CRISPR-Cas fluorescent cleavage assay coupled with recombinase polymerase amplification for sensitive and specific detection of Enterocytozoon hepatopenaei
Enterocytozoon hepatopenaei (EHP) is a parasite that infects pacific whiteleg shrimp, Penaeus vannamei, causing growth retardation and uneven size distributions that lead to severe losses in shrimp productivity. Routine monitoring is crucial to timely prevention and management of EHP, but field-depl...
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doaj-3f829d182260405e9412470c514dac4d2020-11-25T02:52:31ZengElsevierBiotechnology Reports2215-017X2020-09-0127e00485CRISPR-Cas fluorescent cleavage assay coupled with recombinase polymerase amplification for sensitive and specific detection of Enterocytozoon hepatopenaeiSuthasinee Kanitchinda0Jiraporn Srisala1Rungkarn Suebsing2Anuphap Prachumwat3Thawatchai Chaijarasphong4Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, ThailandAquatic Animal Health Research Team (AQHT), Integrative Aquaculture Biotechnology Research Group (AAQG), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Yothi Office, Rama VI Road, Bangkok 10400, ThailandAquatic Animal Health Research Team (AQHT), Integrative Aquaculture Biotechnology Research Group (AAQG), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Yothi Office, Rama VI Road, Bangkok 10400, ThailandCenter of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand; Aquatic Animal Health Research Team (AQHT), Integrative Aquaculture Biotechnology Research Group (AAQG), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Yothi Office, Rama VI Road, Bangkok 10400, ThailandCenter of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand; Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand; Corresponding author.Enterocytozoon hepatopenaei (EHP) is a parasite that infects pacific whiteleg shrimp, Penaeus vannamei, causing growth retardation and uneven size distributions that lead to severe losses in shrimp productivity. Routine monitoring is crucial to timely prevention and management of EHP, but field-deployable diagnostic kits for EHP are still scarce. Here, we proposed the use of recombinase polymerase amplification (RPA) and CRISPR-Cas12a fluorescence assay, henceforth RPA-Cas12a, for detection of EHP. Targeting ptp2 gene, RPA-Cas12a could detect as few as 50 copies of DNA and showed no reactivity with closely related microsporidia. The entire procedure could be performed at a temperature close to 37 °C within 1 h. Naked eye visualization was possible with UV/blue-light excitation or lateral flow detection. Thus, RPA-Cas12a is a rapid, sensitive and specific detection platform that requires no sophisticated equipment and shows promise for on-site surveillance of EHP.http://www.sciencedirect.com/science/article/pii/S2215017X20301272Enterocytozoon hepatopenaeiCRISPR-Cas12aRPALateral flow detection |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Suthasinee Kanitchinda Jiraporn Srisala Rungkarn Suebsing Anuphap Prachumwat Thawatchai Chaijarasphong |
spellingShingle |
Suthasinee Kanitchinda Jiraporn Srisala Rungkarn Suebsing Anuphap Prachumwat Thawatchai Chaijarasphong CRISPR-Cas fluorescent cleavage assay coupled with recombinase polymerase amplification for sensitive and specific detection of Enterocytozoon hepatopenaei Biotechnology Reports Enterocytozoon hepatopenaei CRISPR-Cas12a RPA Lateral flow detection |
author_facet |
Suthasinee Kanitchinda Jiraporn Srisala Rungkarn Suebsing Anuphap Prachumwat Thawatchai Chaijarasphong |
author_sort |
Suthasinee Kanitchinda |
title |
CRISPR-Cas fluorescent cleavage assay coupled with recombinase polymerase amplification for sensitive and specific detection of Enterocytozoon hepatopenaei |
title_short |
CRISPR-Cas fluorescent cleavage assay coupled with recombinase polymerase amplification for sensitive and specific detection of Enterocytozoon hepatopenaei |
title_full |
CRISPR-Cas fluorescent cleavage assay coupled with recombinase polymerase amplification for sensitive and specific detection of Enterocytozoon hepatopenaei |
title_fullStr |
CRISPR-Cas fluorescent cleavage assay coupled with recombinase polymerase amplification for sensitive and specific detection of Enterocytozoon hepatopenaei |
title_full_unstemmed |
CRISPR-Cas fluorescent cleavage assay coupled with recombinase polymerase amplification for sensitive and specific detection of Enterocytozoon hepatopenaei |
title_sort |
crispr-cas fluorescent cleavage assay coupled with recombinase polymerase amplification for sensitive and specific detection of enterocytozoon hepatopenaei |
publisher |
Elsevier |
series |
Biotechnology Reports |
issn |
2215-017X |
publishDate |
2020-09-01 |
description |
Enterocytozoon hepatopenaei (EHP) is a parasite that infects pacific whiteleg shrimp, Penaeus vannamei, causing growth retardation and uneven size distributions that lead to severe losses in shrimp productivity. Routine monitoring is crucial to timely prevention and management of EHP, but field-deployable diagnostic kits for EHP are still scarce. Here, we proposed the use of recombinase polymerase amplification (RPA) and CRISPR-Cas12a fluorescence assay, henceforth RPA-Cas12a, for detection of EHP. Targeting ptp2 gene, RPA-Cas12a could detect as few as 50 copies of DNA and showed no reactivity with closely related microsporidia. The entire procedure could be performed at a temperature close to 37 °C within 1 h. Naked eye visualization was possible with UV/blue-light excitation or lateral flow detection. Thus, RPA-Cas12a is a rapid, sensitive and specific detection platform that requires no sophisticated equipment and shows promise for on-site surveillance of EHP. |
topic |
Enterocytozoon hepatopenaei CRISPR-Cas12a RPA Lateral flow detection |
url |
http://www.sciencedirect.com/science/article/pii/S2215017X20301272 |
work_keys_str_mv |
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