Detection of low numbers of Bacillus anthracis spores in three soils using five commercial DNA extraction methods with and without an enrichment step

Authors


  • Present address
    Antonio J. Luna, CH2M Hill Inc., 4350 W. Cypress St., Suite 600, Tampa, FL 33607-4178, USA.

  • J.S. Gulledge and V.A. Luna contributed equally to this paper.

Vicki Ann Luna, Center for Biological Defense, University of South Florida, College of Public Health, 3602 Spectrum Blvd., Tampa, FL 33612, USA. E-mail: vluna@health.usf.edu

Abstract

Aims:  To (i) compare the limits of detection of Bacillus anthracis spores in three soils (one Florida, one Texas, and one a commercial Garden product) by PCR using DNA extracted with five commercial extraction kits and (ii) examine if removing organic acids or adding an enrichment step utilizing a growth medium will improve the detection limits.

Methods and Results: Bacillus anthracis spores were added to soil aliquots and used immediately with a DNA extraction kit or pretreated to remove organics or incubated overnight in a selective growth medium before the DNA extraction was performed. Using hybridization and PCR assays for capC, pag and lef genes, 105–106B. anthracis spores were detected in untreated Florida soil, 104–107 spores in untreated Texas soil and 106–107 in Garden soil. Pretreatment did not reliably improve detection. DNA from untreated and pretreated soils was suitable for hybridization but not always for PCR. When 101–102 spores were added to the soils and allowed to amplify in a growth medium selective for B. anthracis, DNA extracted using four methods reliably produced PCR acceptable DNA positive for the B. anthracis genes.

Conclusions:  The quality of DNA extracted with commercial kits appears to be influenced by the soil type and pretreatment. Yet, with an enrichment step added, four of five extraction methods produced PCR suitable DNA and detected ≤102 spores.

Significance and Impact of the Study:  The enrichment step could enhance the detection of B. anthracis spores in soils and small samples contaminated with soil.

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