Sensitive detection of CaMV35S based on exponential rolling circle amplification reaction and CRISPR/Cas12a using a portable 3D-printed visualizer

• Published in: Microchemical journal Vol. 205; p. 111313
• Main Authors: Liu, Meilin, Li, Xuheng, Xu, Jingbing, Zhou, Shiying, Deng, Liyuan, Men, Dianhui, Duan, Yi, Huo, Danqun, Hou, Changjun
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.10.2024
• Subjects: CaMV35S; CRISPR/Cas12a trans-cleavage; Exponential Rolling Circle Amplification (E-RCA); Portable detection; Smartphone; Portable detection; Exponential Rolling Circle Amplification (E-RCA); Smartphone; CaMV35S; CRISPR/Cas12a trans-cleavage
• ISSN: 0026-265X
• DOI: 10.1016/j.microc.2024.111313

[Display omitted] •This coupling RCA and Nb.BbvCI achieve an exponential amplification assay.•The E-RCA-CRISPR/Cas12a can detect samples of highly processed food products.•Our cost-effective device gives people a new way to perform real-time testing of GMOs. The biosafety of genetically modified organisms (GMOs) is still a subject of debate among the public, so developing practical and accurate GMOs detection techniques is crucial. However, the existing testing methodologies for GMOs products do not possess the requisite sensitivity and flexibility necessary for effective GMOs surveillance. In this study, our exponential rolling cycle amplification with the CRISPR/Cas12a assay (E-RCA-CRISPR/Cas12a) has successfully enhanced the limit of detection (LOD) for the CaMV35S fragment to 10 aM in a total of 100 min. Rolling cycle amplification can be initiated by the target CaMV35S sequence, utilizing Nb.BbvCI endonuclease nicking activity for the exponential rolling cycle amplification and the trans-cleavage activity of Cas12a for the detection and signal amplification. Our research greatly improves the amplification efficiency through this cyclic process. To reduce the reliance on signal reading devices, we also build a custom 3D-printed visualization vessel and combine a color-reading app on the smartphone to read the fluorescent signals through for sample visualization and analysis. Under ideal circumstances, the limits of detection of our convenient device is 1 fM. We also integrate the exponential rolling circle amplification (E-RCA-CRISPR/Cas12a) assay with the lateral flow strip (LFA) to achieve reading device-free detection with the LOD of 50 fM. Testing the plant and soy sauce samples successfully demonstrated the method’s applicability. The sensing platform thus offers a prototype technique for quick detection of various nucleic acid targets and has optimum sensitivity, specificity, and on-site detection capability.