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

He, Qian; Yu, Dongmei; Bao, Mengdi; Korensky, Grant; Chen, Juhong; Shin, Mingyeong; Kim, Juwon; Park, Myeongkee; Qin, Peiwu; Du, Ke

doi:10.1016/j.bios.2020.112068

Cited by 189 publications

(152 citation statements)

References 38 publications

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“…The sequences of the DNA target and the crRNA are listed in Figure 1 f. The target was selected based on the PAM region in the ASFV-SY18 genome. 21 It has been shown that auto-fluorescence occurs from the chemical treatment of APTES and GA. 27 In our case, we were able to confirm that the phenomenon was specific to GA and APTES ( Figure S1a ) and use it to show that it only occurs on PDMS in the presence of these chemicals ( Figure S1b ). Figure 1 g shows the uniformity of the chemical treatment on the surface, as the intensity of the auto-fluorescence (emission: 525 nm) is uniform throughout.…”

Section: Resultsmentioning

confidence: 99%

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection System for Viral DNA Sensing

Hass

Bao

et al. 2020

ACS Omega

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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, the CRISPR-Cas12a/crRNA complex is injected into the fully enclosed microchannel. With the presence of a double-stranded DNA target, the CRISPR enzyme is activated and denatures the single-stranded DNA reporters from the micropillars. This collateral cleavage releases fluorescence reporters 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 the traditional dye-quencher-labeled probe, thus reducing the fluorescence background presented in the assay. Furthermore, our one-step detection protocol is performed on-chip 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 sensitive and accurate DNA detection within 120 min and paves ways to the next-generation point-of-care diagnostics, responding to emerging and deadly pathogen outbreaks.

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Section: Resultsmentioning

confidence: 99%

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection System for Viral DNA Sensing

Hass

Bao

et al. 2020

ACS Omega

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“…The target was selected based on the PAM region in the ASFV-SY18 genome. 21 It has been shown that auto-fluorescence occurs from the chemical treatment of APTES and glutaraldehyde. 22 In our case, we were able to confirm the phenomenon was specific to glutaraldehyde and APTES ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…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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“…The author used DETECTR to differentiate between different types of human papillomavirus from cultured human cells and clinical samples within 1 h [38]. In addition, another technique combines CRISPR-Cas12 with a fluorescence-based point-of-care (POC) system is recently reported for rapid and detection for accurate African Swine Fever Virus (ASFV) [49]. In this method, Cas12a/crRNA detects and binds to targeting DNA, the Cas12a/ crRNA/DNA complex becomes activated and degrades a fluorescent ssDNA reporter.…”

Section: Type V: Crispr-cas12 Based Diagnosticsmentioning

confidence: 99%

CRISPR-cas systems based molecular diagnostic tool for infectious diseases and emerging 2019 novel coronavirus (COVID-19) pneumonia

Xiang

Qian

Zhang

et al. 2020

Journal of Drug Targeting

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Emerging infectious diseases, the persistent potential for destabilising pandemics, remain a global threat leading to excessive morbidity and mortality. The current outbreak of pneumonia caused by 2019 novel coronavirus (COVID-19) illustrated difficulties in lack of effective drugs for treatment. Accurate and rapid diagnostic tools are essential for early recognition and treatment of infectious diseases, allowing timely implementation of infection control, improved clinical care and other public health measures to stop the spread of the disease. CRISPR-Cas technology speed up the development of infectious disease diagnostics with high rapid and accurate. In this review, we summarise current advance regarding diverse CRISPR-Cas systems, including CRISPR-Cas9, CRISPR-Cas12 and CRISPR-Cas13, in the development of fast, accurate and portable diagnostic tests and highlight the potential of CRISPR-Cas13 in COVID-19 Pneumonia and other emerging infectious diseases diagnosis. ARTICLE HISTORY

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

Cited by 189 publications

References 38 publications

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection System for Viral DNA Sensing

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection System for Viral DNA Sensing

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

CRISPR-cas systems based molecular diagnostic tool for infectious diseases and emerging 2019 novel coronavirus (COVID-19) pneumonia

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