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

我们已经实现了比实时聚合酶链式反应(QPCR)更快、更灵敏的检测，使用了一种新的基于CRISPR的检测技术，用于环境DNA和RNA的快速检测。用设计的CRISPR和qPCR引物分别对同一环境DNA样品进行检测，结果表明CRISPR方法比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，检测灵敏度提高了大约一个数量级。最后，我们实现了从过滤到核酸提取和最终读数的完全现场、一小时的检测过程，并已用于检测几个海洋和淡水鱼物种的环境DNA和环境RNA。这些结果已汇总并提交同行审议。我们计划通过缩短反应时间、实现定量检测以及简化操作步骤来进一步优化该方法，使非专业人员能够使用环境DNA和RNA进行物种检测。

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.