Mesophilic Argonaute-based isothermal detection of SARS-CoV-2
Coronavirus disease (COVID-19), caused by SARS-CoV-2 infection and its mutations, has spread rapidly all over the world and still requires sensitive detection to distinguish mutations. CRISPR-based diagnosis has been regarded as a next-generation detection method; however, it has some limitations, s...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-07-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.957977/full |
| _version_ | 1811312536569511936 |
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| author | Xiao Li Huarong Dong Xiang Guo Fei Huang Xiaoyi Xu Nuolan Li Yu Yang Tianbao Yao Yan Feng Qian Liu |
| author_facet | Xiao Li Huarong Dong Xiang Guo Fei Huang Xiaoyi Xu Nuolan Li Yu Yang Tianbao Yao Yan Feng Qian Liu |
| author_sort | Xiao Li |
| collection | DOAJ |
| description | Coronavirus disease (COVID-19), caused by SARS-CoV-2 infection and its mutations, has spread rapidly all over the world and still requires sensitive detection to distinguish mutations. CRISPR-based diagnosis has been regarded as a next-generation detection method; however, it has some limitations, such as the need for specific recognition sequences and multiple enzymes for multiplex detection. Therefore, research on the exploration and development of novel nucleases helps to promote specific and sensitive diagnoses. Prokaryotic Argonaute (Ago) proteins exert directed nuclease activity that can target any sequence. Recently, thermophilic Agos have been developed as new detection techniques achieving multiplexity for multiple targets using a single enzyme, as well as accurate recognition of single-base differential sequences. In this study, to overcome the requirement for high reaction temperature of thermophilic Ago-based methods, we expanded the mining of mesophilic Agos to achieve CRISPR-like isothermal detection, named mesophilic Ago-based isothermal detection method (MAIDEN). The principle of MAIDEN uses mesophilic Ago cleavage combined with reverse transcription, which can provide single-strand DNA as a substrate and allow cleavage of fluorescence probes to sense SARS-CoV-2 at moderate temperature. We first mined and optimized the mesophilic Ago and the fluorescence reporter system and then selected a compatible reverse transcription reaction. Furthermore, we optimized MAIDEN into a one-step reaction that can detect SARS-CoV-2 RNA at the nanomolar concentration at a constant temperature of 42°C within 60 min. Therefore, MAIDEN shows advantageous portability and easy-to-implement operation, avoiding the possibility of open-lid contamination. Our study was the first attempt to demonstrate that mesophilic Agos can be harnessed as diagnostic tools, and MAIDEN was easily extended to detect other pathogens in a rapid and efficient manner. |
| first_indexed | 2024-04-13T10:38:03Z |
| format | Article |
| id | doaj.art-5e2962bc3df4406a99fe6d3b5afee3ed |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-13T10:38:03Z |
| publishDate | 2022-07-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-5e2962bc3df4406a99fe6d3b5afee3ed2022-12-22T02:49:59ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2022-07-011310.3389/fmicb.2022.957977957977Mesophilic Argonaute-based isothermal detection of SARS-CoV-2Xiao Li0Huarong Dong1Xiang Guo2Fei Huang3Xiaoyi Xu4Nuolan Li5Yu Yang6Tianbao Yao7Yan Feng8Qian Liu9State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, ChinaState Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, ChinaState Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, ChinaState Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, ChinaState Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, ChinaState Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, ChinaState Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, ChinaDepartment of Cardiology, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, ChinaState Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, ChinaState Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, ChinaCoronavirus disease (COVID-19), caused by SARS-CoV-2 infection and its mutations, has spread rapidly all over the world and still requires sensitive detection to distinguish mutations. CRISPR-based diagnosis has been regarded as a next-generation detection method; however, it has some limitations, such as the need for specific recognition sequences and multiple enzymes for multiplex detection. Therefore, research on the exploration and development of novel nucleases helps to promote specific and sensitive diagnoses. Prokaryotic Argonaute (Ago) proteins exert directed nuclease activity that can target any sequence. Recently, thermophilic Agos have been developed as new detection techniques achieving multiplexity for multiple targets using a single enzyme, as well as accurate recognition of single-base differential sequences. In this study, to overcome the requirement for high reaction temperature of thermophilic Ago-based methods, we expanded the mining of mesophilic Agos to achieve CRISPR-like isothermal detection, named mesophilic Ago-based isothermal detection method (MAIDEN). The principle of MAIDEN uses mesophilic Ago cleavage combined with reverse transcription, which can provide single-strand DNA as a substrate and allow cleavage of fluorescence probes to sense SARS-CoV-2 at moderate temperature. We first mined and optimized the mesophilic Ago and the fluorescence reporter system and then selected a compatible reverse transcription reaction. Furthermore, we optimized MAIDEN into a one-step reaction that can detect SARS-CoV-2 RNA at the nanomolar concentration at a constant temperature of 42°C within 60 min. Therefore, MAIDEN shows advantageous portability and easy-to-implement operation, avoiding the possibility of open-lid contamination. Our study was the first attempt to demonstrate that mesophilic Agos can be harnessed as diagnostic tools, and MAIDEN was easily extended to detect other pathogens in a rapid and efficient manner.https://www.frontiersin.org/articles/10.3389/fmicb.2022.957977/fullmesophilic AgoRNA virusesisothermal detectionone-step methodSARS-CoV-2 |
| spellingShingle | Xiao Li Huarong Dong Xiang Guo Fei Huang Xiaoyi Xu Nuolan Li Yu Yang Tianbao Yao Yan Feng Qian Liu Mesophilic Argonaute-based isothermal detection of SARS-CoV-2 Frontiers in Microbiology mesophilic Ago RNA viruses isothermal detection one-step method SARS-CoV-2 |
| title | Mesophilic Argonaute-based isothermal detection of SARS-CoV-2 |
| title_full | Mesophilic Argonaute-based isothermal detection of SARS-CoV-2 |
| title_fullStr | Mesophilic Argonaute-based isothermal detection of SARS-CoV-2 |
| title_full_unstemmed | Mesophilic Argonaute-based isothermal detection of SARS-CoV-2 |
| title_short | Mesophilic Argonaute-based isothermal detection of SARS-CoV-2 |
| title_sort | mesophilic argonaute based isothermal detection of sars cov 2 |
| topic | mesophilic Ago RNA viruses isothermal detection one-step method SARS-CoV-2 |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.957977/full |
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