Identification of Microbial Populations Assimilating Nitrogen from RDX in Munitions Contaminated Military Training Range Soils by High Sensitivity Stable Isotope Probing

The leaching of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) from particulates deposited in live-fire military training range soils contributes to significant pollution of groundwater. In situ microbial degradation has been proposed as a viable method for onsite containment of RDX. However, there is only a single report of RDX degradation in training range soils and the soil microbial communities involved in RDX degradation were not identified. Here we demonstrate aerobic RDX degradation in soils taken from a target area of an Eglin Air Force Base bombing range, C52N Cat’s Eye, (Eglin, Florida U.S.A.). RDX-degradation activity was spatially heterogeneous (found in less than 30% of initial target area field samples) and dependent upon the addition of exogenous carbon sources to the soils. Therefore, biostimulation (with exogenous carbon sources) and bioaugmentation may be necessary to sustain timely and effective in situ microbial biodegradation of RDX. High sensitivity stable isotope probing analysis of extracted soils incubated with fully labeled ¹⁵N-RDX revealed several organisms with ¹⁵N-labeled DNA during RDX-degradation, including <i>xplA</i>-bearing organisms. <i>Rhodococcus</i> was the most prominent genus in the RDX-degrading soil slurries and was completely labeled with ¹⁵N-nitrogen from the RDX. <i>Rhodococcus</i> and <i>Williamsia</i> species isolated from these soils were capable of using RDX as a sole nitrogen source and possessed the genes <i>xplB</i> and <i>xplA</i> associated with RDX-degradation, indicating these genes may be suitable genetic biomarkers for assessing RDX degradation potential in soils. Other highly labeled species were primarily Proteobacteria, including: <i>Mesorhizobium</i> sp., <i>Variovorax</i> sp., and <i>Rhizobium</i> sp

Semiquantitative Nucleic Acid Test with Simultaneous Isotachophoretic Extraction and Amplification

Nucleic acid amplification tests (NAATs) provide high diagnostic accuracy for infectious diseases and quantitative results for monitoring viral infections. The majority of NAATs require complex equipment, cold chain dependent reagents, and skilled technicians to perform the tests. This largely confines NAATs to centralized laboratories and can significantly delay appropriate patient care. Low-cost, point-of-care (POC) NAATs are especially needed in low-resource settings to provide patients with diagnosis and treatment planning in a single visit to improve patient care. In this work, we present a rapid POC NAAT with integrated sample preparation and amplification using electrokinetics and paper substrates. We use simultaneous isotachophoresis (ITP) and recombinase polymerase amplification (RPA) to rapidly extract, amplify, and detect target nucleic acids from serum and whole blood in a paper-based format. We demonstrate simultaneous ITP and RPA can consistently detect 5 copies per reaction in buffer and 10 000 copies per milliliter of human serum with no intermediate user steps. We also show preliminary extraction and amplification of DNA from whole blood samples. Our test is rapid (results in less than 20 min) and made from low-cost materials, indicating its potential for detecting infectious diseases and monitoring viral infections at the POC in low resource settings

Semiquantitative Nucleic Acid Test with Simultaneous Isotachophoretic Extraction and Amplification

Nucleic acid amplification tests (NAATs) provide high diagnostic accuracy for infectious diseases and quantitative results for monitoring viral infections. The majority of NAATs require complex equipment, cold chain dependent reagents, and skilled technicians to perform the tests. This largely confines NAATs to centralized laboratories and can significantly delay appropriate patient care. Low-cost, point-of-care (POC) NAATs are especially needed in low-resource settings to provide patients with diagnosis and treatment planning in a single visit to improve patient care. In this work, we present a rapid POC NAAT with integrated sample preparation and amplification using electrokinetics and paper substrates. We use simultaneous isotachophoresis (ITP) and recombinase polymerase amplification (RPA) to rapidly extract, amplify, and detect target nucleic acids from serum and whole blood in a paper-based format. We demonstrate simultaneous ITP and RPA can consistently detect 5 copies per reaction in buffer and 10 000 copies per milliliter of human serum with no intermediate user steps. We also show preliminary extraction and amplification of DNA from whole blood samples. Our test is rapid (results in less than 20 min) and made from low-cost materials, indicating its potential for detecting infectious diseases and monitoring viral infections at the POC in low resource settings

Schematic illustrating the plasma collection disc in operation.

The schematic illustrates a single PCD. Photos on the left show the PCD with the the filter disc placed on a manifold for multiple samples and the addition of 35 μL of whole blood to each disc (top), a PDC detail view prior to whole blood being added (middle), and the discs after blood has been absorbed and finally the plasma collection disc exposed after the whole blood treated filter has been removed (bottom). The disc is removed with sterile forceps and is ready for use or can be stored until required.</p

Summary statistics and cross-laboratory coefficients of variation (CV) for plasma, DBS and PCD.

Summary statistics and cross-laboratory coefficients of variation (CV) for plasma, DBS and PCD.</p

The comparison of results for plasma, DBS and the volume adjusted PCD eluates using <i>R</i>_<i>s</i> and recovery of expected plasma values.

The comparison of results for plasma, DBS and the volume adjusted PCD eluates using Rs and recovery of expected plasma values.</p

Sample panels and experiment design.

Sample panels and experiment design.</p

Fig 4 -

A. Bland Altman plots of the difference in results derived from wet plasma versus DBS types measured using the 7-plex assay. Average of the wet plasma and dry sample type (x-axes) are plotted against the difference between measurements from the 7-Plex assay for paired wet plasma and dry sample types (y-axes). B. Bland Altman plots of the difference in results derived from wet plasma versus the adjusted PCD. For both Figures, the horizontal lines indicate line of perfect agreement (green), mean (purple), and ± 2standard deviations of the difference (red). AGP, α-1-acid glycoprotein; CRP, C-reactive protein; FER, ferritin; HRP2, histidine rich protein 2; RBP4, retinol binding protein 4; sTfR, soluble transferrin receptor; Tg, thyroglobulin; DBS, dried blood spot.</p

Fig 3 -

A. Lin’s concordance correlation coefficient (CCC) plots comparing the analyte measurements in paired wet plasma (x-axes) and DBS samples (y-axes). AGP, α-1-acid glycoprotein; CRP, C-reactive protein; FER, ferritin; HRP2, histidine rich protein 2; RBP4, retinol binding protein 4; sTfR, soluble transferrin receptor; Tg, thyroglobulin; DBS, dried blood spot; rho_c, CCC. B, Lin’s CCC plots comparing the analyte measurements in paired wet plasma (x-axes) and the adjusted PCD (y-axes). AGP, α-1-acid glycoprotein; CRP, C-reactive protein; FER, ferritin; HRP2, histidine rich protein 2; RBP4, retinol binding protein 4; sTfR, soluble transferrin receptor; Tg, thyroglobulin; DBS, dried blood spot; rho_c, CCC.</p

Contrived blood samples spiked with a range of elevated levels of AGP, CRP, HRP2 and sTfR.

Contrived blood samples spiked with a range of elevated levels of AGP, CRP, HRP2 and sTfR.</p