Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a contaminant of concern at many military sites in the US. One approach that may be used to clean up these sites is bioremediation, and six functional genes, diaA, nfsI, pnrB, xenA, xenB, and xplA, have been linked to RDX degradation to date. Quantitative polymerase chain reaction (qPCR) is typically used to detect these genes in environmental samples, but the primer sets previously used do not have good theoretical coverage of the known gene sequences, so they may yield false negative results. To address this, new primer sets were designed with the EcoFunPrimer tool based on sequences collected by the Functional Gene Pipeline and Repository and verified based on residues and motifs. These primer sets were then used to quantify the RDX functional genes in RDX-contaminated groundwater before and after biostimulation, RDX-contaminated sediment and uncontaminated samples (Red Cedar River water and agricultural soils) using the SmartChip Real-Time PCR System. The newly designed primer sets improved upon the theoretical coverage of the previously published ones, and this corresponded to more detections in the environmental samples. All genes except diaA, were detected in the environmental samples, with xenA and xenB being the most predominant. In the sediment samples, nfsI was the only gene detected. The primer sets designed in this study may be used for more reliable detection of the RDX functional genes at contaminated sites. However, additional work with high throughput sequencing is required to confirm the specificity of these assays.
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