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. 2022 Nov 3;13:977382. doi: 10.3389/fmicb.2022.977382

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Copyright © 2022 Zhu, Xing, Yang, Li, Wang, Zhou, Zhang, Ren, Liu, He, Shen, Du 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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Development of dual-gene detection system for SFTSV. (A, B) Both ssDNA reporter (FAM-BHQ) and ssRNA reporter (VIC-BHQ) were added in the detection of Cas12a or Cas13a, and observed the changes in the two kinds of fluorescence intensity. Only the ssDNA reporter was cleaved by the activated Cas12a, and only the ssRNA reporter was cleaved by the activated Cas13a. (C) The schematic diagram of the dual-gene detection system for SFTSV with LbaCas12a and LwaCas13a. The L gene was detected in the FAM channel and M gene in the VIC channel. (D) Determination of the sensitivity of the multiplexed detection system using synthetic RNA standards after the condition optimization. The detectable signal at 10 cp/μl for Cas12a channel was weaker and unstable. Values represent mean ± SD (n = 3). Statistical significance for comparison was determined using Two-way ANOVA and significance was considered as ns ≥ 0.05, *p < 0.05, **p < 0.01, ****p < 0.0001. (E) The results of SFTSV detection in 22 clinical samples (12 positives and 10 negatives) using the multiplexed detection system. The L gene was detected by Cas13a using Cas13-L-crRNA1 and the M gene was detected by Cas12-M-crRNA8. Values represent mean ± SD (n = 3).