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. 2019 Nov 8;9:16374. doi: 10.1038/s41598-019-52922-y

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© The Author(s) 2019

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Performance evaluation of the ADE-filter system for large sample volumes in various sample matrices. The total amount of pathogenic bacteria was controlled at the same sample level for all of the parallel experiments. (a) Performance of the ADE–DMS system in both tube and filter formats. Here, 1 mL of 10² CFU/mL Brucella ovis in PBS samples were used. The RNA isolation process followed that described in the sections “Tube-based pathogen enrichment and nucleic acid extraction” and “Filter-based pathogen enrichment and nucleic acid extraction.” Error bars indicate the standard deviation from the mean based on at least three independent experiments. The p-values were evaluated by Student’s t-test (^★p < 0.05). (b) RNA isolated by the ADE-filter system from different volume samples (1 mL to 50 mL) containing 10⁴ CFU of Brucella ovis in PBS. For the 1 mL (10⁴ CFU/mL) samples, the kit used 200 μL of the 1 mL samples whereas the filter-based assay used the entire 1 mL samples because of its different capacity. “Abs” refers to the absolute NA references extracted from 100 μL (10⁵ CFU/mL) samples, which contained the same amount of pathogens as the 1 mL of 10⁴ CFU/mL samples. All of the samples were diluted from the same stock solution. (c) RNA isolated by the ADE-filter system from different sample matrices containing 10³ CFU of Brucella ovis in all of the 10 mL PBS, urine, and serum samples. No C_T values were obtained from the amplification of no-template controls (NTC) in any of these experiments. Error bars indicate standard deviation from the mean based on at least three independent experiments. The p-values were evaluated by Student’s t-test (^★p < 0.05, ^★★p < 0.01; different colors indicate the matched samples compared with the kit). (d) Robust testing with 96-well filter plates. DNA isolated by the ADE 96-well filter plate from the sample containing 10³ CFU of Brucella ovis in the 1 mL PBS sample. Eight samples were treated simultaneously. Error bars indicate standard deviation from the mean based on at least three independent experiments.