Reverse Transcription Recombinase Polymerase Amplification Coupled with CRISPR-Cas12a for Facile and Highly Sensitive Colorimetric SARS-CoV-2 Detection DOI Open Access

Wei S. Zhang,

Jianbin Pan, Feng Li

et al.

Analytical Chemistry, Journal Year: 2021, Volume and Issue: 93(8), P. 4126 - 4133

Published: Feb. 11, 2021

The outbreak of the pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for an urgent unmet need developing a facial and cost-effective detection method. requirement well-trained personnel sophisticated instrument current primary mean (reverse transcription polymerase chain reaction, RT-PCR) may hinder practical application worldwide. In this regard, reverse recombinase amplification (RT-RPA) coupled with CRISPR-Cas12a colorimetric assay is proposed SARS-CoV-2 detection. methodology we have described herein utilizes DNA-modified gold nanoparticles (AuNPs) as universal readout can specifically target ORF1ab N regions genome. After virus genome amplified through RT-RPA, resulting abundant dsDNA will bind activate Cas12a. Under trans-cleavage degradation, capped DNA substrate be hydrolyzed gradually from AuNPs, demonstrating change in surface plasmon resonance (SPR), which facially monitored UV-vis absorbance spectroscopy naked eye observation. high efficiency RT-RPA Cas12a process bring sensitivity our method to 1 copy viral sequence per test. Notably, under dual variations inspecting isothermal activation process, false positive events other beta coronavirus members effectively avoided thus significantly improve specificity. Furthermore, reliability validated standard clinical samples hospital laboratory department. Through integration inherently specificity RPA-coupled system intrinsic simplicity AuNP-based assay, increases testing availability SARS-CoV-2.

Language: Английский

SHERLOCK: nucleic acid detection with CRISPR nucleases DOI
Max J. Kellner, Jeremy Koob, Jonathan S. Gootenberg

et al.

Nature Protocols, Journal Year: 2019, Volume and Issue: 14(10), P. 2986 - 3012

Published: Sept. 23, 2019

Language: Английский

Citations

1201
Disposable Sensors in Diagnostics, Food, and Environmental Monitoring DOI Creative Commons
Can Dincer, Richard C. Bruch, Estefanía Costa‐Rama

et al.

Advanced Materials, Journal Year: 2019, Volume and Issue: 31(30)

Published: May 15, 2019

Abstract Disposable sensors are low‐cost and easy‐to‐use sensing devices intended for short‐term or rapid single‐point measurements. The growing demand fast, accessible, reliable information in a vastly connected world makes disposable increasingly important. areas of application such numerous, ranging from pharmaceutical, agricultural, environmental, forensic, food sciences to wearables clinical diagnostics, especially resource‐limited settings. capabilities can extend beyond measuring traditional physical quantities (for example, temperature pressure); they provide critical chemical biological (chemo‐ biosensors) that be digitized made available users centralized/decentralized facilities data storage, remotely. These features could pave the way new classes systems health, food, environmental monitoring democratize across globe. Here, brief insight into materials basics (methods transduction, molecular recognition, amplification) is provided followed by comprehensive overview currently used medical analysis. Finally, views on how field will continue its evolution discussed, including future trends, challenges, opportunities.

Language: Английский

Citations

755
Diagnostics for SARS-CoV-2 infections DOI Creative Commons
Bhavesh D. Kevadiya, Jatin Machhi, Jonathan Herskovitz

et al.

Nature Materials, Journal Year: 2021, Volume and Issue: 20(5), P. 593 - 605

Published: Feb. 15, 2021

Language: Английский

Citations

731
Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay DOI Creative Commons
Xiong Ding, Kun Yin, Ziyue Li

et al.

