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The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate.\n            <jats:italic>Bacillus atrophaeus<\/jats:italic>\n            (vegetative cells and spores) and\n            <jats:italic>Serratia marcescens<\/jats:italic>\n            were used as surrogates for\n            <jats:italic>Bacillus anthracis<\/jats:italic>\n            (anthrax) and\n            <jats:italic>Yersinia pestis<\/jats:italic>\n            (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and\n            <jats:italic>recA<\/jats:italic>\n            gene for\n            <jats:italic>B. atrophaeus<\/jats:italic>\n            and the\n            <jats:italic>gyrB<\/jats:italic>\n            ,\n            <jats:italic>wzm<\/jats:italic>\n            , and\n            <jats:italic>recA<\/jats:italic>\n            genes for\n            <jats:italic>S. marcescens<\/jats:italic>\n            . All assays showed high specificity when tested against 5 ng of closely related\n            <jats:italic>Bacillus<\/jats:italic>\n            and\n            <jats:italic>Serratia<\/jats:italic>\n            nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for\n            <jats:italic>B. atrophaeus<\/jats:italic>\n            genomic DNA using the ITS and\n            <jats:italic>B. atrophaeus recA<\/jats:italic>\n            Q-PCR assays was 7.5 fg per PCR. The limits of detection of\n            <jats:italic>S. marcescens<\/jats:italic>\n            genomic DNA using the\n            <jats:italic>gyrB<\/jats:italic>\n            ,\n            <jats:italic>wzm<\/jats:italic>\n            , and\n            <jats:italic>S. marcescens recA<\/jats:italic>\n            Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of\n            <jats:italic>B. atrophaeus<\/jats:italic>\n            vegetative cells and spores was linear (\n            <jats:italic>R<\/jats:italic>\n            <jats:sup>2<\/jats:sup>\n            &gt; 0.98) over a 7-log-unit dynamic range down to 10\n            <jats:sup>1<\/jats:sup>\n            <jats:italic>B. atrophaeus<\/jats:italic>\n            cells or spores. Quantification of\n            <jats:italic>S. marcescens<\/jats:italic>\n            (\n            <jats:italic>R<\/jats:italic>\n            <jats:sup>2<\/jats:sup>\n            &gt; 0.98) was linear over a 6-log-unit dynamic range down to 10\n            <jats:sup>2<\/jats:sup>\n            <jats:italic>S. marcescens<\/jats:italic>\n            cells. The developed Q-PCR assays are highly specific and sensitive and can be used for monitoring the fate and transport of the BW surrogates\n            <jats:italic>B. atrophaeus<\/jats:italic>\n            and\n            <jats:italic>S. marcescens<\/jats:italic>\n            in building debris and leachate.\n          <\/jats:p>","DOI":"10.1128\/aem.00779-07","type":"journal-article","created":{"date-parts":[[2007,8,25]],"date-time":"2007-08-25T01:08:40Z","timestamp":1188004120000},"page":"6557-6565","update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":46,"title":["Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate"],"prefix":"10.1128","volume":"73","author":[{"given":"Pascal E.","family":"Saikaly","sequence":"first","affiliation":[{"name":"Department of Civil, Construction, and Environmental Engineering, North Carolina State University, Raleigh, North Carolina 27695"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Morton A.","family":"Barlaz","sequence":"additional","affiliation":[{"name":"Department of Civil, Construction, and Environmental Engineering, North Carolina State University, Raleigh, North Carolina 27695"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"suffix":"III","given":"Francis L.","family":"de los Reyes","sequence":"additional","affiliation":[{"name":"Department of Civil, Construction, and Environmental Engineering, North Carolina State University, Raleigh, North Carolina 27695"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0022-2836(05)80360-2"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.172.1.342-349.1990"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1021\/ac990347o"},{"key":"e_1_3_2_5_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.70.8.4740-4747.2004"},{"key":"e_1_3_2_6_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.67.6.2564-2570.2001"},{"key":"e_1_3_2_7_2","doi-asserted-by":"publisher","DOI":"10.1023\/A:1026555830014"},{"key":"e_1_3_2_8_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.02660-05"},{"key":"e_1_3_2_9_2","doi-asserted-by":"publisher","DOI":"10.1021\/ac0488098"},{"key":"e_1_3_2_10_2","doi-asserted-by":"publisher","DOI":"10.1099\/00207713-52-2-531"},{"key":"e_1_3_2_11_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.70.6.3664-3672.2004"},{"key":"e_1_3_2_12_2","doi-asserted-by":"publisher","DOI":"10.1128\/AEM.69.11.6597-6604.2003"},{"key":"e_1_3_2_13_2","doi-asserted-by":"publisher","DOI":"10.1046\/j.1472-765x.2001.00966.x"},{"key":"e_1_3_2_14_2","doi-asserted-by":"publisher","DOI":"10.1099\/00207713-42-1-166"},{"key":"e_1_3_2_15_2","first-page":"95","volume":"41","year":"1999","unstructured":"Hall, T. 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