Novel Sample Preparation Method for Safe and Rapid Detection of Bacillus anthracis Spores in Environmental Powders and Nasal Swabs

Abstract

Bacillus anthracis spores have been used as a biological weapon in the United States. We wanted to develop a safe, rapid method of sample preparation that provided safe DNA for the detection of spores in environmental and clinical specimens. Our method reproducibly detects B. anthracis in samples containing <10 spores.Bacillus anthracis spores, a recent threat, can remain dormant for years while retaining full virulence (5,10,13,20,21,22; S. Endicott, E. Hagerman, and M. Furmanski, Letter, JAMA 284:561-562, 2000). Powders and environmental samples, the most common nonclinical specimens, and nasopharyngeal swabs are submitted to designated laboratories in the national Laboratory Response Network (LRN), which has facilities to work safely on the specimens (6,19,23). High numbers of specimen can overwhelm a laboratory's capability and capacity to perform the tests in a timely fashion. From October to December 2001, the Florida Department of Health's three LRN laboratories each received hundreds of samples per day, which revealed the need for a method to render the samples harmless so the DNA could be safely extracted under biological safety level 2 conditions, to alleviate the bottleneck, and to decrease the turnaround time to the final result. Specimens may contain Ͻ10 spores, and the isolation and identification of B. anthracis may take days (5, 15). The purpose of this work was to develop a method of sample preparation that would provide safe DNA for the detection of Յ10 B. anthracis spores.Previous studies used sonication probes in open tubes and cartridges to hold samples, a method that necessitated the use of a biological safety level 3 environment and sterilization between each sample preparation (2, 3, 7). Dang et al. demonstrated that DNA from autoclaved spores was usable for PCR assays (9). No one explored the sensitivity of these methods or if combinations of methods could be used.(A preliminary report of this work has been presented previously [V. A. Luna, M.

Bacillus anthracis Virulent Plasmid pX02 Genes Found in Large Plasmids of Two Other Bacillus Species

“…Total DNA was extracted from the original environmental specimens and used as template in a PCR assay for Ba813, an intergenic chromosomal marker for B. anthracis, as previously described (19,22). In addition, the PCR assays for the pX01 and pX02 plasmids and chromosomal marker were repeated following LRN procedures (31).…”

“…After more tests demonstrated that none of these were B. anthracis, the 19 isolates were given to the Center for Biological Defense for further examination. By in-house real-time PCR assays, 14 of the 19 isolates and respective powders tested positive only for the Ba813 chromosomal element, which is considered to be unique to B. anthracis (19,22,23). These were not studied further.…”

In silico and in vitro evaluation of PCR-based assays for the detection ofBacillus anthracischromosomal signature sequences

“…30 These genetic markers provide limited specificity and require additional timeconsuming and labor-intensive post-PCR analysis steps. Other areas of the chromosome have also been investigated as potential DNA-targets for identification purposes, including the so-called BA813 [31][32][33][34][35][36][37][38] and BA5510 sequences, 19 genes bclB, 39 sap, 40,41 saspB, 5,42 and sspE, 22,43 the B-type small acid-soluble spore protein gene (SASP), 44 a glycosyltransferase group 1 family protein, 45 a protein showing similarities with an abhydrolase, 18 and several DNA loci located on prophage regions, 17 i.e., BA5345, 21 BA5357, 46 and PL3. 47 Although most of these regions have been claimed to be anthrax-specific, B. cereus strains sometimes yield false positive results.…”

“…Assays mentioned by the World Health Organization (WHO) 31,40,44 were also included in the ring trial, as well as a hydrolysis probe assay 35 that targets the often used BA813 marker [31][32][33][34][35][36][37][38] (Table 3). The latter marker has shown in silico cross-reactions toward the near-neighbor strains in use in this trial and was included for this reason.…”

Performance Assessment of Three Commercial Assays for Direct Detection of Bacillus anthracis Spores

“…When areas contain numbers of spores lower than the test assay detection limit, the resulting false-negative findings may lead first responders to employ relaxed safety precautions. Although the assays tested are selfcontained and easy to use in the field, their sensitivity fails to meet that of our laboratory experiments (e.g., Ͻ10 spores on environmental swab samples) (2).…”