SPADA Book - April 11, 2017

DRAFT ENVIRONMENTAL ORGANISMS PANEL, VERSION 4

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Part 2: Environmental Panel Organisms

2.1 3 4 Airborne soil particles may constitute a significant challenge to the analysis of collected aerosol samples 5 by p olymerase chain reaction ( PCR ) assays. Soils contain genomic materials or nucleic acid fragments of 6 countless archaebacterial, bacterial, and eukaryotic organisms. Some of the more common soil 7 organisms can be anticipated. Soils may also contain unanticipated inhibitors that interfere with 8 extraction, denaturation, polymerization, or annealing reactions. Therefore, determining the effect of a 9 variety of representative soils on the PCR assay is an important first step in establishing the specificity of 10 the primers/probes, and the robustness of a PCR assay in the presence of interfering compounds. 11 12 13 Using the primers/probe, and amplicon sequences specific for any given assay evaluate each regional 14 soil type*† for any signs of positive response. 15 16 Samples of each regional soil type* should be spiked at 2x, 5x and 10x AMDL with the archetype 17 organism (usually specified in the SMPR for AMDL testing, such as strain CO92 for Yersinia pestis ) and 18 then the samples evaluated for inhibition. Inhibition testing should be done using intact target 19 organisms so that potential interference with the DNA extraction can be determined. 23 † See section 2.2 “Bioinformatics Analysis of Signature Sequences” on probing all available data bases 24 including those containing soil metagenome sequences generated from specific regions of operations (if 25 available) for In Silico Analysis and further validation of the signature sequences. Soil Testing 20 21 22 * Arizona Test Dust is available as a baseline starting point.

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