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. 2021 Jun 8;11:12017. doi: 10.1038/s41598-021-91336-7

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Limit of detection determination of multiplex RPA assays developed for specific detection of genus Clavibacter in general and C. nebraskensis. Line 1: detects Clavibacter species/subspecies strains; line 2 detects C. nebraskensis strains, and; line C is a control line. The primer/probe sets for genus Clavibacter and C. nebraskensis are labeled with Biotin/DIG and Biotin/FAM, respectively. A tenfold genomic DNA serial dilution (10 ng–10 fg) was used to perform the sensitivity assays. (A) Limit of detection determination using pure C. nebraskensis (A6094) DNA; detected until 100 fg and 10 pg using Clavibacter and C. nebraskensis specific primers/probe set, respectively. (B) Spiked multiplex test performed by adding 1 µL of corn plant sap in tenfold serially diluted C. nebraskensis (A6094) genomic DNA; detected until 1000 fg and 10 pg using Clavibacter and C. nebraskensis specific primers/probe set, respectively. (C) Detection using tenfold serially diluted C. michiganensis (A2058) purified genomic DNA; detected until 10 pg by Clavibacter genus-specific primers/probe—no detection with C. nebraskensis specific primers/probe set.