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. 2022 Nov 18;13:1070940. doi: 10.3389/fmicb.2022.1070940

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Copyright © 2022 Lin, Zheng, Li, Wang, Wei, Xie, Lu, Tian, Wang, Qu and Liu.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Optimization of the reaction conditions for CALIBURN-v2. (A) Schematic presentation of CALIBURN-v2 for closed-lid pathogen detection, showing reaction conditions to be optimized. (B,C) The effects of incubation time of RT-RPA (B) and Cas12a (C) reaction on the final signal readout. (D) Screening the optimal concentrations for RNase H. (E) Comparison of the assay efficiency using different Cas12a reaction buffers. (F–H) Effect of Cas12a reaction temperature (F), the mixing ratio between RT-RPA and Cas12a reaction (G), and the sequences of ssDNA reporters (H) on the assay performance. (I) Comparison of the sensitivity of the assay before and after optimization. The black dotted line represents the threshold, which is set as the mean of NTC plus 3-fold standard deviation (SD). Target, SARS-CoV-2 IVT N gene RNA (1 nM). NTC, non-template control. The data from three biological replicates are shown as mean ± SD.