Reston Microbiology Laboratory (RML) Fee-For-Service Capabilities

The following analysis are available:

• Bacterial cell counts

• DNA extraction and quantification of the amphibian pathogen Batrachochytrium dendrobatidis (chytrid fungus)

• qPCR standards for quantification of the amphibian pathogen Batrachochytrium dendrobatidis (chytrid fungus)

• DNA extraction from water and sediment samples

• qPCR for total Bacteria and Archaea, and microbial functional groups

If you are interested in using our services please see the procedure for requesting analysis and required forms below.

PRICES FOR EACH TYPE OF ANALYSIS (FY 2017)

*Non-USGS orders, credit card payments only

Bacterial Cell Counts

USGS: $50 (per sample)

Non-USGS: $60 (per sample)

Batrachochytrium dendrobatidis qPCR Analyses of Environmental Samples (Water Filters)

USGS: $60 (per sample)

Non-USGS: $72 (per sample)

Batrachochytrium dendrobatidis qPCR Analyses of Swabs

USGS: $22 (per sample)

Non-USGS: $27 (per sample)

Batrachochytrium dendrobatidis Plasmid qPCR Standard (Transgenic E. coli Culture)

USGS: $17 (per sample)

Non-USGS: $20 (per sample)

DNA Extraction +2 qPCR Analyses from Soil or Sediment Samples

USGS: $200 (per sample)

Non-USGS: $235 (per sample)

DNA extraction +2 qPCR Analyses from Water Filters

USGS: $125 (per sample)

Non-USGS: $150 (per sample)

Additional qPCR Analyses from Extracted Sample

USGS: $25 (per sample)

Non-USGS: $30 (per sample)

Procedure for Requesting Services

• Please request analysis and agree on a payment option in advance by emailing Denise Akob (dakob@usgs.gov).

• The submitter should fill out the Sample Submission Excel Form (an individual file is needed for each type of analysis). The file should be emailed to dakob@usgs.gov and a hard copy should be included with each shipment.

• Contact RML in advance for to verify they can be received. The shipping address will be provided at that time.

• If you ship your samples in a cooler and want your cooler returned, please provide your FedEx account number, otherwise the cooler will not be returned.

• Data will not be released until payment has been received.

• Laboratory services provided for USGS programs and personnel are typically paid via a journal voucher (JV).

• If laboratory services are provided to other federal, state, and county agencies, these analytical costs are higher due to USGS overhead. Prior to accepting samples a User Agreement Form must be completed and be submitted for Bureau approval (normally approved within 1 week). Payments from non-USGS agencies are preferably made via credit card and should also be accompanied by a completed RML Order Form. Contact Denise Akob (dakob@usgs.gov) for more information.

• If you are a USGS group and would like to request and pay in advance for analyses of samples to be collected next fiscal year please contact RMEML and fill out the Advance Request for Analytical Services Form.

Batrachochytrium dendrobatidis qPCR analyses of swabs or water samples

We analyze swab or water samples for the presence of the Chytrid fungus Batrachochytrium dendrobatidis (Bd). We accept samples that come directly from swabbing amphibians using a MW113 Sterile Swab fine tip (Medical Wire & Equipment Co (Bath) Ltd, Cat. No. MW113). Please contact the lab for information regarding procedures for swabbing amphibians. Water samples filtered using Sterivex GV 0.22 µm filter units (Cat. No. SVGV010RS) with lower end sealed with HemataSEAL sealant (Cat. No. 260-100) and upper end sealed with Male luer lock plug (Cat. No. EW-45503-56) from amphibian habitats are also analyzed; note that no other filter types are accepted. Detailed protocols for preparing samples are available upon request. We will perform DNA extraction on each water sample or swab followed by quantitative PCR (qPCR). qPCR is running using a SYBR green assay with ITS1-3chytr and 5.8Schytr primers (Boyle et al., 2004). Samples are run in triplicate. Presence-absence data, as well as quantitative estimates of pathogen copy number per swab or liter of water (if positive) are provided in the data returned to you.

qPCR standards for quantifying Batrachochytrium dendrobatidis

Standards for quantifying the Chytrid fungus Batrachochytrium dendrobatidis (Bd) using qPCR are available for purchase. The standard is provided as a culture of transgenic E. coli that contains a plasmid with the Bd gene. If purified and linearized plasmid is needed please contact the lab for pricing and availability.

DNA Extraction & quantitative PCR Analyses from Environmental Samples

We accept environmental samples such as water, soil, and sediment for quantifying microbial groups and potential metabolism (functional genes). Water samples should be collected by filtering through a Sterivex GV 0.22 µm filter unit (Cat. No. SVGV010RS), then stored at -20ºC (frozen) prior to shipping. Soil or sediment samples (~10 g) should be collected in sterile bags or tubes and stored at -20ºC (frozen) prior to shipping. We will perform DNA extraction on each type of sample followed by quantitative PCR to identify:

• Total Bacteria are quantified using the structural gene 16S rRNA with the primer set 46f/519r (Brunk et al., 1996; Lane 1991)

• Total Archaea are quantified using the 16S rRNA structural gene with the primer set 671f/15256r (Brunk et al., 1996)

• Sulfate-reducing bacteria (SRB) are quantified using the functional gene dsrB (dissimilatory sulfite reductase beta-subunit) with the primer set dsrp2060f/dsr4r (Geets et al., 2006; Wagner et al., 1998).

