Potential application of CRISPR-Cas12a fluorescence assay coupled with rapid nucleic acid amplification for detection of white spot syndrome virus in shrimp
© 2019 Elsevier B.V. White spot disease (WSD), caused by white spot syndrome virus (WSSV), is among the most severe diseases of cultivated shrimp. Here, the CRISPR-Cas12a system coupled with nucleic acid amplification was optimized for the detection of WSSV. The CRISPR-Cas12a system was used to spec...
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th-mahidol.497172020-01-27T14:21:23Z Potential application of CRISPR-Cas12a fluorescence assay coupled with rapid nucleic acid amplification for detection of white spot syndrome virus in shrimp Thawatchai Chaijarasphong Thanyawit Thammachai Ornchuma Itsathitphaisarn Kallaya Sritunyalucksana Rungkarn Suebsing Mahidol University Thailand National Center for Genetic Engineering and Biotechnology Agricultural and Biological Sciences © 2019 Elsevier B.V. White spot disease (WSD), caused by white spot syndrome virus (WSSV), is among the most severe diseases of cultivated shrimp. Here, the CRISPR-Cas12a system coupled with nucleic acid amplification was optimized for the detection of WSSV. The CRISPR-Cas12a system was used to specifically cleave the WSSV amplicons, simultaneously releasing a quenched reporter molecule resulting in fluorescence that could be detected with a simple UV transilluminator or a microplate reader. This specific cleavage accompanied by fluorescence simultaneously revealed the presence of the amplicon and confirmed its identity, preventing false positive test results from non-specific amplicons. When coupled with PCR or recombinase polymerase amplification (RPA), the Cas12a platform was capable of detecting as few as 200 copies WSSV per reaction and displayed no cross-reactivity with other shrimp DNA viruses. The method was also un-interfered by the presence of large amounts of unrelated background DNA. Moreover, the RPA-Cas12a protocol from start to finish could be performed at a constant temperature near 37 °C and required <1 h, without the need for complex equipment. Overall, our results demonstrated that the CRISPR-Cas12a method is robust, specific, confirmatory, user-friendly and potentially adaptable for in-field diagnosis of shrimp diseases. 2020-01-27T07:21:23Z 2020-01-27T07:21:23Z 2019-10-15 Article Aquaculture. Vol.512, (2019) 10.1016/j.aquaculture.2019.734340 00448486 2-s2.0-85071650221 https://repository.li.mahidol.ac.th/handle/123456789/49717 Mahidol University SCOPUS https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85071650221&origin=inward |
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Agricultural and Biological Sciences Thawatchai Chaijarasphong Thanyawit Thammachai Ornchuma Itsathitphaisarn Kallaya Sritunyalucksana Rungkarn Suebsing Potential application of CRISPR-Cas12a fluorescence assay coupled with rapid nucleic acid amplification for detection of white spot syndrome virus in shrimp |
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© 2019 Elsevier B.V. White spot disease (WSD), caused by white spot syndrome virus (WSSV), is among the most severe diseases of cultivated shrimp. Here, the CRISPR-Cas12a system coupled with nucleic acid amplification was optimized for the detection of WSSV. The CRISPR-Cas12a system was used to specifically cleave the WSSV amplicons, simultaneously releasing a quenched reporter molecule resulting in fluorescence that could be detected with a simple UV transilluminator or a microplate reader. This specific cleavage accompanied by fluorescence simultaneously revealed the presence of the amplicon and confirmed its identity, preventing false positive test results from non-specific amplicons. When coupled with PCR or recombinase polymerase amplification (RPA), the Cas12a platform was capable of detecting as few as 200 copies WSSV per reaction and displayed no cross-reactivity with other shrimp DNA viruses. The method was also un-interfered by the presence of large amounts of unrelated background DNA. Moreover, the RPA-Cas12a protocol from start to finish could be performed at a constant temperature near 37 °C and required <1 h, without the need for complex equipment. Overall, our results demonstrated that the CRISPR-Cas12a method is robust, specific, confirmatory, user-friendly and potentially adaptable for in-field diagnosis of shrimp diseases. |
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Mahidol University |
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Mahidol University Thawatchai Chaijarasphong Thanyawit Thammachai Ornchuma Itsathitphaisarn Kallaya Sritunyalucksana Rungkarn Suebsing |
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Thawatchai Chaijarasphong Thanyawit Thammachai Ornchuma Itsathitphaisarn Kallaya Sritunyalucksana Rungkarn Suebsing |
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Thawatchai Chaijarasphong |
title |
Potential application of CRISPR-Cas12a fluorescence assay coupled with rapid nucleic acid amplification for detection of white spot syndrome virus in shrimp |
title_short |
Potential application of CRISPR-Cas12a fluorescence assay coupled with rapid nucleic acid amplification for detection of white spot syndrome virus in shrimp |
title_full |
Potential application of CRISPR-Cas12a fluorescence assay coupled with rapid nucleic acid amplification for detection of white spot syndrome virus in shrimp |
title_fullStr |
Potential application of CRISPR-Cas12a fluorescence assay coupled with rapid nucleic acid amplification for detection of white spot syndrome virus in shrimp |
title_full_unstemmed |
Potential application of CRISPR-Cas12a fluorescence assay coupled with rapid nucleic acid amplification for detection of white spot syndrome virus in shrimp |
title_sort |
potential application of crispr-cas12a fluorescence assay coupled with rapid nucleic acid amplification for detection of white spot syndrome virus in shrimp |
publishDate |
2020 |
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https://repository.li.mahidol.ac.th/handle/123456789/49717 |
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1763490699798380544 |