Development of Quantitative Real-Time PCR Assays for the Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate

Department of Civil, Construction, and Environmental Engineering, North Carolina State University, North Carolina 27695

^* To whom correspondence should be addressed. Email: pesaikal{at}ncsu.edu.

	Abstract

Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. 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. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR Green Q-PCR assays were the 16S-23S intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia non-target 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 (C_t) 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 B. atrophaeus genomic DNA using the ITS and recAB Q-PCR assays was 7.5 fg per PCR reaction. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and recAS Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR reaction, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R²>0.98) over a 7-log dynamic range down to 10¹ B. atrophaeus cells or spores. Quantification of S. marcescens (R²>0.98) was linear over a 6-log dynamic range down to 10² S. marcescens 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 B. atrophaeus and S. marcescens in building debris and leachate.