Mr Jiwei Yang1, Mr Shoma Matsushita2, Mr Fei Xia4, Dr Susumu Yoshizawa3, Dr Shoji Oda1, Dr Wataru Iwasaki1
1 Department of Integrated Biosciences, Graduate School of Frontier Sciences, the University of Tokyo, Kashiwa, Japan, 2Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, the University of Tokyo, Kashiwa, Japan, 3Atmosphere and Ocean Research Institute, the University of Tokyo, Kashiwa, Japan, 4Faculty of Environment and Information Studies, Keio University, Fujisawa, Japan
High-resolution monitoring of endangered or invasive species by environmental DNA (eDNA) is needed as a critical tool in the fight against biodiversity loss. Here, we utilized the CRISPR-Cas13a system (SHERLOCK) to achieve more sensitive eDNA detection than the widely used real-time PCR (qPCR) method, which has imperfect sensitivity and requires thermal cycling equipment. We introduced a SHERLOCK sensing platform, utilizing the collateral cleavage of Cas13a nucleases, to detect eDNA of European Carp (Cyprinus carpio) in two field ponds in Japan and performed side-by-side comparisons with qPCR. Our results demonstrate that SHERLOCK enables a detection limit down to 1.68 copy/µL and a higher detection rate than qPCR across different filtered volumes of water samples: 300mL (100% vs. 88.9%) and 50mL (76.9% vs. 15.4%). In addition, we confirmed that reverse transcription of eRNA further increases the sensitivity of SHERLOCK. We also achieved eDNA on-site detection by SHERLOCK within 37 minutes using a one-minute heat-free extraction step. The outstanding sensitivity and portability of SHERLOCK can increase the effectiveness of ecological monitoring via eDNA and provide a new approach for rapid in-situ analysis of species of conservation concern.
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