Contamination-free visual detection of CaMV35S promoter amplicon using CRISPR/Cas12a coupled with a designed reaction vessel: Rapid, specific and sensitive

Wu, Hui; He, Jinsong; Zhang, Fang; Ping, Jianfeng; Wu, Jian

doi:10.1016/j.aca.2019.10.042

Cited by 66 publications

(39 citation statements)

References 26 publications

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“…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%

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

119

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.

show abstract

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

119

129

View full text Add to dashboard Cite

show abstract

“…In addition, related applications in plants have also been proposed. For instance, Cas12a is engineered to detect the virus of grapevine and CaMV35S promoter in genetically modified soybeans ( Li et al, 2019b ; Wu et al, 2019 ). Zhang et al (2020) first utilized the CRISPR/Cas12a system to realize the fast detection of the rice blast pathogen and GMO in rice.…”

Section: Discussionmentioning

confidence: 99%

Recombinase Polymerase Amplification/Cas12a-Based Identification of Xanthomonas arboricola pv. pruni on Peach

et al. 2021

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Peach bacterial spot caused by Xanthomonas arboricola pv. pruni (Xap) is a devastating disease worldwide and frequently causes massive economic losses. In recent years, it has become a pandemic outbreak in most peach production areas of China, especially on precocious peaches in the middle reach of the Yangtze River. Rapid, user-friendly detection is extremely important to make the correct diagnosis and develop suitable control strategies. In this study, we described a recombinase polymerase amplification (RPA)/Cas12a-based system that combines RPA and CRISPR/Cas12a for Xap identification. A total of three crRNAs were designed to target a highly conserved ABC transporter ATP-binding protein-encoding gene ftsX to make specific detection of Xap. Results showed that crRNA 2 and crRNA 3 could get consistent detection for Xap. To realize the visualization of detection results, we additionally introduced FQ-reporter and FB-reporter. The developed method was highly sensitive and could detect as low as 10–18 M Xap gDNA with a mini-UV torch, corresponding to 1.63 copies/μl or 8.855 fg/μl gDNA of Xap, while with lateral flow strips, the sensitivity was 10–17 M. In addition, this method could specifically detect Xap from other closely related bacteria or pathogens associated with peach diseases. Furthermore, this method could make correct identification for Xap with crude DNA using NaOH-based extraction (3 min) directly from diseased peach samples. Considering that the developed method could get results within 2 h and could be performed at 37°C (body temperature), it is promising to be applied for Xap diagnosis and monitoring in fields.

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“…To enable one-pot CRISPR reactions dehydrated CRISPR may for example be attached to the lid of a reaction tube for enhanced temperature stability ( Wu et al, 2020b ) ( Qian et al, 2019 ). Wu et al showed in 2020 their design of a lid where the Cas12a solution was separated from the reaction solution by a sealed film ( Wu et al, 2020a ). After the LAMP amplification step, the lid could be tightened, allowing a needle to puncture the sealed film ( Fig.…”

Section: Challenges In Poc Crispr Sensing Systemsmentioning

confidence: 99%

Section: Challenges In Poc Crispr Sensing Systemsmentioning

confidence: 99%

Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities

Dongen

Berendsen

Steenbergen

et al. 2020

Biosensors and Bioelectronics

262

191

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With the trend of moving molecular tests from clinical laboratories to on-site testing, there is a need for nucleic acid based diagnostic tools combining the sensitivity, specificity and flexibility of established diagnostics with the ease, cost effectiveness and speed of isothermal amplification and detection methods. A promising new nucleic acid detection method is Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease (Cas)-based sensing. In this method Cas effector proteins are used as highly specific sequence recognition elements that can be combined with many different read-out methods for on-site point-of-care testing. This review covers the technical aspects of integrating CRISPR/Cas technology in miniaturized sensors for analysis on-site. We start with a short introduction to CRISPR/Cas systems and the different effector proteins and continue with reviewing the recent developments of integrating CRISPR sensing in miniaturized sensors for point-of-care applications. Finally, we discuss the challenges of point-of-care CRISPR sensing and describe future research perspectives.

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Contamination-free visual detection of CaMV35S promoter amplicon using CRISPR/Cas12a coupled with a designed reaction vessel: Rapid, specific and sensitive

Cited by 66 publications

References 26 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

Recombinase Polymerase Amplification/Cas12a-Based Identification of Xanthomonas arboricola pv. pruni on Peach

Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities

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