Rapid and Fully Microfluidic Ebola Virus Detection with CRISPR-Cas13a

Qin, Peiwu; Park, Myeongkee; Alfson, Kendra J.; Tamhankar, Manasi; Carrión, Ricardo; Patterson, Jean L.; Griffiths, Anthony John; He, Qian; Yıldız, Ahmet; Mathies, Richard A.; Du, Ke

doi:10.1021/acssensors.9b00239

Cited by 224 publications

(179 citation statements)

References 38 publications

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“…SHERLOCK can achieve a visual readout by combining with lateral flow readout (Gootenberg et al, ). The CRISPR‐Cas13a‐based detection has been successfully applied to detect Zika virus (ZIKV), Dengue virus (DENV), Avian influenza A (H7N9) virus, Ebola virus and other molecules such as miRNA and N1‐methyladenosine (Chen et al, ; Liu et al, ; Myhrvold et al, ; Qin et al, ; Shan, Zhou, Huang, & Xing, ). In this study, the enhanced Cas13a detection by combining RPA, with T7 transcription and the collateral effect of CRISPR‐Cas13a was developed for sensitive, specific, equipment‐free and visual detection of PRRSV targeting the conserved PRRSV M gene.…”

Section: Introductionmentioning

confidence: 99%

Visual detection of porcine reproductive and respiratory syndrome virus using CRISPR‐Cas13a

Chang

Deng

et al. 2019

Transbound Emerg Dis

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Porcine reproductive and respiratory syndrome virus (PRRSV) has varied constantly and circulated in the pig industry worldwide. The prevention and control of porcine reproductive and respiratory syndrome (PRRS) is complicated. A visual, sensitive and specific diagnostic method is advantageous to the control of PRRS. The collateral cleavage activity of LwCas13a is activated to degrade non‐targeted RNA, when crRNA of LwCas13a bond to target RNA. The enhanced Cas13a detection is the combination of collateral cleavage activity of LwCas13a and recombinase polymerase amplification (RPA). In this study, the enhanced Cas13a detection for PRRSV was established. The novel method was an isothermal detection at 37°C, and the detection can be used for real‐time analysis or visual readout. The detection limit of the enhanced Cas13a detection was 172 copies/μl, and there were no cross‐reactions with porcine circovirus 2, porcine parvovirus, classical swine fever virus and pseudorabies virus. The enhanced Cas13a detection can work well in clinical samples. In summary, a visual, sensitive and specific nucleic acid detection method based on CRISPR‐Cas13a was developed for PRRSV.

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Section: Introductionmentioning

confidence: 99%

Visual detection of porcine reproductive and respiratory syndrome virus using CRISPR‐Cas13a

Chang

Deng

et al. 2019

Transbound Emerg Dis

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“…Fluorescence Quantification: To quantify the fluorescence intensity of the ssDNA reporter probes, a custom designed fluorometer was used and has been reported before. 21,50 Briefly, a continuous wave laser with an emission peak at 488 nm (Sapphire 488 LP, Coherent) was aligned under a reservoir filled with fluorescent molecules. The fluorescence signal was collected by an off-axis parabolic mirror and a fiber coupled mini USB spectrometer (USB 2000+, Ocean Optics).…”

Section: Discussionmentioning

confidence: 99%

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) System for Rapid Viral DNA Sensing

Bao

et al. 2020

Preprint

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A fully Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) system is developed for viral DNA detection. This powerful system is patterned with high-aspect ratio micropillars to enhance reporter probe binding. After surface modification and probe immobilization, CRISPR Cas12a/crRNA complex is injected into the fully enclosed system. With the presence of double-stranded DNA target, the CRISPR enzyme is activated and non-specifically cleaves the ssDNA reporters initially immobilized on the micropillars. This collateral cleavage releases fluorescence dyes into the assay, and the intensity is linearly proportional to the target DNA concentration ranging from 0.1 to 10 nM. Importantly, this system does not rely on traditional dye-quencher labeled probe thus eliminating the fluorescence background presented in the assay. Furthermore, our one-step detection protocol is performed at isothermal conditions (37°C) without using complicated and time-consuming off-chip probe hybridization and denaturation. This miniaturized and fully packed IMPACT chip demonstrates rapid, sensitive, and simple nucleic acid detection and is an ideal candidate for the next generation molecular diagnostic platform for point-of-care (POC) applications, responding to emerging and deadly pathogen outbreaks.

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“…Clustered regularly interspaced short palindromic repeats (CRISPR) technology has emerged in recent years as a powerful tool to modify defective genes within living organisms for disease treatment (Canver et al, 2015;Dever et al, 2016;Knott et al, 2017;Schwank et al, 2013;Tambe et al, 2018) The recent discovery of CRISPR Cas12a and Cas13a, which possess the capability of recognizing a nucleic acid target, followed by the indiscriminate cleavage of nucleic acid reporters, has been used for rapid detection of pathogens East-Seletsky et al, 2017Gootenberg et al, 2017;Qin et al, 2019) Rather than using precisely controlled heating cycles in PCR, CRISPR-based detection can be operated at physiological temperature or even room temperature (Kundert et al, 2019;Malzahn et al, 2019;Qin et al, 2019), thus significantly simplifying the detection procedure. The non-specific cleavage by these CRISPR-Cas enzymes occur at a very high turnover rate, enabling accurate detection of low-concentration targets.…”

Section: Introductionmentioning

confidence: 99%

High-throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system

Bao

et al. 2019

Preprint

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Here we report the development of a high throughput, all-solution phase, and isothermal detection system to detect African Swine Fever Virus (ASFV). CRISPR-Cas12a programmed with a CRISPR RNA (crRNA) is used to detect ASFV target DNA. Upon ASFV DNA binding, the Cas12a/crRNA/ASFV DNA complex becomes activated and degrades a fluorescent single stranded DNA (ssDNA) reporter present in the assay. We combine this powerful CRISPR-Cas assay with fluorescence-based point-of-care (POC) system we developed for rapid and accurate virus detection. Without nucleic acid amplification, a detection limit of 1 pM is achieved within 2 hrs. In addition, the ternary Cas12a/crRNA/ASFV DNA complex is highly stable at physiological temperature and continues to cleave the ssDNA reporter even after 24 hrs of incubation, resulting in an improvement of the detection limit to 100 fM. We show that this system is very specific and can differentiate nucleic acid targets with closely matched sequences. The high sensitivity and selectivity of our system enables the detection of ASFV in femtomolar range. Importantly, this system features a disposable cartridge and a sensitive custom designed fluorometer, enabling compact, multiplexing, and simple ASFV detection, intended for low resource settings.

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Rapid and Fully Microfluidic Ebola Virus Detection with CRISPR-Cas13a

Cited by 224 publications

References 38 publications

Visual detection of porcine reproductive and respiratory syndrome virus using CRISPR‐Cas13a

Visual detection of porcine reproductive and respiratory syndrome virus using CRISPR‐Cas13a

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) System for Rapid Viral DNA Sensing

High-throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system

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