RT-RPA-PfAgo System: A Rapid, Sensitive, and Specific Multiplex Detection Method for Rice-Infecting Viruses
The advancement in CRISPR-Cas biosensors has transmuted the detection of plant viruses owing to their rapid and higher sensitivity. However, false positives and restricted multiplexing capabilities are still the challenges faced by this technology, demanding the exploration of novel methodologies. I...
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MDPI AG
2023-10-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/13/10/941 |
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| author | Yan Liu Wenqiang Xia Wei Zhao Peiying Hao Zhengliang Wang Xiaoping Yu Xuping Shentu Kai Sun |
| author_facet | Yan Liu Wenqiang Xia Wei Zhao Peiying Hao Zhengliang Wang Xiaoping Yu Xuping Shentu Kai Sun |
| author_sort | Yan Liu |
| collection | DOAJ |
| description | The advancement in CRISPR-Cas biosensors has transmuted the detection of plant viruses owing to their rapid and higher sensitivity. However, false positives and restricted multiplexing capabilities are still the challenges faced by this technology, demanding the exploration of novel methodologies. In this study, a novel detection system was developed by integrating reverse transcriptome (RT) techniques with recombinase polymerase isothermal amplification (RPA) and <i>Pyrococcus furiosus</i> Argonaute (PfAgo). The RT-RPA-PfAgo system enabled the simultaneous detection of rice ragged stunt virus (RRSV), rice grassy stunt virus (RGSV), and rice black streaked dwarf virus (RBSDV). Identifying targets via guide DNA without being hindered by protospacer adjacent motif sequences is the inherent merit of PfAgo, with the additional advantage of it being simple, cost-effective, and exceptionally sensitive, with detection limits between 3.13 and 5.13 copies/µL, in addition to it effectively differentiating between the three distinct viruses. The field evaluations were also in accordance with RT-PCR methods. The RT-RPA-PfAgo system proved to be a robust, versatile, highly specific, and sensitive method with great potential for practicality in future plant virus diagnostics. |
| first_indexed | 2024-03-10T21:23:39Z |
| format | Article |
| id | doaj.art-8c79ac66af3f468e836c7a601460b9cd |
| institution | Directory Open Access Journal |
| issn | 2079-6374 |
| language | English |
| last_indexed | 2024-03-10T21:23:39Z |
| publishDate | 2023-10-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj.art-8c79ac66af3f468e836c7a601460b9cd2023-11-19T15:51:38ZengMDPI AGBiosensors2079-63742023-10-01131094110.3390/bios13100941RT-RPA-PfAgo System: A Rapid, Sensitive, and Specific Multiplex Detection Method for Rice-Infecting VirusesYan Liu0Wenqiang Xia1Wei Zhao2Peiying Hao3Zhengliang Wang4Xiaoping Yu5Xuping Shentu6Kai Sun7Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, ChinaInstitute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, ChinaZhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, ChinaZhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, ChinaZhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, ChinaZhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, ChinaZhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, ChinaZhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, ChinaThe advancement in CRISPR-Cas biosensors has transmuted the detection of plant viruses owing to their rapid and higher sensitivity. However, false positives and restricted multiplexing capabilities are still the challenges faced by this technology, demanding the exploration of novel methodologies. In this study, a novel detection system was developed by integrating reverse transcriptome (RT) techniques with recombinase polymerase isothermal amplification (RPA) and <i>Pyrococcus furiosus</i> Argonaute (PfAgo). The RT-RPA-PfAgo system enabled the simultaneous detection of rice ragged stunt virus (RRSV), rice grassy stunt virus (RGSV), and rice black streaked dwarf virus (RBSDV). Identifying targets via guide DNA without being hindered by protospacer adjacent motif sequences is the inherent merit of PfAgo, with the additional advantage of it being simple, cost-effective, and exceptionally sensitive, with detection limits between 3.13 and 5.13 copies/µL, in addition to it effectively differentiating between the three distinct viruses. The field evaluations were also in accordance with RT-PCR methods. The RT-RPA-PfAgo system proved to be a robust, versatile, highly specific, and sensitive method with great potential for practicality in future plant virus diagnostics.https://www.mdpi.com/2079-6374/13/10/941virus detectionplant pathogensRT-RPAPfAgorice virus |
| spellingShingle | Yan Liu Wenqiang Xia Wei Zhao Peiying Hao Zhengliang Wang Xiaoping Yu Xuping Shentu Kai Sun RT-RPA-PfAgo System: A Rapid, Sensitive, and Specific Multiplex Detection Method for Rice-Infecting Viruses Biosensors virus detection plant pathogens RT-RPA PfAgo rice virus |
| title | RT-RPA-PfAgo System: A Rapid, Sensitive, and Specific Multiplex Detection Method for Rice-Infecting Viruses |
| title_full | RT-RPA-PfAgo System: A Rapid, Sensitive, and Specific Multiplex Detection Method for Rice-Infecting Viruses |
| title_fullStr | RT-RPA-PfAgo System: A Rapid, Sensitive, and Specific Multiplex Detection Method for Rice-Infecting Viruses |
| title_full_unstemmed | RT-RPA-PfAgo System: A Rapid, Sensitive, and Specific Multiplex Detection Method for Rice-Infecting Viruses |
| title_short | RT-RPA-PfAgo System: A Rapid, Sensitive, and Specific Multiplex Detection Method for Rice-Infecting Viruses |
| title_sort | rt rpa pfago system a rapid sensitive and specific multiplex detection method for rice infecting viruses |
| topic | virus detection plant pathogens RT-RPA PfAgo rice virus |
| url | https://www.mdpi.com/2079-6374/13/10/941 |
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