Development of a rapid on-site detection method for largemouth bass virus based on RPA-CRISPR/Cas12a system
IntroductionLargemouth bass virus (LMBV), the causative agent of largemouth bass ulcerative syndrome, poses a significant economic threat to the aquaculture industry. Rapid, simple, and reliable detection methods are essential for the timely identification of LMBV infections, enabling effective prev...
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Frontiers Media S.A.
2025-05-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1599006/full |
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| author | Na Li Chunmei Dai Yuelin Mao Yimeng Zhang Huiyuan Yan Huijiao Wang Yinghui Qin Baicheng Huang Xinjie Wang Lunguang Yao |
| author_facet | Na Li Chunmei Dai Yuelin Mao Yimeng Zhang Huiyuan Yan Huijiao Wang Yinghui Qin Baicheng Huang Xinjie Wang Lunguang Yao |
| author_sort | Na Li |
| collection | DOAJ |
| description | IntroductionLargemouth bass virus (LMBV), the causative agent of largemouth bass ulcerative syndrome, poses a significant economic threat to the aquaculture industry. Rapid, simple, and reliable detection methods are essential for the timely identification of LMBV infections, enabling effective prevention and control measures.MethodsIn this study, a detection platform utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system was developed for LMBV. CRISPR RNA (crRNA) and recombinase polymerase amplification (RPA) primers were designed to target the highly conserved region of the major capsid protein (MCP) gene. Additionally, a one-pot method and lyophilization strategy were optimized for field applications.ResultsThe RPA-CRISPR/Cas12a system achieved a sensitivity of 50 copies/reaction within 40 minutes, without requiring specialized equipment, and exhibits high specificity for LMBV. Validation with 42 clinical samples of suspected LMBV infection demonstrated 100% concordance among the RPA-CRISPR/Cas12a method, quantitative PCR and lateral flow strip assay. The one-pot method and lyophilization strategy demonstrated consistent detection results with two-step RPA-CRISPR methods in clinical sample testing, offering more convenient and stable application characteristics for on-site detection.DiscussionThis study establishes an efficient process for detecting LMBV nucleic acids in fish clinical samples, culminating in a CRISPR-based fluorescent readout, offering significant advantages for viral diagnosis and monitoring. |
| format | Article |
| id | doaj-art-76800dadef0546389acc2eae7c0b7d09 |
| institution | OA Journals |
| issn | 1664-302X |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Microbiology |
| spelling | doaj-art-76800dadef0546389acc2eae7c0b7d092025-08-20T01:55:30ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2025-05-011610.3389/fmicb.2025.15990061599006Development of a rapid on-site detection method for largemouth bass virus based on RPA-CRISPR/Cas12a systemNa Li0Chunmei Dai1Yuelin Mao2Yimeng Zhang3Huiyuan Yan4Huijiao Wang5Yinghui Qin6Baicheng Huang7Xinjie Wang8Lunguang Yao9Henan Provincial Engineering and Technology Center of Health Products for Livestock and Poultry, Henan Field Observation and Research Station of Headwork Wetland Ecosystem of The Central Route of South-to-North Water Diversion Project, School of Life Science, Nanyang Normal University, Nanyang, ChinaHenan Provincial Engineering and Technology Center of Health Products for Livestock and Poultry, Henan Field Observation and Research Station of Headwork Wetland Ecosystem of The Central Route of South-to-North Water Diversion Project, School of Life Science, Nanyang Normal University, Nanyang, ChinaHenan Provincial Engineering and Technology Center of Health Products for Livestock and Poultry, Henan Field Observation and Research Station of Headwork Wetland Ecosystem of The Central Route of South-to-North Water Diversion Project, School of Life Science, Nanyang Normal University, Nanyang, ChinaHenan Provincial Engineering and Technology Center of Health Products for Livestock and Poultry, Henan Field Observation and Research Station of Headwork Wetland Ecosystem of The Central Route of South-to-North Water Diversion Project, School of Life Science, Nanyang Normal University, Nanyang, ChinaHenan Provincial Engineering and