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.
journal_name
Appl Environ Microbioljournal_title
Applied and environmental microbiologyauthors
Saikaly PE,Barlaz MA,de Los Reyes FL 3rddoi
10.1128/AEM.00779-07subject
Has Abstractpub_date
2007-10-01 00:00:00pages
6557-65issue
20eissn
0099-2240issn
1098-5336pii
AEM.00779-07journal_volume
73pub_type
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.41.4.849-856.1981
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
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journal_title:Applied and environmental microbiology
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pub_type: 杂志文章,随机对照试验
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更新日期:2020-11-24 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.55.9.2365-2371.1989
更新日期:1989-09-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.49.3.556-562.1985
更新日期:1985-03-01 00:00:00
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pub_type: 杂志文章
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journal_title:Applied and environmental microbiology
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更新日期:1982-07-01 00:00:00
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更新日期:1981-03-01 00:00:00
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更新日期:1996-12-01 00:00:00
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
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journal_title:Applied and environmental microbiology
pub_type: 杂志文章
doi:10.1128/AEM.31.2.213-215.1976
更新日期:1976-02-01 00:00:00
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pub_type: 杂志文章
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