Rapid detection of environmental RNA and absolute quantification using internal strand RNA

使用内链 RNA 快速检测环境 RNA 并进行绝对定量

• 批准号: 22KJ0945
• 负责人: YANG Jiwei
• 金额: $ 1.09万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for JSPS Fellows
• 财政年份: 2023
• 资助国家: 日本
• 起止时间: 2023-03-08 至 2024-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-22KJ0945/
• 关键词: Rapid detection; On-site detection; Environmental DNA; Environmental RNA; Real-time PCR; CRISPR; RNA-Seq; Messenger RNA

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.

我们使用基于 CRISPR 的新型检测技术实现了比实时 PCR (qPCR) 更快、更灵敏的检测，用于环境 DNA 和 RNA 快速检测的第一部分。我们分别使用为鲤鱼设计的 CRISPR 和 qPCR 引物检查了相同环境 DNA 的样本，结果表明 CRISPR 比 qPCR 方法具有更高的灵敏度。此外，通过添加逆转录酶，我们可以同时检测环境DNA和RNA，将检测灵敏度提高约一个数量级。最后，我们实现了从过滤到核酸提取和最终读数的完全基于现场的一小时检测流程，并已针对多种海洋和淡水鱼类的环境DNA和环境RNA的检测进行了测试。这些结果已被总结并提交同行评审。我们计划进一步优化该方法，减少反应时间，实现定量检测，简化操作步骤，使非专业人员也能利用环境DNA和RNA进行物种检测。

项目成果

Environmental DNA detection by CRISPR-Cas13

CRISPR-Cas13 环境 DNA 检测

• 发表时间: 2023
• 作者: YANG Jiwei

1st Australian & New Zealand eDNA Conference

澳大利亚第一名

• 发表时间: 2023