Technology Center of Health Products for Livestock and Poultry, Henan Field Observation and Research Station of Headwork Wetland Ecosystem of The Central Route of South-to-North Water Diversion Project, School of Life Science, Nanyang Normal University, Nanyang, ChinaHenan Provincial Engineering and Technology Center of Health Products for Livestock and Poultry, Henan Field Observation and Research Station of Headwork Wetland Ecosystem of The Central Route of South-to-North Water Diversion Project, School of Life Science, Nanyang Normal University, Nanyang, ChinaHenan Provincial Engineering and Technology Center of Health Products for Livestock and Poultry, Henan Field Observation and Research Station of Headwork Wetland Ecosystem of The Central Route of South-to-North Water Diversion Project, School of Life Science, Nanyang Normal University, Nanyang, ChinaZhejiang Lab, Hangzhou, ChinaGenome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, ChinaHenan Provincial Engineering and Technology Center of Health Products for Livestock and Poultry, Henan Field Observation and Research Station of Headwork Wetland Ecosystem of The Central Route of South-to-North Water Diversion Project, School of Life Science, Nanyang Normal University, Nanyang, ChinaIntroductionLargemouth bass virus (LMBV), the causative agent of largemouth bass ulcerative syndrome, poses a significant economic threat to the aquaculture industry. Rapid, simple, and reliable detection methods are essential for the timely identification of LMBV infections, enabling effective prevention and control measures.MethodsIn this study, a detection platform utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system was developed for LMBV. CRISPR RNA (crRNA) and recombinase polymerase amplification (RPA) primers were designed to target the highly conserved region of the major capsid protein (MCP) gene. Additionally, a one-pot method and lyophilization strategy were optimized for field applications.ResultsThe RPA-CRISPR/Cas12a system achieved a sensitivity of 50 copies/reaction within 40 minutes, without requiring specialized equipment, and exhibits high specificity for LMBV. Validation with 42 clinical samples of suspected LMBV infection demonstrated 100% concordance among the RPA-CRISPR/Cas12a method, quantitative PCR and lateral flow strip assay. The one-pot method and lyophilization strategy demonstrated consistent detection results with two-step RPA-CRISPR methods in clinical sample testing, offering more convenient and stable application characteristics for on-site detection.DiscussionThis study establishes an efficient process for detecting LMBV nucleic acids in fish clinical samples, culminating in a CRISPR-based fluorescent readout, offering significant advantages for viral diagnosis and monitoring.https://www.frontiersin.org/articles/10.3389/fmicb.2025.1599006/fulllargemouth bass virusMCP generecombinase polymerase amplificationCRISPR/Cas12adetection |
| spellingShingle | Na Li Chunmei Dai Yuelin Mao Yimeng Zhang Huiyuan Yan Huijiao Wang Yinghui Qin Baicheng Huang Xinjie Wang Lunguang Yao Development of a rapid on-site detection method for largemouth bass virus based on RPA-CRISPR/Cas12a system Frontiers in Microbiology largemouth bass virus MCP gene recombinase polymerase amplification CRISPR/Cas12a detection |
| title | Development of a rapid on-site detection method for largemouth bass virus based on RPA-CRISPR/Cas12a system |
| title_full | Development of a rapid on-site detection method for largemouth bass virus based on RPA-CRISPR/Cas12a system |
| title_fullStr | Development of a rapid on-site detection method for largemouth bass virus based on RPA-CRISPR/Cas12a system |
| title_full_unstemmed | Development of a rapid on-site detection method for largemouth bass virus based on RPA-CRISPR/Cas12a system |
| title_short | Development of a rapid on-site detection method for largemouth bass virus based on RPA-CRISPR/Cas12a system |
| title_sort | development of a rapid on site detection method for largemouth bass virus based on rpa crispr cas12a system |
| topic | largemouth bass virus MCP gene recombinase polymerase amplification CRISPR/Cas12a detection |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1599006/full |
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