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.

与实时PCR（QPCR）相比，使用基于CRISPR的新检测技术，我们在快速检测环境DNA和RNA的第一部分中实现了更快，更敏感的检测。我们使用设计的CRISPR和QPCR引物分别检查了相同的环境DNA的样品，并分别研究了cyprinus carpio的样品，并表明CRISPR具有比qPCR方法更高的灵敏度。另外，通过添加逆转录酶，我们可以同时检测环境DNA和RNA，从而提高了检测灵敏度大约一个数量级。最后，我们已经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过程，该过程已通过几种海洋和淡水鱼类的环境DNA和环境RNA进行了测试。这些结果已汇总并提交了同行评审。我们计划通过减少反应时间，实现定量检测并简化操作步骤来进一步优化此方法，以使非专业人士使用环境DNA和RNA进行物种检测。

项目成果

1st Australian & New Zealand eDNA Conference

澳大利亚第一名

• 发表时间: 2023

Environmental DNA detection by CRISPR-Cas13

CRISPR-Cas13 环境 DNA 检测

• 发表时间: 2023
• 作者: YANG Jiwei