Potential application of CRISPR-Cas12a fluorescence assay coupled with rapid nucleic acid amplification for detection of white spot syndrome virus in shrimp

Chaijarasphong, Thawatchai; Thammachai, Thanyawit; Itsathitphaisarn, Ornchuma; Sritunyalucksana, Kallaya; Suebsing, Rungkarn

doi:10.1016/j.aquaculture.2019.734340

Cited by 77 publications

(66 citation statements)

References 37 publications

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“…The CRISPR Cas12a assay is isothermal and amplification-free, making it a possible substitute for PCR. Furthermore, the detection sensitivity of Cas12a assay can easily be extended to attomolar level by adding an isothermal recombinase polymerase amplification (RPA) (Chaijarasphong et al, 2019). Unlike PCR which requires thermal cycles, the Cas12a assay and RPA reaction are carried out at 37°C by mixing the reagents together without the need of precise temperature control.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…These techniques are also based on the collateral cleavage activity of ssDNA probes. The potential role of this protein was also reported in the diagnosis of white spot syndrome virus in shrimps (Chaijarasphong et al, 2019). Cas12based diagnostics play an important role in the direct diagnosis of DNA viruses without undergoing RNA transcription, which makes it popular for the diagnosis of both DNA and RNA viruses ( Table 1).…”

Section: Cas12-based Diagnosticsmentioning

confidence: 94%

Next-Generation Molecular Diagnostics Development by CRISPR/Cas Tool: Rapid Detection and Surveillance of Viral Disease Outbreaks

Srivastava

Upadhyay

Srivastava

2020

Front. Mol. Biosci.

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Virus disease spreads effortlessly mechanically or through minute insect vectors that are extremely challenging to avoid. Emergence and reemergence of new viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), H1N1 influenza virus, avian influenza virus, dengue virus, Citrus tristeza virus, and Tomato yellow leaf curl virus have paralyzed the economy of many countries. The cure for major viral diseases is not feasible; however, early detection and surveillance of the disease can obstruct their spread. Therefore, advances in the field of virus diagnosis and the development of new point-of-care testing kits become necessary globally. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is an emerging technology for gene editing and diagnostics development. Several rapid nucleic acid diagnostic kits have been developed and validated using Cas9, Cas12, and Cas13 proteins. This review summarizes the CRISPR/Cas-based next-generation molecular diagnostic techniques and portability of devices for field-based utilization.

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“…SHERLOCK technique is a detection method which combines isothermal amplification with CRISPR-mediated detection [ [104] , [105] , [106] , [107] ], and the technology reduces reliance on equipment. At present, this method is often used for virus detection [ [108] , [109] , [110] ].…”

Section: Detection Methodsmentioning

confidence: 99%

“…This detection method not only has good sensitivity and specificity, but also does not require complex instruments. However, this method requires DNA extraction and amplification, and the operation of the instrument is complex, and patients cannot self-detect, which increases the risk of contact and infection of medical staff [ 107 ].…”

Section: Detection Methodsmentioning

confidence: 99%

Current methods and prospects of coronavirus detection

Deng

Liu

et al. 2021

Talanta

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Potential application of CRISPR-Cas12a fluorescence assay coupled with rapid nucleic acid amplification for detection of white spot syndrome virus in shrimp

Cited by 77 publications

References 37 publications

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

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

Next-Generation Molecular Diagnostics Development by CRISPR/Cas Tool: Rapid Detection and Surveillance of Viral Disease Outbreaks

Current methods and prospects of coronavirus detection

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