## 3 Results
### Optimization of DNA Extraction
We found that we could capture DNA from our NaOH lysates using the Mag-Bind RXN Pure Plus, a commercial preparation of paramagnetic beads in unspecified binding buffer. We compared paramagnetic bead purification versus direct dilution for extraction from duplicate samples of broth cultures of three Gram-positive and three Gram-negative bacteria (Figure 3). For these comparisons, the elution from the paramagnetic beads used the same volume as the final volume from direct dilution. The qPCR Ct values for the extractions from the Gram-negative species were nearly equivalent with both methods, while for the Gram-positive, the paramagnetic bead capture gave consistently lower Ct values indicative of reduced PCR inhibition. The NaOH extraction and magnetic bead capture method was employed on selected air samples from days 42, 44, and 48-56, and all gave positive qPCR-HRM results for 16S rDNA (Table 1), demonstrating that this method is highly effective for extraction from these air samples. The NaOH extraction and magnetic bead capture method was then used for a second BCO challenge experiment in which for days 21-37 we compared computed total  
Figure 2: Average Ct values from qPCR-HRM for boil (gray) vs. NaOH extraction (green) for duplicate samples from broth cultures of _Staphylococcus agents_, _Enterococcus escorum_, or three isolates of _Enterococcus faecalis_. Overnight cultures were pelleted and suspended in 10 volumes of sterile water. Duplicate 100 aliquots were treated by boiling (10 min at 100 \({}^{\circ}\)C in PCR machine) or NaOH (addition of 11 uL 1 M NaOH for 10 min at room temperature). The samples were then diluted with 400 uL Te and assayed in triplicate by qPCR. Error bars are standard deviation. There was no amplification for _E. faecalis_ 1570 for the boil extract. Ct values for controls: 2 ng 908 DNA—\(19.6\pm 0.7\); H\({}_{2}\)O—No Ct.  
CFU to the Ct values for days 21-37 of air sampling (Table 2). The total CFU increases through day 37 and the qPCR Ct values decrease from days 21 to 37 as expected with increasing bacterial load in the air. Therefore, the system we describe using a portable pump and sterile impinger is a low-cost, effective system that can not only collect viable bacteria but also be used to efficiently extract for qPCR analysis of total bacteria or be subjected to species-specific qPCR to detect particular bacteria of interest. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Day** & **Total CFU** & **Average Ct \(\pm\) SEM** \\ \hline
21 & 340 & 22.3 \(\pm\) 0.4 \\
21 & 1520 & 20.5 \(\pm\) 0.2 \\
23 & 1260 & 19.5 \(\pm\) 0.2 \\
23 & 2010 & 18.3 \(\pm\) 0.1 \\
25 & 2080 & 16.0 \(\pm\) 0.01 \\
25 & 3640 & 17.4 \(\pm\) 0.1 \\
27 & 4580 & 20.1 \(\pm\) 0.01 \\
27 & 3650 & 19.3 \(\pm\) 0.01 \\
37 & 11,200 & 16.0 \(\pm\) 0.1 \\
37 & 12,800 & 15.8 \(\pm\) 0.02 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Air samples, bacterial counts, and qPCR Ct values during a 56-day lameness trial in which all samples were collected in 20 min and extracted using 100 mM NaOH and paramagnetic bead capture. Columns are as in Table 1 except that for the qPCR, which displays the average of triplicate Ct values with SEM. All qPCR-HRM assays included positive controls of purified 908 DNA and negative controls of H\({}_{2}\)O, which performed as expected. Figure 3: Average Ct values from qPCR-HRM for NaOH extracts from broth cultures of 3 Grampositive (_Staphylococcus aureus_ 1516, _Staphylococcus agnetis_ 908, _Enterococcus faecalis_ 1558) and 3 Gramnegative (_Escherichia coli_ 1409, _Escherichia fergusoni_ 1412, _Salmonella enterica_ 1414) species. Extracts were processed by paramagnetic bead capture (open bars) or by dilution with 4 volumes of Te (gray bars). Final samples for each method were adjusted to the same volume and assayed by qPCR-HRM in triplicate. Error bars are standard deviation. Ct values for controls: 2 ng 908 DNA—12.6 \(\pm\) 1.1; H\({}_{2}\)O—31.5 \(\pm\) 1.0 (no melt product for 16S product).