Quantifying Bacterial Diversity via qPCR

qPCR assays targeting genes for NTM, Pseudomonas, and total bacteria were performed by previously published methods (34 (link), 53 (link), 54 (link)) as detailed in Text S6. All DNA samples were processed in triplicate with negative controls and standards. Assays were conducted with 96-well polypropylene plates on a CFX96 real-time quantitative thermocycler (Bio-Rad, Hercules, CA). Each 10-μl reaction mixture contained 5 μl of Fast EvaGreen qPCR master mix (Biotium, Fremont, CA), 0.8 μl of each primer (0.4 μM; IDT, Coralville, IA), 2.4 μl of water, and 1 μl of template DNA (~0.2 ng ⋅ μl⁻¹). PCR conditions for NTM and total 16S rRNA gene assays consisted of 40 cycles, whereas the Pseudomonas assay was performed with 35 cycles. Melting curve analysis of the PCR products was conducted following each assay to confirm that the fluorescence signal originated from specific PCR products and not from primer dimers or other artifacts. For all qPCR assays, a linear relationship between the log of the standards’ DNA copy number and the calculated threshold cycle value across the specified concentration range was confirmed (R² value of >0.99 in all cases). Amplification efficiencies, calculated by the method described by Pfaffl (55 (link)), varied from 1.8 to 2.0 across all assays, and these values are consistent with those reported in other studies (54 (link), 56 (link)).