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. 2022 Mar 25;14(4):682. doi: 10.3390/v14040682

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© 2022 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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Overall summary of two-step RT-qPCR pan-orthohantavirus detection tool. 1. S segment sequences from both New and Old World orthohantaviruses were compiled and aligned through NCBI and CLUSTAL Omega. Little to no completely conserved regions existed; therefore, degenerate primers were designed. 2. Samples (plasmids, in vitro cultured virus, and lung tissue from wild-caught rodents) were used to validate primer selection. 3. Two-step RT-qPCR was performed followed by DNA gel electrophoresis. Sanger sequencing was also used for additional confirmation in positive lung tissue samples from wild-caught rodents.