Quantifying Denitrification Genes in Samples

The abundance of denitrification genes, including periplasmic nitrate reductase (napA), membrane-bound nitrate reductase (narG), nitrite reductase (nirS and nirk), nitric oxide reductase (cnorB and qnorB) and nitrous oxide reductase (nosZ), were detected using quantitative polymerase chain reaction (qPCR). A 20-μL reaction mixture was prepared with 1 μL template DNA, 10 μL SYBR Green I PCR master mix (Bio-Rad, U.S.), 1 μL forward and reverse primers and 7 μL sterile water. The qPCR was conducted using a CFX96 Touch real-time PCR Detection System (Bio-Rad, U.S.) according to our previous study [9 (link)]. Each qPCR amplification for each gene comprised a total of 40 cycles. The primer sequences are provided in Table B in S1 File. Three biological replicates were used.

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Han B., Zhang S., Zhang L., Liu K., Yan L., Wang P., Wang C, & Pang S. (2018). Characterization of microbes and denitrifiers attached to two species of floating plants in the wetlands of Lake Taihu. PLoS ONE, 13(11), e0207443.

Publication 2018

SYBR Green I PCR master mix CFX96 Touch real-time PCR Detection System

Biological Denitrification Gene amplification Genes Membrane Nirs Nitrate Nitrate reductase Nitric oxide reductase Nitrite reductase Nitrous oxide reductase Periplasmic Periplasmic reductase Polymerase chain reaction Primer Sterile Sybr green i Touch

Corresponding Organization : Hohai University

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Variable analysis

independent variables

The abundance of denitrification genes, including periplasmic nitrate reductase (napA), membrane-bound nitrate reductase (narG), nitrite reductase (nirS and nirk), nitric oxide reductase (cnorB and qnorB) and nitrous oxide reductase (nosZ)

dependent variables

The measured abundance of denitrification genes using quantitative polymerase chain reaction (qPCR)

control variables

The 20-μL reaction mixture composition (1 μL template DNA, 10 μL SYBR Green I PCR master mix, 1 μL forward and reverse primers, 7 μL sterile water)
The qPCR protocol using a CFX96 Touch real-time PCR Detection System according to the previous study [9]
The number of qPCR amplification cycles (40 cycles)

controls

No positive or negative controls were explicitly mentioned in the protocol.

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