Characterization of Research Support of Genome Editing Technologies and Transition to Clinical Trials.
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