Rapid, sensitive, and visual detection of Clonorchis sinensis with an RPA-CRISPR/Cas12a-based dual readout portable platform.

Clonorchis sinensis is a food-borne parasite that parasitizes the liver and bile ducts of humans and many animals. This parasite exerts a high burden due to diverse hepatobiliary morbidities (e.g., cholangitis, cholecystitis, cholelithiasis, and cholangiocarcinoma), and an effective detection strategy is urgently needed. CRISPR/Cas12a exhibits nonspecific trans-cleavage activity upon binding to its specific target and has been widely used for nucleic acid detection. In this study, an RPA-CRISPR/Cas12a-based dual readout portable detection platform was established, which shows high sensitivity (one copy/μl) and specificity (no cross-reactivity with common pathogens) by rapid preamplification and combines lateral flow strips and visual fluorescence for visualization of results by the naked eye within 1 h. Moreover, 50 human fecal swabs and 50 fish flesh samples were detected by this platform and nested PCR. The CRISPR/Cas12a-based dual readout portable platform showed 10.0 % (5/50) C. sinensis-positive samples in human fecal swabs and 28.0 % (14/50) in fish flesh, which was consistent with the results of nested PCR. The results demonstrate that our portable platform has the advantages of stability, sensitivity, accuracy, and low equipment requirements. Furthermore, we provide novel point-of-care testing (POCT) for clinical use in remote rural and resource-constrained areas.