Figure 4.

Evaluation of the ADE-filter system for RNA isolation based on the cycle threshold (C_T) values of real-time quantitative PCR (qPCR), showing the effect of common syringe filter materials on the ADE-filter system’s RNA isolation efficiency. (a) The investigation of filter membranes included (a) different pore sizes (0.2, 0.45, and 1 μm) and (b) different materials, including cellulose acetate (CA), polyethersulfone (PES), polyvinylidene fluoride (PVDF), and polytetrafluoroethylene (PTFE). All of the RNA templates were isolated from a sample containing 10⁵ CFU/mL of Brucella ovis in PBS. (c) Comparison of a naked filter with the proposed ADE–DMS-assisted filter system for producing RNA templates from pathogens captured on a filter and from non-captured pathogens in solution. Captured pathogens on filter refers to tested samples being subjected to the assay following the protocol described in the section “Filter-based pathogen enrichment and NA extraction.” NA was isolated from the pathogens captured by the assay. The non-captured pathogens in filtered solutions indicated that NA was isolated from the remaining solutions after the assay treatment. NA was isolated from the pathogens not captured by the assay. “Abs” refers to absolute NA references extracted from 100 μL of 10⁶ CFU/mL samples, which contained the same amount of pathogens as the 1 mL of 10⁵ CFU/mL samples. All of the samples were diluted from the same stock solution. (d) The qPCR performance of isolated RNA templates from both a commercial kit (Kit) and the syringe-based ADE system (ADE-Filter) using Brucella ovis in PBS. No C_T values were obtained from the amplification of no-template controls (NTC) in any of these experiments. 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, ^★★p < 0.01, ^★★★p < 0.001; different colors indicate the matched samples compared with the kit).