## 3 Results
### PCR Method Selection for Each Gene  
Each PCR reaction was analyzed using nine PCR methods (#1-9), which were expected to amplify _amo_ (six _amoA_, two _amoB_, and one _amoC_). For each PCR method expected to amplify _amoA_ and _amoB_, 10-13 and 6-8 amplicons among 25 surface soil DNAs were amplified to secure candidate positive controls. However, the PCR method that was expected to amplify _amoC_ did not form an amplicon of a specific size. Among the six _amoA_ PCR methods, weak amplification or multi-bands were formed in methods #3 and #5. PCR method #2 formed an excellent reaction; however, methods #1, #3, and #6 were selected due to their amplicon size (approximately 110 bp). Among the two _amoB_ PCR methods, #1 showed weak amplification and multi-bands; therefore, #2 was selected (Supplementary Figure S1A). After comparing the detection sensitivity of PCR methods #1, #3, and #6 for _amoA_ amplification, the detection sensitivity levels of 10\({}^{0}\)-10\({}^{-4}\) were analyzed from six surface soil DNAs. The average sensitivity values were \(-\)1.17, \(-\)1.50, and \(-\)1.67 for #1, #3, and #6, respectively. PCR method #2 for _amoB_ showed a 10\({}^{-1}\) detection sensitivity (Supplementary Figure S1B). \begin{table}
\begin{tabular}{c c c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**\# Loss**} & \multirow{2}{*}{** \begin{tabular}{}**\end{tabular} **} & \multirow{2}{*}{** \begin{tabular}{}**\end{tabular} **} & \multirow{2}{*}{** \begin{tabular}{}**\end{tabular} **} & \multicolumn{4}{c}{** \begin{tabular}{}**\end{tabular} **} & \multicolumn{4}{c}{** \begin{tabular}{}**\end{tabular} **} & \multicolumn{4}{c}{** \begin{tabular}{}**\end{tabular} **} & \multicolumn{4}{c}{**\ \begin{tabular}{}**\end{tabular} **} & \multicolumn{4}{c}{** \begin{tabular}{}**\end{tabular} **} \\ \hline \hline
**\# Loss**  
We analyzed seven PCR methods expected to amplify _norB_ (five _cnorB_ and two _qnorB_). Among these, PCR methods #1-4, to amplify _cnorB_, formed a band of a specific size in the bacterial nucleic acid, thereby securing a positive control. However, methods #1, #3, and #4 did not amplify the _cnorB_-specific nucleic acids from the samples. By contrast, method #2 amplified approximately 64% of the samples (16/25); therefore, only a short length (approximately 177 bp) was selected. PCR method #1, used to amplify _qnorB_, showed no reaction, whereas, in method #2, a specific band was formed from one bacterial and eight sample DNAs (Supplementary Figure S2). Among the three and two PCR primer sets for _hao_ and _hzo_, respectively, an amplification band of a specific size did not appear in the 25 surface soil total DNA samples (Supplementary Figures S3 and S4). Among the five PCR methods to amplify _hzs_, methods #1-4 did not form specific bands from the sample nucleic acids. By contrast, the expected band sizes were observed for method #5 from six sample DNAs, yielding a detection sensitivity of \(10^{-1}\) (Supplementary Figure S5). A comparison of the four PCR methods for _napA_ amplification demonstrated that method #3 did not show amplification in the bacteria and sample nucleic acids, and method #1 showed excellent performance but with an amplicon size of approximately 152 bp. PCR methods #2 and #4 were selected due to their detection sensitivity levels of \(10^{0}\)-\(10^{-1}\) (average, \(-0.33\)) and \(10^{-1}\)-\(10^{-3}\) (average, \(-2.00\)) and their amplification of approximately 60% of the samples (15/25) by method #4 and approximately 52% of the samples (13/25) by method #4 (Supplementary Figure S6). Among the four PCR methods expected to amplify _narG_, methods #2 and #3 did not show amplification in the bacteria or sample nucleic acids. Method #4 showed excellent performance but with an amplicon size of approximately 173 bp. By contrast, method #1 was expected to have an amplicon size of 524 bp, showed a response to four bacterial DNAs, and amplified approximately 56% of the samples (14/25), with a detection sensitivity of approximately \(10^{-3}\) (Supplementary Figure S7).