Publication: 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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Issued Date
2019-10-15
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00448486
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2-s2.0-85071650221
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Mahidol University
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SCOPUS
Bibliographic Citation
Aquaculture. Vol.512, (2019)
Suggested Citation
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. Aquaculture. Vol.512, (2019). doi:10.1016/j.aquaculture.2019.734340 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/49717
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Title
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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Abstract
© 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.