Experimental PCR Data on Soil DNA Extracts

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Frequently anticipated questions:


What does this data set describe?

Title: Experimental PCR Data on Soil DNA Extracts
Abstract:
Bacillus species and B. anthracis presence/absence data were determined in 4,770 soil samples collected across the contiguous United States, in cooperation with the U.S. Environmental Protection Agency (EPA). Polymerase Chain Reaction (PCR) data for Bacillus species and B. anthracis rpoB gene PCR amplicon detection were reported as non-detect (n), low (l), medium (m), and high (h). Results for both pag and lef genes of the pX01 plasmid were reported by the University of South Florida's Center for Biological Defense. This data was recorded as negative or positive for each of the genes and included the following combinations: neg/neg, pos/neg, neg/pos, and pos/pos. Data for the pX02 plasmid were recorded as negative (neg) or positive (pos).
Supplemental_Information:
Total screened sample supplementary information: This data release also includes a separate file, banthracis_sampling_supplementary_data.csv, detailing the total number of samples screened in each state and the number and percent of those that were positive for the presence of Bacillus species. The table headers are as follow: "State" (the state the samples were taken in); "# samples screened"; “# Bacillus sp. positive" (within each state); and “% Bacillus sp. positive" (from each state). A summary of presumptive PCR positives results of the pX01 pag, pX01 lef, and pX02 markers show that all three were found to be positive in 10 samples: 3 in Alabama, 1 in Kansas, 1 in Maine, 1 in Missouri, 1 in New York, 2 in Utah, and 1 in West Virginia. There were 66 sample sites where all three genetic markers were found to be negative: 2 in Alabama, 2 in Arkansas, 2 in Arizona, 1 in Florida, 7 in Georgia, 13 in Idaho, 1 in Indiana, 4 in Louisiana, 1 in Michigan, 5 in Minnesota, 1 in Montana, 1 in Nevada, 3 in Ohio, 7 in Oklahoma, 3 in Oregon, 1 in Pennsylvania, 6 in South Carolina, 1 in Texas, 2 in Utah, 1 in Virginia, and 2 in Washington.
  1. How might this data set be cited?
    Griffin, Dale W., and Douglas, Steven H., 20160602, Experimental PCR Data on Soil DNA Extracts: U.S. Geological Survey Data Release doi:10.5066/F7WW7FRJ, U.S. Geological Survey, St. Petersburg, FL.

    Online Links:

    This is part of the following larger work.

    Douglas, Steven H., Unknown, Presence of Microbes and the Distribution of Climatic, Environmental, and Geochemical Variables.

  2. What geographic area does the data set cover?
    West_Bounding_Coordinate: -124.4019
    East_Bounding_Coordinate: -67.5201
    North_Bounding_Coordinate: 48.9835
    South_Bounding_Coordinate: 25.1376
  3. What does it look like?
  4. Does the data set describe conditions during a particular time period?
    Beginning_Date: 2007
    Ending_Date: 2013
    Currentness_Reference:
    ground condition
  5. What is the general form of this data set?
    Geospatial_Data_Presentation_Form: Tabular digital data
  6. How does the data set represent geographic features?
    1. How are geographic features stored in the data set?
      This is a Point data set.
    2. What coordinate system is used to represent geographic features?
  7. How does the data set describe geographic features?
    banthracis detection survey data and banthracis sampling supplementary data
    These .csv and .xlsx files contain tabular data results for the rpoB gene PCR amplicon detection analysis of Bacillus species and B. anthracis. The supplementary sampling data file describes the sample number, total count, and percent of positive Bacillus sp. detected in each state. (Source: U.S. Geological Survey)
    id
    Numerical sample ID assigned to each sample by the analyzing laboratory. (Source: None)
    Range of values
    Minimum:8
    Maximum:31081
    Units:None
    state
    state abbreviation (Source: None) State Names
    state_site
    State abbreviation and id fields were combined, in order to merge the data in Microsoft Access. (Source: U.S. Geological Survey)
    Range of values
    Minimum:AL 10016
    Maximum:WY 9966
    Units:None
    bacillus_s
    PCR results for Bacillus species (Source: U.S. Geological Survey)
    ValueDefinition
    n,l,m,hRange of n = not detected, l = low, m = medium, h = high
    banthpcr
    PCR results for Bacillus anthracis rpoB gene detection. (Source: U.S. Geological Survey)
    ValueDefinition
    n, l, m, hRange of n = not detected, l = low, m = medium, h = high
    px01paglef
    PCR results for Bacillus anthracis PX01 pag and lef markers (Source: U.S. Geological Survey)
    ValueDefinition
    neg/neg, neg/pos, pos/neg, pos/posNegative or positive
    px02
    PCR results for Bacillus antracis PX02 marker (Source: U.S. Geological Survey)
    ValueDefinition
    neg/neg, neg/pos, pos/neg, pos/posNegative or positive
    paglefpx02
    PCR results for Bacillus anthracis PX01 pag, lef, and PX02 markers (Source: U.S. Geological Survey)
    ValueDefinition
    neg/neg/neg, neg/neg/pos, pos/neg/pos, neg/pos/pos, pos/pos/posNegative or positive

Who produced the data set?

