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 rRNA 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
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
B. atrophaeus
genomic DNA using the ITS and
B. atrophaeus recA
Q-PCR assays was 7.5 fg per PCR. The limits of detection of
S. marcescens
genomic DNA using the
gyrB
,
wzm
, and
S. marcescens recA
Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of
B. atrophaeus
vegetative cells and spores was linear (
R
2
> 0.98) over a 7-log-unit dynamic range down to 10
1
B. atrophaeus
cells or spores. Quantification of
S. marcescens
(
R
2
> 0.98) was linear over a 6-log-unit dynamic range down to 10
2
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.