Experimental PCR Data on Soil DNA Extracts

This dataset provides an estimate of the abundance and spatial distribution of chemical elements and minerals in soils of the conterminous United States and represents a baseline for soil geochemistry and mineralogy against which future outbreak data may be analyzed. Methodology Methodology_Type Lab Methodology_Description Sample sites: Using a Generalized Random Tessellation Stratified (GRTS) design for sample site selection, ~4,800 soil samples were collected across the contiguous United States at a density of 1 site per ~1600 km2 and 4,770 of these were screened for the presence of Bacillus species and B. anthracis (1-5). Sample collection: Sterilized 50 ml tubes and aseptic techniques were used for collection of P-horizon soils as previously described in Smith et al, 2009 (1). The target collection depth interval was 0-5 cm but overall ranged from 0 - 40 cm, due to some site characteristics such as heavy detritus cover. All samples were shipped from various field locations to the USGS microbiology laboratory in St. Petersburg, Florida and then stored by refrigeration until analyzed. DNA extraction: Approximately 0.25 g of soil was transferred from each 50 ml collection tube and weighed using sterile techniques, using a plastic weigh-boat and a bench-top scale. DNA was extracted from the samples using the PowerSoil DNA Isolation Kit and protocols (MO BIO Laboratories, Inc., Carlsbad, CA). Three microliters (µl) of kit eluent (total eluent volume per sample 100 µl) was utilized for each PCR template. For Bacillus species, Bacillus anthracis and pX02 virulence plasmid screening, the multiplex PCR primers utilized were previously described in Ko et al, 2003: BA-RF (5'-GACGATCATYTWGGAAACCG-3'), BA-RR (5'-GGNGTYTCRATYGGACACAT-3'), Ba-SF (5'-TTCGTCCTGTTATTGCAG-3'), Cap-S (5'-ACGTATGGTGTTTCAAGATTCATG-3'), and Cap-R (5'-ATTTTCGTCTCATTCTACCTCACC-3') (6). These primers amplify a 359 base-pair region of rpoB gene (encodes the RNA polymerase ß-subunit) that is specific for Bacillus species at the genus level (BA-RF/BA-RR primer pair), a 208 base-pair region of the same gene that is specific for B. anthracis (inclusion of the additional forward primer Ba-SF), and a 291 base-pair region of the cap gene for pX02 detection (6). Master-mix recipe per reaction was: 10 µl of QIAGEN HotStarTaq Plus Master Mix Kit (QIAGEN, Valencia, CA), 2 µl of the CoralLoad concentrate (component of the HotStarTaq Kit), 1 µl of each of the five primers (10 µm working stock), and 3 µl of template. Tempcycler reaction profile was: Fifteen minutes at 95 degrees Celsius (C),followed by 30 cycles of setting the temperature at 95 C for 30 s, then 45 C for 30 s, and then 72 C for 1 min. At the end of those 30 cycles, a final extension at 72 C for 10 min followed by a hold at 4 C. PCR amplicons were visualized using 10 µl of the reaction volume and SYBRGold stained gel electrophoresis. Samples that produced amplicons for both rpoB markers were logged as presumptively positive for B. anthracis with or without the presence of the cap marker (due to cap amplicon signal strength issues (7)). Confirmation of PCR B. anthracis rpoB positive samples: Samples that were rpoB PCR positive for B. anthracis were sent to the University of South Florida Center for Biological Defense (USF-CBD) for confirmation. USF-CBD is an affiliate member of the Southeast Regional Center of Excellence for Emerging Infections and Biodefense, which was established by the National Institutes of Health and the U.S. Department of Homeland Security in 2003. Approximately 5 g of each potentially positive soil sample was transferred to a pre-sterilized 50 ml tube, using sterile techniques and sent to USF-CBD for analyses. USF-CBD utilizes published and in-house designed primers and probes and dot-blot assay as described in Luna et al, 2006 to screen the samples for the presence of the pOX1 (pag and lef markers) and pOX2 (cap marker) plasmid virulence genes (8). PCR reaction controls: Bacillus atrophaeus DNA obtained from a liquid culture extract was utilized for PCR positive control reactions. Negative control template was PCR grade water. Control template spike volumes were 3 µl of water for the negative control and 2 µl of water and 1 µl of DNA for the positive control. References:(1)Smith DB, Woodruff LG, O'Leary RM, Cannon WF, Garrett RG, Kilburn JE, Goldhaber MB. 2009. Pilot studies for the North American Soil Geochemical Landscapes Project - Site selection, sampling protocols, analytical methods, and quality control protocols. Applied Geochemistry 24:1357-1368; (2)Smith DB, Cannon WF, Woodruff LG. 2011. A national-scale geochemical and mineralogical survey of soils of the conterminous United States. Applied Geochemistry 26:S250-S255; (3)Stevens DL, Olsen AR. 2004. Spatially balanced sampling of natural resources. Journal of the American Statistical Association 99:262-278; (4)Stevens DL, Olsen AR. 2003. Variance estimation for spatially balanced samples of environmental resources. Environmetrics 14:593-610; (5)Stevens DL, Olsen AR. 1999. Spatially restricted surveys over time for aquatic resources. Journal of Agricultural Biological and Environmental Statistics 4:415-428; (6)Ko KS, Kim JM, Kim JW, Jung BY, Kim W, Kim IJ, Kook YH. 2003. Identification of Bacillus anthracis by rpoB sequence analysis and multiplex PCR. Journal of Clinical Microbiology 41:2908-2914; (7)Griffin, D.W., V. Luna, T. Petrosky, and S.A. Morman. 2009. A Bacillus anthracis survey of North American soil using several long-range transects, a group of post-Katrina New Orleans soils samsples, and multiplex-PCR. Applied Geochemistry. 24:1464-1471; (8)Luna VA, King DS, Peak KK, Reeves F, Heberlein-Larson L, Veguilla W, Heller L, Duncan KE, Cannons AC, Amuso P, Cattani J. 2006. Bacillus anthracis virulent plasmid pXO2 genes found in large plasmids of two other Bacillus species. Journal of Clinical Microbiology 44:2367-2377.