A new method to detect red spotted grouper neuro necrosis virus (RGNNV) based on CRISPR/Cas13a
Red-spotted grouper nervous necrosis virus (RGNNV) is a serious pathogen that causes high mortality in many species of fish in marine aquaculture. Larvae and juveniles are more easily infected by RGNNV, and the cumulative mortality can reach 100% post-infection. To date, a simple, convenient, and lo...
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| Published in | Aquaculture Vol. 555; p. 738217 |
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| Main Authors | , , , , , , , , , |
| Format | Journal Article |
| Language | English |
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Elsevier B.V
30.06.2022
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| Abstract | Red-spotted grouper nervous necrosis virus (RGNNV) is a serious pathogen that causes high mortality in many species of fish in marine aquaculture. Larvae and juveniles are more easily infected by RGNNV, and the cumulative mortality can reach 100% post-infection. To date, a simple, convenient, and low complexity method to detect this virus does not exist. Therefore, we developed a rapid detection method for RGNNV based on the CRISPR-Cas13a system. We optimized each of the four parts of the detection system: selection of efficient CRISPR-derived RNA(crRNA), reaction temperature, control of reporter probe content, and reaction concentration ratio of Cas13a to crRNA. Our detection system was sensitive enough to detect single-stranded RGNNV at a minimum of 102 fM. We evaluated the effectiveness of the detection system by testing it on different NNVs and RGNNV-infected RGNNV fish from different regions of China and found that the system had high specificity. Overall, our results showed that the CRISPR-Cas13a detection system was a robust, specific, and validated method for RGNNV detection. Thus, it provided a theoretical basis for the field detection of RGNNV.
•Engineered Crispr/Cas13a system successfully dedect RGNNV.•2.Designed and selected specific crRNA that accurately target RGNNV.•3.Our detection system was sensitive enough to detect RGNNV at a minimum of 102 fM |
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| AbstractList | Red-spotted grouper nervous necrosis virus (RGNNV) is a serious pathogen that causes high mortality in many species of fish in marine aquaculture. Larvae and juveniles are more easily infected by RGNNV, and the cumulative mortality can reach 100% post-infection. To date, a simple, convenient, and low complexity method to detect this virus does not exist. Therefore, we developed a rapid detection method for RGNNV based on the CRISPR-Cas13a system. We optimized each of the four parts of the detection system: selection of efficient CRISPR-derived RNA(crRNA), reaction temperature, control of reporter probe content, and reaction concentration ratio of Cas13a to crRNA. Our detection system was sensitive enough to detect single-stranded RGNNV at a minimum of 102 fM. We evaluated the effectiveness of the detection system by testing it on different NNVs and RGNNV-infected RGNNV fish from different regions of China and found that the system had high specificity. Overall, our results showed that the CRISPR-Cas13a detection system was a robust, specific, and validated method for RGNNV detection. Thus, it provided a theoretical basis for the field detection of RGNNV.
•Engineered Crispr/Cas13a system successfully dedect RGNNV.•2.Designed and selected specific crRNA that accurately target RGNNV.•3.Our detection system was sensitive enough to detect RGNNV at a minimum of 102 fM |
| ArticleNumber | 738217 |
| Author | Liang, Kaishan Zhou, Xiaoming Yang, Min Shan, Jinhong Duan, Xuzhuo Zhao, Huihong Qin, Qiwei Huang, Fengqi Jia, Xianze Wang, Qing |
| Author_xml | – sequence: 1 givenname: Fengqi surname: Huang fullname: Huang, Fengqi organization: College of Marine Sciences, South China Agricultural University, Guangzhou 510642, China – sequence: 2 givenname: Jinhong surname: Shan fullname: Shan, Jinhong organization: College of Marine Sciences, South China Agricultural University, Guangzhou 510642, China – sequence: 3 givenname: Kaishan surname: Liang fullname: Liang, Kaishan organization: College of Marine Sciences, South China Agricultural University, Guangzhou 510642, China – sequence: 4 givenname: Min surname: Yang fullname: Yang, Min organization: College of Marine Sciences, South China Agricultural University, Guangzhou 510642, China – sequence: 5 givenname: Xiaoming surname: Zhou fullname: Zhou, Xiaoming organization: MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China – sequence: 6 givenname: Xuzhuo surname: Duan fullname: Duan, Xuzhuo organization: College of Marine Sciences, South China Agricultural University, Guangzhou 510642, China – sequence: 7 givenname: Xianze surname: Jia fullname: Jia, Xianze organization: College of Marine Sciences, South China Agricultural University, Guangzhou 510642, China – sequence: 8 givenname: Huihong surname: Zhao fullname: Zhao, Huihong organization: College of Marine Sciences, South China Agricultural University, Guangzhou 510642, China – sequence: 9 givenname: Qiwei surname: Qin fullname: Qin, Qiwei email: qinqw@scau.edu.cn organization: College of Marine Sciences, South China Agricultural University, Guangzhou 510642, China – sequence: 10 givenname: Qing surname: Wang fullname: Wang, Qing email: wangqing@scau.edu.cn organization: College of Marine Sciences, South China Agricultural University, Guangzhou 510642, China |
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| Keywords | CRISPR-Cas13a Grouper RGNNV Nucleic acid detection |
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| Title | A new method to detect red spotted grouper neuro necrosis virus (RGNNV) based on CRISPR/Cas13a |
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