# Comparison of PCR Primers for Analyzing Dentirifying Microorganisms in the Hyporheic Zone
## 3 Results
### Comparison of PCR Primers for Denitrification Associated Genes  
The specific amplification of six denitrification-associated genes (_narG_, _napA_, _nirS_, _nirK_, _norB_ (_cnorB_ and _qnorB_), and _nosZ_) from 11 type strains was determined using PCR primer sets. For denitrification-associated _narG_, primer sets 2, 5, and 3 formed a specific band in six type strains, two type strains, and one type strain, respectively, whereas only non-specific bands in some of the type strains were observed for primer sets 1 and 4. For _napA_, only primer set 1 formed a specific band in three strains and no reaction was observed for primer set 2. For _nirS_, primer set 1 formed a specific band in five strains but multiple non-specific bands were formed, making differentiation difficult. Although specific reactions were observed for primer sets 2 and 5 in four strains, several non-specific bands were observed with primer set 5. For _nirK_, sets 1 and 2, sets 3 and 5, and set 4 formed a specific band in four strains, three strains, and two strains, respectively, and multiple non-specific bands of strong intensity were observed for primer sets 1, 3, 4, and 5. For _norB_, primer sets 2 and 3 and primer set 1 formed a specific band in four strains and three strains for _cnorB_, respectively, whereas primer sets 4 and 5 formed a specific band in three strains and two strains, respectively, for _qnorB_. Additionally, multiple bands and non-specific bands were observed for five sets. For _nosZ_, primer sets 4, 1, 2, and primer sets 3 and 5 formed a specific band in eleven strains, ten strains, seven strains, and three strains, respectively. Multiple bands were observed for primer set 1 and non-specific bands were observed in several reactions for primer set 4 (Table 2). In addition, the PCR time for each denitrification-associated gene was approximately 60-140 min (see the Supplementary Materials).  
\begin{table}
\begin{tabular}{c c c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**Gene**} & \multirow{2}{*}{**Primer Set No.**} & \multicolumn{10}{c}{**Dentirifying Bacteria (Type Strain)**} \\ \cline{3-11}  & & **1** & **2** & **3** & **4** & **5** & **6** & **7** & **8** & **9** & **10** & **11** & **Remark** \\ \hline \multirow{4}{*}{_narG_} & 1 & & & & & & & & & & & Non-specific band \#2 \\  & 2 & \(\bullet\) & \(\bullet\) & & & & & & & & & \\  & 3 & & & & & & & & & & & \\  & 4 & & & & & & & & & & & Non-specific band \\  & 5 & \(\bullet\) & \(\bullet\) & & & & & & & & & \\ \hline \multirow{2}{*}{_napa_} & 1 & \(\bullet\) & ND & \(\bullet\) & & & & & & & & Multi-band \\  & 2 & & & & & & & & & & & & Multi-band \\ \hline \multirow{4}{*}{_nipS_} & 1 & & \(\bullet\) & & & & \(\bullet\) & & & & & Multi-band \\  & 2 & & \(\bullet\) & \(\bullet\) & & & & & & & Multi-band \\  & 3 & & & & & & & & & & Multi-band \\  & 4 & & & & & & & & & & Multi-band \\  & 5 & \(\bullet\) & & & & & & & & & & Multi-band \\ \hline \multirow{4}{*}{_nirK_} & 1 & \(\bullet\) & \(\bullet\) & \(\bullet\) & \(\bullet\) & ND & & & & Multi-band \\  & 2 & \(\bullet\) & & \(\bullet\) & \(\bullet\) & & & & & & & Multi-band \\  & 3 & \(\bullet\) & & \(\bullet\) & \(\bullet\) & & & & & & Multi-band \\  & 4 & & & & & & & & & & ND & Multi-band \\  & 5 & \(\bullet\) & & & & & & & & & & Multi-band \\ \hline \multirow{4}{*}{_norgB_} & 1 & & \(\bullet\) & \(\bullet\) & & & & & & ND & \(\bullet\) & Multi-band \\  & 2 & & \(\bullet\) & & & & & & & \(\bullet\) & Non-specific band \\  & 3 & & \(\bullet\) & ND & & \(\bullet\) & & & & & \(\bullet\) & Non-specific band (nor B) \\  & 4 & \(\bullet\) & & & & & & & & & Multi-band \\  & 5 & \(\bullet\) & & & & & & & & & Non-specific band (_nor B_) \\ \hline \multirow{4}{*}{_nosZ_} & 1 & \(\bullet\) & \(\bullet\) & \(\bullet\) & \(\bullet\) & \(\bullet\) & \(\bullet\) & \(\bullet\) & \(\bullet\) & Multi-band \\  & 2 & \(\bullet\) & \(\bullet\) & \(\bullet\) & & & & & & & \(\bullet\) & Multi-band \\ \cline{1-1}  & 4 & \(\bullet\) & \(\bullet\) & \(\bullet\) & \(\bullet\