Nature Communications, Journal Year: 2020, Volume and Issue: 11(1)

Published: Sept. 18, 2020

The recent outbreak of novel coronavirus (SARS-CoV-2) causing COVID-19 disease spreads rapidly in the world. Rapid and early detection SARS-CoV-2 facilitates intervention prevents spread. Here, we present an All-In-One Dual CRISPR-Cas12a (AIOD-CRISPR) assay for one-pot, ultrasensitive, visual detection. By targeting SARS-CoV-2's nucleoprotein gene, two CRISPR RNAs without protospacer adjacent motif (PAM) site limitation are introduced to develop AIOD-CRISPR detect nucleic acids with a sensitivity few copies. We validate by using clinical swab samples obtain consistent results RT-PCR assay. Furthermore, low-cost hand warmer (~$0.3) is used as incubator within 20 min, enabling instrument-free, at point care. Thus, our method has significant potential provide rapid, sensitive, one-pot point-of-care SARS-CoV-2.

Language: Английский

Citations

720
Cytokines: From Clinical Significance to Quantification DOI
Chao Liu, Dewei Chu, Kourosh Kalantar‐zadeh

et al.

Advanced Science, Journal Year: 2021, Volume and Issue: 8(15)

Published: June 10, 2021

Abstract Cytokines are critical mediators that oversee and regulate immune inflammatory responses via complex networks serve as biomarkers for many diseases. Quantification of cytokines has significant value in both clinical medicine biology the levels provide insights into physiological pathological processes can be used to aid diagnosis treatment. their significance introduced from perspective pro‐ anti‐inflammatory effects. Factors affecting quantification biological fluids, native different body sample processing storage conditions, sensitivity freeze‐thaw, soluble cytokine receptors discussed. In addition, recent advances vitro vivo assays, biosensors based on signal outputs intracellular extracellular protein expression summarized. Various platforms high‐sensitivity reliable measurement scenarios discussed, commercially available assays compared. A discussion challenges development advancement technologies aim achieve real‐time multiplex analysis point‐of‐care situations applicable biomedical research practice

Language: Английский

Citations

616
Exploring the Trans‐Cleavage Activity of CRISPR‐Cas12a (cpf1) for the Development of a Universal Electrochemical Biosensor DOI
Yifan Dai, Rodrigo A. Somoza, Liu Wang

et al.

Angewandte Chemie International Edition, Journal Year: 2019, Volume and Issue: 58(48), P. 17399 - 17405

Published: Oct. 1, 2019

An accurate, rapid, and cost-effective biosensor for the quantification of disease biomarkers is vital development early-diagnostic point-of-care systems. The recent discovery trans-cleavage property CRISPR type V effectors makes a potential high-accuracy bio-recognition tool. Herein, CRISPR-Cas12a (cpf1) based electrochemical (E-CRISPR) reported, which more portable than optical-transduction-based biosensors. Through optimizing in vitro activity Cas12a, E-CRIPSR was used to detect viral nucleic acids, including human papillomavirus 16 (HPV-16) parvovirus B19 (PB-19), with picomolar sensitivity. aptamer-based E-CRISPR cascade further designed detection transforming growth factor β1 (TGF-β1) protein clinical samples. As demonstrated, could enable portable, diagnostic

Language: Английский

Citations

529
Ultrasensitive and Highly Specific Lateral Flow Assays for Point-of-Care Diagnosis DOI Creative Commons
Yilin Liu, Li Zhan, Zhenpeng Qin

et al.

