# Comparison of PCR Primers for Analyzing Dentirifying Microorganisms in the Hyporheic Zone
## 5 Conclusions  
The hydrogeological patterns in hyporheic zones affect all microbial communities, including denitrifiers. In this study, we performed comparative analyses and sample testing of PCR primer sets for each denitrification-associated gene for the analysis of denitrifying microorganisms in environmental samples extracted from hyporheic zones. The specific amplification of six denitrification-associated genes using PCR primer sets was compared.  
Thereafter, the PCR primer sets that were determined to be suitable were selected for each denitrification-associated gene and tested using sixteen environmental samples (three groundwater, three surface water, and ten hyporheic water samples). The specific amplification was determined using PCR primer sets for denitrification-associated genes and nucleic acid from eleven type strains. _NosZ_ was found to be the most frequently amplified gene from the nucleic acid of type strains, with a specific band formed in all eleven type strains. The specific band amplification and PCR time of the type strains were analyzed to select one PCR primer set for each gene. The selected PCR primer sets were used to analyze 16 aqueous environmental samples in which denitrifying microorganisms were expected to be present, and the results showed that specific bands of _narG_, _mirS_, and _nosZ_ were most frequently observed. Therefore, we believe that microorganisms with _narG_, which is involved in the reduction of nitrate to nitrite; _mirS_, which is involved in the reduction of nitrite to nitrite oxide; and _nosZ_, which is involved in the reduction of nitrous oxide to nitrogen gas were the most abundant in the aqueous environmental samples used in this study.  
_NosZ_ was the most frequently amplified gene from the nucleic acid of type strains, with a specific band formed in all eleven type strains. The specific band amplification and PCR time of the type strains were analyzed to select one PCR primer set for each gene. However, this case report showed that for the samples from different water environments, even the best-performing primer pair for each gene failed to amplify more than about 50% of a selection of denitrifying strains, except that for _nosZ_. Further studies on denitrification genes related to water environment samples, such as hyporheic zones, are needed.  
The selected PCR primer sets were used to analyze three groundwater, three stream water, and ten hyporheic water samples in which denitrifying microorganisms were expected to be present, and the results showed that specific bands of _narG_, _napA_, _mirS_, and _nosZ_ were most frequently observed in the hyporheic zone water samples. We expect that the PCR primer sets selected for the denitrification-associated genes in this study may be used to evaluate denitrification by denitrifying microorganisms in response to seasonal and regional changes.  
The following are available online at [[http://www.mdpi.com/2076-3417/10/12/4172/s1](http://www.mdpi.com/2076-3417/10/12/4172/s1)]([http://www.mdpi.com/2076-3417/10/12/4172/s1](http://www.mdpi.com/2076-3417/10/12/4172/s1)), Table S1: PCR primer information of denitrification-associated genes.  
**Funding:** This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (grant numbers 2019R1A6A1A03033167, 2019R1I1A2A01057002).  
**Conflicts of Interest:** The author declares that they have no known competing financial interests or personal relationships that could influence the work reported in this paper.