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Journal of Clinical Microbiology, August 2002, p. 2897-2902, Vol. 40, No. 8
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.8.2897-2902.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Detection of Bacillus anthracis DNA by LightCycler PCR

Constance A. Bell,1,{dagger} James R. Uhl,1 Ted L. Hadfield,2 John C. David,2 Richard F. Meyer,3 Thomas F. Smith,1 and Franklin R. Cockerill III1,4*

Division of Clinical Microbiology and Division of Infectious Diseases, Mayo Clinic, Rochester, Minnesota 55905,1 Armed Forces Institute of Pathology, Washington, D.C. 20306,2 Centers for Disease Control and Prevention, Atlanta, Georgia 303333

Received 2 April 2002/ Returned for modification 15 April 2002/ Accepted 26 April 2002

Anthrax is a zoonotic disease that is also well recognized as a potential agent of bioterrorism. Routine culture and biochemical testing methods are useful for the identification of Bacillus anthracis, but a definitive identification may take 24 to 48 h or longer and may require that specimens be referred to another laboratory. Virulent isolates of B. anthracis contain two plasmids (pX01 and pX02) with unique targets that allow the rapid and specific identification of B. anthracis by PCR. We developed a rapid-cycle real-time PCR detection assay for B. anthracis that utilizes the LightCycler instrument (LightCycler Bacillus anthracis kit; Roche Applied Science, Indianapolis, Ind.). PCR primers and probes were designed to identify gene sequences specific for both the protective antigen (plasmid pX01) and the encapsulation B protein (plasmid pX02). The assays (amplification and probe confirmation) can be completed in less than 1 h. The gene encoding the protective antigen (pagA) was detected in 29 of 29 virulent B. anthracis strains, and the gene encoding the capsular protein B (capB) was detected in 28 of 29 of the same strains. Three avirulent strains containing only pX01 or pX02, and therefore only pagA or pagB genes, could be detected and differentiated from virulent strains. The assays were specific for B. anthracis: the results were negative for 57 bacterial strains representing a broad range of organisms, including Bacillus species other than anthracis (n = 31) and other non-Bacillus species (n = 26). The analytical sensitivity demonstrated with target DNA cloned into control plasmids was 1 copy per µl of sample. The LightCycler Bacillus anthracis assay appears to be a suitable method for rapid identification of cultured isolates of B. anthracis. Additional clinical studies are required to determine the usefulness of this test for the rapid identification of B. anthracis directly from human specimens.

* Corresponding author. Mailing address: Mayo Clinic, Division of Microbiology, 200 1st St. SW, Rochester, MN 55905. Phone: (507) 284-2901. Fax: (507) 284-4272. E-mail: cockerill.franklin{at}mayo.edu.

{dagger} Present address: Tripler Army Medical Center, Honolulu, Hawaii.

Journal of Clinical Microbiology, August 2002, p. 2897-2902, Vol. 40, No. 8
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.8.2897-2902.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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Copyright © 2002 by the American Society for Microbiology. All rights reserved.