ACS Nano, Journal Year: 2021, Volume and Issue: 15(3), P. 3593 - 3611

Published: Feb. 19, 2021

Lateral flow assays (LFAs) are paper-based point-of-care (POC) diagnostic tools that widely used because of their low cost, ease use, and rapid format. Unfortunately, traditional commercial LFAs have significantly poorer sensitivities (μM) specificities than standard laboratory tests (enzyme-linked immunosorbent assay, ELISA: pM-fM; polymerase chain reaction, PCR: aM), thus limiting impact in disease control. In this Perspective, we review the evolving efforts to increase sensitivity specificity LFAs. Recent work improve through assay improvement includes optimization kinetics signal amplification by either reader systems or additional reagents. Together, these produced with ELISA-level (pM-fM). addition, sample preamplification can be applied both nucleic acids (direct amplification) other analytes (indirect prior LFA testing, which lead PCR-level (aM) sensitivity. However, strategies also detection time complexity, inhibits large-scale POC use Perspectives achieve future (<30 min), ultrasensitive (PCR-level), "sample-to-answer" diagnostics provided. case specificity, recent research focused on high-affinity molecules reduce nonspecific binding. Furthermore, novel highly specific molecules, such as CRISPR/Cas systems, integrated into diagnosis produce not only but diagnostics. summary, continuing improvements, may soon offer performance at is competitive techniques while retaining a

Language: Английский

Citations

421
Multiplexed CRISPR technologies for gene editing and transcriptional regulation DOI Creative Commons
Nicholas S. McCarty, Alicia E. Graham, Lucie Studená

et al.

Nature Communications, Journal Year: 2020, Volume and Issue: 11(1)

Published: March 9, 2020

Abstract Multiplexed CRISPR technologies, in which numerous gRNAs or Cas enzymes are expressed at once, have facilitated powerful biological engineering applications, vastly enhancing the scope and efficiencies of genetic editing transcriptional regulation. In this review, we discuss multiplexed technologies describe methods for assembly, expression processing synthetic guide RNA arrays vivo. Applications that benefit from including cellular recorders, circuits, biosensors, combinatorial perturbations, large-scale genome rewiring metabolic pathways, highlighted. We also offer a glimpse emerging challenges emphasize experimental considerations future studies.

Language: Английский

Citations

398
CRISPR/Cas13a‐Powered Electrochemical Microfluidic Biosensor for Nucleic Acid Amplification‐Free miRNA Diagnostics DOI Creative Commons
Richard C. Bruch,

Julia Baaske,

Claire Chatelle

et al.

Advanced Materials, Journal Year: 2019, Volume and Issue: 31(51)

Published: Oct. 30, 2019

Abstract Noncoding small RNAs, such as microRNAs, are becoming the biomarkers of choice for multiple diseases in clinical diagnostics. A dysregulation these microRNAs can be associated with many different diseases, cancer, dementia, and cardiovascular conditions. The key effective treatment is an accurate initial diagnosis at early stage, improving patient's survival chances. In this work, first clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a‐powered microfluidic, integrated electrochemical biosensor on‐site detection introduced. Through unique combination, quantification potential tumor markers microRNA miR‐19b miR‐20a realized without any nucleic acid amplification. With a readout time 9 min overall process less than 4 h, limit 10 p m achieved, using measuring volume 0.6 µL. Furthermore, feasibility platform to detect serum samples children, suffering from brain demonstrated. validation obtained results standard quantitative real‐time polymerase chain reaction method shows ability CRISPR‐powered system low‐cost, easily scalable, target amplification‐free tool based

Language: Английский

Citations

386
Cas12aVDet: A CRISPR/Cas12a-Based Platform for Rapid and Visual Nucleic Acid Detection DOI
Bei Wang, Rui Wang,

Daqi Wang

et al.

Analytical Chemistry, Journal Year: 2019, Volume and Issue: 91(19), P. 12156 - 12161

Published: Aug. 28, 2019

A rapid and sensitive method is crucial for nucleic acid detection. Recently, RNA-guided CRISPR/Cas12a nuclease-based methods present great promise In the methods, however, DNA amplification subsequent Cas12a cleavage separated whole process takes as long 2 h. Most importantly, uncapping operation increases risk of aerosol contamination. this study, we propose a CRISPR/Cas12a-based named "Cas12aVDet" By integrating recombinase polymerase (RPA) with in single reaction system, detection can be accomplished 30 min contamination avoided. The signal observed by naked eye under blue light. This could detect at molecule level demonstrated 100% accuracy mycoplasma detection, presenting potential variety applications.

Language: Английский

Citations

359