## 3 Results
### Optimization of DNA Extraction
The results with our PCR buffer formulation (Methods) showed no inhibition at NaOH concentrations of 0 to 5.6 mM NaOH. The average Ct values increased by about one cycle with 7.5 mM NaOH and by approximately four with 10 mM NaOH. We then tested 25, 50, 75, and 100 mM NaOH in duplicate for 10 min room temperature (RT) extraction of 4 \(\times\) 10\({}^{3}\) CFU of broth-grown _S. agnetis_ 908 (see Methods). After incubation, each sample was diluted with four volumes of SPW, and qPCR-HRM was performed in triplicate on 2 \(\upmu\)L of the diluted sample (final NaOH concentrations in the PCR were therefore 0.5, 1, 1.5, and 2 mM). The qPCR results in Figure 1 demonstrate that all four concentrations of NaOH liberated DNA but that 75 and 100 mM yielded lower Ct values, consistent with more complete lysis. We chose 100 mM as an appropriately effective concentration for subsequent extractions where fivefold dilution would reduce the concentration to 20 mM NaOH, with the subsequent addition of 2 \(\upmu\)L to a 20 \(\upmu\)L PCR, which would result in a final concentration of 2 mM NaOH in the PCR or less than half the minimum inhibitory concentration. We compared RT extraction to extraction at 37 \({}^{\circ}\)C and extraction for 5, 10, 20, or 30 min and found no difference for time or temperature. We added water-washed _S. agnetis_ 908 from broth overnight cultures in 10-fold dilutions estimated at 8 \(\times\) 10\({}^{-2}\) to 8 \(\times\) 10\({}^{5}\) CFU in 2 \(\upmu\)L directly to 20 \(\upmu\)L qPCR. Specific amplification of the 16S amplicon resulted from the reactions from 8 \(\times\) 10\({}^{2}\) to 8 \(\times\) 10\({}^{5}\), but the Ct values were higher for \(\geq\)8 \(\times\) 10\({}^{4}\) CFU, consistent with PCR inhibition. When we instead used a similar dilution series for extraction with 100 mM NaOH followed by fivefold dilution, there was specific amplification with input of as little as 10 CFU and no inhibition even with input of estimated 10\({}^{7}\) CFU. Thus, NaOH extraction produced a specific qPCR-HRM product from fewer input cells and minimized PCR inhibition from the addition of more than one hundred times more cells. We then compared the boil and NaOH extraction methods for an _E. ecororum_ isolate (1415) and three different isolates of _E. faecalis_ (1558, 1570, and 1582), which had previously been problematic for boil extractions for PCR. We included _S. agnetis_ 908 as a control. Figure 1: Average Ct values for different NaOH concentrations (mM) for extraction from duplicate aliquots (light gray vs. dark gray) of 4 \(\times\) 10\({}^{3}\) CFU of an _S. agnetis_ 908 broth culture followed by dilution with 4 volumes of Te and then qPCR-HRM. Data plotted are average for triplicate qPCR. Error bars are standard deviation. Ct values for controls: 0.25 ng 908 DNA—11.8 \(\pm\) 0.5; H\({}_{2}\)O—No Ct. Equivalent volumes of cell extracts from each method were added to the qPCR-HRM. The results in Figure 2 show that the NaOH extraction produced Ct values lower than those from the boil. For _E. faecalis_ 1570, there was no 16S amplification from the boil extract, while the NaOH with dilution method was positive for 16S PCR. Based on \(\Delta\)Ct calculations [37] using the positive control of purified _S. agnetis_ 908 genomic DNA, we computed the _E. faecalis_ 1570 NaOH extract from 20 uL of overnight culture and dilution to a final volume of 500 uL to a 3 pg/uL. Similar calculations for the NaOH extracts for the other cultures were as follows: _S. agnetis_ 908: 680 pg/uL; _E. cecorum_ 1415: 71 pg/uL; _E. faecalis_ 1558: 16 pg/uL; and _E. faecalis_ 1572: 5 pg/uL. The NaOH yield for _E cecorum_ 1415 was more than 30\(\times\) higher than that of the boil method. We speculated that the NaOH extraction method would be much more useful if it could be combined with a simple enrichment step. Others have shown that genomic DNA could be captured on silica spin columns from ethanol-NaOH lysis solutions [24].