2022 Fiscal Year Annual Research Report
環境RNAのオンサイト検出法の開発と内部標準RNAによる絶対定量
| Project/Area Number |
22J14328
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| Allocation Type | Single-year Grants |
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| Research Institution | The University of Tokyo |
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Principal Investigator |
YANG Jiwei 東京大学, 新領域創成科学研究科, 特別研究員(DC2)
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| Project Period (FY) |
2022-04-22 – 2024-03-31
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| Keywords | Rapid detection / On-site detection / Environmental DNA / Environmental RNA / Real-time PCR / CRISPR / RNA-Seq / Messenger RNA |
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| Outline of Annual Research Achievements |
We have achieved faster and more sensitive detection than real-time PCR (qPCR) using a new CRISPR-based detection technology for the first part of the rapid detection of environmental DNA and RNA. We examined samples of the same environmental DNA using the designed CRISPR and qPCR primers for Cyprinus carpio separately and showed that CRISPR possessed higher sensitivity than the qPCR method. Additionally, by adding reverse transcriptase, we can simultaneously detect environmental DNA and RNA, improving the detection sensitivity by approximately one order of magnitude. Finally, we have achieved a fully field-based, one-hour detection process from filtration to nucleic acid extraction and final readouts, which has been tested for the detection of environmental DNA and environmental RNA from several marine and freshwater fish species. These results have been summarized and submitted for peer-review. We plan to further optimize this method by reducing reaction time, achieving quantitative detection, and simplifying the operation steps to enable non-professionals to use environmental DNA and RNA for species detection.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The study's first part has reached the goal of achieving CRISPR-based on-site rapid detection. For the second part of our study, we focused on the quantitative analysis of environmental RNA using RNA internal standards. Our planned field experiment of constructing experimental tanks was restricted due to the lack of workforce and required materials caused by the COVID-19 pandemic. In addition, it is difficult to find appropriate closed field sites to investigate the diffusion and degradation of environmental DNA and RNA of the reared fish.
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| Strategy for Future Research Activity |
For the second part of quantitative analysis of environmental RNA, we have modified our experimental plan first to understand the sources of environmental messenger RNA in fish, i.e., which tissues contribute to the environmental messenger RNA found in the environment and the proportion of different tissues. Additionally, we aim to identify which environmental messenger RNA of fish in the water is relatively stable and whether these genes can be used as indicators to reflect the physiological status of fish. To achieve this, we are collecting water samples of fish at different temperatures and tissue samples from different organs of fish, which will be extracted and analyzed using RNA-Seq.
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