• Geobacteraceae are quantified based on group specific 16S rRNA gene-based primers. Geobacter are known iron-reducing bacteria (FeRB) and quantification of this group can provide a proxy for the abundance of FeRB in the environment. The primers Geo494f and Geo825r published by Anderson et al., 1998 and Holmes et al., 2002 are used for this SYBR-based assay.

• Ammonia-oxidizing bacteria are quantified using the functional gene amoA (the ammonia monooxygenase functional gene of Bacteria) using the primer set amo1f/2r from Rotthauwe et al., 1997.

• Vinyl chloride reducers are quantified using a TaqMan assay targeting the vcrA gene, encoding the vinyl chloride reductase A enzyme. The TaqMan assay uses primers vcr1022f and vcr1093r and probe vcr1042probe published by Ritalahti et al., 2006.

• Dehalococcoides are quantified using group-specific 16S rRNA gene primers dhc730f and dhc1350r published by Bunge et al., 2003.

• Methanogens are quantified using the function gene mcrA, encoding the methyl coenzyme M reductase enzyme, using primer sets mcrAf and mcrAr (Luton et al., 2002).

• Methanomicrobiales are quantified using group-specific 16S rRNA gene primers mbl471f and mbl754r published by Duhamel & Edwards 2006.

• Methanosarcina are quantified using group-specific 16S rRNA gene primers msarc180f and msarc511r (Duhamel & Edwards 2006).

• Methanosaeta are quantified using a TaqMan assay targeting group-specific 16S rRNA gene primers. The assay uses primers msaeta702f and msaeta862r and probe mst753f published by Yu et al., 2005.

Quantitative estimates of each group analyzed as copy number per g of soil/sediment or per liter of water are provided in the data returned to you.

Additional analyses are available upon request.

References of Methods Used

Anderson, R. T., Rooney-Varga, J. N., Gaw, C. V., and Lovley, D. R. (1998) Anaerobic benzene oxidation in the Fe(III) reduction zone of petroleum-contaminated aquifers. Environmental, Science & Technology 32(9):1222–1229.

Brunk, C. F., E. Avaniss-Aghajani, and C. A. Brunk (1996) A computer analysis of primer and probe hybridization potential with bacterial smallsubunit rRNA sequences. Applied and Environmental Microbiology 62:872–879.

Bunge, M., Adrian, L., Kraus, A., Opel, M., Lorenz, W.G., Andreesen, J.R., Görisch, H. and U. Lechner (2003) Reductive dehalogenation of chlorinated dioxins by an anaerobic bacterium. Nature 421:357–360.

Duhamel, M. and E. A. Edwards (2006) Microbial composition of chlorinated ethene-degrading cultures dominated by Dehalococcoides. FEMS Microbiology Ecology 58(3):538-549.

Geets, J., B. Borremans, L. Diels, D. Springael, J. Vangronsveld, D. van der Lelie, and K. Vanbroekhoven (2006) DsrB gene-based DGGE for community and diversity surveys of sulfate-reducing bacteria. Journal of Microbiological Methods 66:194-205.

Holmes, D., Finneran, K., O'Neil, R., and Lovley, D. (2002) Enrichment of members of the family Geobacteraceae associated with stimulation of dissimilatory metal reduction in uranium-contaminated aquifer sediments. Applied and Environmental Microbiology 68(5), 2300–2306.

Lane, D. J. (1991) 16S/23S rRNA sequencing, p. 115–148. In E. Stackebrandt and M. Goodfellow (ed.), Nucleic acid techniques in bacterial systematics. Wiley, New York, NY.

Luton P. E., Wayne J. M., Sharp R. J., and Riley P. W. (2002) The mcrA gene as an alternative to 16S rRNA in the phylogenetic analysis of methanogen populations in landfill. Microbiology 148:3521–353.

Ritalahti, K.M., Amos, B.K., Sung, Y., Wu, Q., Koenigsberg, S.S., and Löffler, F.E. (2006) Quantitative PCR targeting 16S rRNA and reductive dehalogenase genes simultaneously monitors multiple Dehalococcoides strains. Applied and Environmental Microbiology 72: 2765-2774.

Rotthauwe, J.-H., Witzel, K.-P., and Liesack, W. (1997) The ammonia monooxygenase structural gene amoA as a functional marker: Molecular fine-scale analysis of natural ammonia-oxidizing populations. Applied and Environmental Microbiology 63:4704-4712.

Wagner, M., Roger, A. J., Flax, J. L., Brusseau, G. A., and Stahl, D. A. (1998) Phylogeny of dissimilatory sulfite reductases supports an early origin of sulfate respiration. Journal of Bacteriology 180(11):2975–2982.

Yu, Y., Lee, C., Kim, J., and Hwang, S. (2005) Group specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnology and Bioengineering 89: 670–679