  1. Who are the originators of the data set? (may include formal authors, digital compilers, and editors)
    • Dale W. Griffin
    • Steven H. Douglas
  2. Who also contributed to the data set?
    USEPA
  3. To whom should users address questions about the data?
    Dale Griffin
    U.S. Geological Survey
    600 4th St. S
    St. Petersburg, FL
    USA

    727-502-8075 (voice)
    dgriffin@usgs.gov

Why was the data set created?

The goal of this dataset is to plot the results from the respective soil survey, including presence/absence of pathogens of interest. These data and environmental variables such as (but not limited to) geochemical make-up of the soil, ambient meteorological conditions, soil moisture content, land use, etc. are used to help predict persistence and natural occurrence of these agents in the environment.

How was the data set created?

  1. From what previous works were the data drawn?
    DS801 (source 1 of 1)
    Smith, David, 20131025, Geochemical and Mineralogical Data for Soils of the Conterminous United States: U.S. Geological Survey, Denver, CO.

    Online Links:

    Type_of_Source_Media: Online
    Source_Contribution:
    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.
  2. How were the data generated, processed, and modified?
    Date: 01-Jan-2014 (process 1 of 2)
    Lab PCR results for Bacillus sp. genetic marker tests and sample identification information were entered into the spreadsheet as separate columns for each test.
    Date: 27-Mar-2018 (process 2 of 2)
    Keywords section of metadata optimized by adding theme keyword thesauri and associated keywords. Person who carried out this activity:
    U.S. Geological Survey
    Attn: Arnell S. Forde
    Geologist
    600 4th Street South
    St. Petersburg, FL

    727-502-8000 (voice)
    aforde@usgs.gov
  3. What similar or related data should the user be aware of?

How reliable are the data; what problems remain in the data set?

  1. How well have the observations been checked?
    The data included in this dataset represent geochemical and mineralogical analyses of soil samples collected in support of the USGS Soil Geochemical Landscapes Project (http://minerals.cr.usgs.gov/projects/soil_geochemical_landscapes/). A written protocol was established prior to the initiation of the field work as several crews from the U.S. Geological Survey, state geological surveys, and the Natural Resources Conservation Service (NRCS) were expected to be involved with the collection of samples.
  2. How accurate are the geographic locations?
    No formal positional accuracy tests were conducted.
  3. How accurate are the heights or depths?
    No formal positional accuracy tests were conducted.
  4. Where are the gaps in the data? What is missing?
    This dataset is considered complete for the information presented, as described in the abstract. Users are advised to read the rest of the metadata record carefully for additional details.
  5. How consistent are the relationships among the observations, including topology?
    The dataset was constructed by processing data collected in the field, recorded in field sheets, and from laboratory-based chemical and mineralogical analyses. The following criteria were chosen for the reporting of the data: (1) Each sample site has a unique identifier (SiteID), (2) each sample site has a set of geographic coordinates (latitude and longitude), (3) each sample collected in the field and analyzed for chemistry in the lab has a unique lab number, and (4) each analytical determination is linked to a valid, unique lab number.

How can someone get a copy of the data set?

Are there legal restrictions on access or use of the data?
Access_Constraints:
Latitude and longitude data for microbiological sampling were removed for reasons of national security. Information requests will be reviewed on a case by case basis. Please contact Dale Griffin (dgriffin@usgs.gov) for more details. This dataset reflects the conditions at the time the sample was taken. Site specific data may be influenced by sample method detection limit, temperature, and nutrient and water availability. These data are experimental in nature and should not be used, without taking these uncertainties into consideration.
Use_Constraints:
Although these data have been processed successfully on a computer system at the U.S. Geological Survey (USGS), no warranty expressed or implied is made regarding the display or utility of the data on any other system or for general or scientific purposes, nor shall the act of distribution constitute any such warranty. The USGS or the U.S. Government shall not be held liable for improper or incorrect use of the data described and/or contained herein.
  1. Who distributes the data set? (Distributor 1 of 1)
    Dale Griffin
    U.S. Geological Survey
    600 4th St. S
    St. Petersburg, FL
    USA

    727-502-8075 (voice)
    dgriffin@usgs.gov
  2. What's the catalog number I need to order this data set?
  3. What legal disclaimers am I supposed to read?
    The U.S. Environmental Protection Agency, through its Office of Research and Development, collaborated on the research described herein under an Interagency Agreement with the U.S. Geological Survey (IA #DW 14957748 and DW 92401101). This document has been subjected to the Agency's review and has been approved for publication. This report was generated using references (secondary data) that could not be evaluated for accuracy, precision, representativeness, completeness, or comparability and therefore no assurance can be made that the data extracted from these publications meet EPA's stringent quality assurance requirement. The contents of this document reflect the views of the contributors and do not necessarily reflect the views of the Agency. Mention of trade names or commercial products in this document or in the literature referenced in this document does not constitute endorsement or recommendation for use.
  4. How can I download or order the data?

Who wrote the metadata?

Dates:
Last modified: 27-Mar-2018
Metadata author:
Steven Douglas
Cherokee Nation Technologies contracted to U.S. Geological Survey, Coastal and Marine Science Center, St. Petersburg, FL
600 4th St S
St Petersburg, FL
USA

727-502-8185 (voice)
sdouglas@usgs.gov
Metadata standard:
Content Standard for Digital Geospatial Metadata (FGDC-STD-001-1998)

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