Abstract
The cytochrome cd1-containing nitrite reductase, nirS, plays an important role in biological denitrification. Consequently, investigating the presence and abundance of nirS is a commonly used approach to understand the distribution and potential activity of denitrifying bacteria, in addition to denitrifier communities. Herein, a rapid method for detecting nirS gene with loop-mediated isothermal amplification (LAMP) was developed, using Pseudomonas aeruginosa PAO1 (P. aeruginosa PAO1) as model microorganism to optimize the assay. The LAMP assay relied on a set of four primers that were designed to recognize six target sequence sites, resulting in high target specificity. The limit of detection for the LAMP assay under optimized conditions was 1.87 pg/reaction of genomic DNA, which was an order of magnitude lower than that required by conventional PCR assays. Moreover, it was validated that P. aeruginosa PAO1 cells as well as genomic DNA could be directly used as template. Only 1 h was needed from the addition of bacterial cells to the reaction to the verification of amplification success. The nirS gene of P. aeruginosa PAO1 in spiked seawater samples could be detected with both DNA-template based LAMP assay and cell-template based LAMP assay, demonstrating the practicality of in-field use.
Highlights
Denitrification that involves the reduction of nitrate to gaseous forms is a globally important process with relevance to many e nvironments[1,2,3]
Using 0.19 ng genomic DNA of P. aeruginosa as template, Loop-mediated isothermal amplification (LAMP) amplification of nirS at 63 °C for 60 min resulted in amplification products of various size, as indicated by gel electrophoresis and the presence of many sized bands in a reproducible ladder-like pattern (Fig. 3a), which was the same phenomena obtained somewhere[20,23,24,33,34]
NirS gene is absent in S. aureus and E. coli genomes, but present in those of P. aeruginosa[47], which is consistent with other r eports[35,42]
Summary
Denitrification that involves the reduction of nitrate to gaseous forms is a globally important process with relevance to many e nvironments[1,2,3]. The key step in denitrification is the reduction of nitrite to nitric oxide that is catalyzed by two structurally different, but functionally equivalent, forms of nitrite reductase encoded by the nirK and nirS genes[2,3,7]. (3) Sensitivity is less affected by substances that usually inhibit PCR r eactions[22,23] These advantages suggest that simple assays could be developed using LAMP with elimination of the most cumbersome steps of sample pretreatment including DNA extraction and p urification[24,25,26]. The LAMP assays were used to detect nirS gene in seawater samples spiked with genomic DNA or P. aeruginosa PAO1 cells
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