Cas12aVDet: A CRISPR/Cas12a-Based Platform for Rapid and Visual Nucleic Acid Detection

Wang, Bei; Wang, Rui; Wang, Daqi; Wu, Jian; Li, Jixi; Wang, Jin; Liu, Huihui; Wang, Yongming

doi:10.1021/acs.analchem.9b01526

Cited by 320 publications

(226 citation statements)

References 21 publications

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“…Compared to previously reported CRISPR-based nucleic acid detection, 14,16,18,23,24,25 our versatile and robust AIOD-CRISPR has some distinctive advantages. First, AIOD-CRISPR system is a true single reaction system.…”

Section: Discussionmentioning

confidence: 94%

“…First, AIOD-CRISPR system is a true single reaction system. All the components are prepared prior to incubation, thoroughly circumventing the preamplification of target nucleic acids, 14 physical separation of Cas enzyme, 25 or the requirement of specially designed tubes or devices 26 . Second, AIOD-CRISPR is a true isothermal nucleic acid detection method.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus

Ding

Yin

et al. 2020

Preprint

122

129

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A recent outbreak of novel coronavirus (SARS-CoV-2), the causative agent of COVID-19, has spread rapidly all over the world. Human immunodeficiency virus (HIV) is another deadly virus and causes acquired immunodeficiency syndrome (AIDS). Rapid and early detection of these viruses will facilitate early intervention and reduce disease transmission risk. Here, we present an All-In-One Dual CRISPR-Cas12a (termed "AIOD-CRISPR") assay method for simple, rapid, ultrasensitive, one-pot, and visual detection of coronavirus SARS-CoV-2 and HIV virus. In our AIOD CRISPR assay, a pair of crRNAs was introduced to initiate dual CRISPR-Cas12a detection and improve detection sensitivity. The AIOD-CRISPR assay system was successfully utilized to detect nucleic acids (DNA and RNA) of SARS-CoV-2 and HIV with a sensitivity of few copies. Also, it was evaluated by detecting HIV-1 RNA extracted from human plasma samples, achieving a comparable sensitivity with real-time RT-PCR method. Thus, our method has a great potential for developing next-generation point-of-care molecular diagnostics.

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Section: Discussionmentioning

confidence: 94%

Section: Discussionmentioning

confidence: 99%

All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus

Ding

Yin

et al. 2020

Preprint

122

129

View full text Add to dashboard Cite

show abstract

“…However, a fluorescence reporter was used in the study, which restricted its application in the laboratories 21 . A recent study developed a blue lightbased readout 29 . Though this readout does not rely on laboratory equipment, it is less applicable in the field.…”

Section: Discussionmentioning

confidence: 99%

“…However, the fluorescence readout of Cas12a-based assay requires lab instruments. According to a recent report, the detection signal can be observed by naked eye under blue light 29 . This procedure is not suitable for detection in the field, as it requires a centrifuge, and its signal can be interfered in biological samples.…”

Section: Introductionmentioning

confidence: 99%

Rapid detection of African swine fever virus using Cas12a-based portable paper diagnostics

Chen

et al. 2020

Cell Discov

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African swine fever virus (ASFV) is a dsDNA virus responsible for a severe, highly contagious, and lethal disease affecting both domestic and wild pigs. ASFV has brought enormous economic loss to a number of countries, and effective vaccine and therapy are still lacking. Therefore, a rapid, sensitive, and field-deployable detection of ASFV is important for disease surveillance and control. Herein, we developed a Cas12a-mediated portable paper assay to rapidly and precisely detect ASFV. We identified a robust set of crRNAs that recognized the highly conserved region of essential ASFV genes. The Cas12a-mediated detection assay showed low tolerance for mismatch mutations, and no cross-reactivity against other common swine pathogens. We further developed a paper-based assay to allow instrument-free detection of ASFV. Specifically, we applied gold nanoparticle-antibody conjugate to engineer homemade strips and combined it with Cas12a-mediated ASFV detection. This portable paper, instrument-free diagnostics, faithfully detected ASFV in swine samples, showing comparable sensitivity to the traditionally instrumentdependent qPCR method. Taking together, we developed a highly sensitive, instant, and economic Cas12a-mediated paper diagnostics of ASFV, with a great application potential for monitoring ASFV in the field.

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“…37,38 To extend the detection limit, our system can be integrated with isothermal amplification methods such as recombinase polymerase amplification for sensitive "one pot" target amplification and CRISPR reaction, without the background issues seen in traditional "one-pot" methods. 17,39 An advantage of the IMPACT chip compared to other detection apparatuses such as the SHERLOCK test strip is that it is a fully enclosed system without extra packaging need. 40,41 This is advantageous as the treated micropillars are sealed before molecular diagnostics.…”

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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Cas12aVDet: A CRISPR/Cas12a-Based Platform for Rapid and Visual Nucleic Acid Detection

Cited by 320 publications

References 21 publications

All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus

All-in-One Dual CRISPR-Cas12a (AIOD-CRISPR) Assay: A Case for Rapid, Ultrasensitive and Visual Detection of Novel Coronavirus SARS-CoV-2 and HIV virus

Rapid detection of African swine fever virus using Cas12a-based portable paper diagnostics

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

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