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J Med Microbiol 55 (2006), 551-559; DOI: 10.1099/jmm.0.46350-0
© 2006 Society for General Microbiology
ISSN 1473-5644

Using real-time PCR to specifically detect Burkholderia mallei

Melanie P. Ulrich1, David A. Norwood1, Deanna R. Christensen1 and Ricky L. Ulrich2

Diagnostic Systems Division1 and Bacteriology Division2 , United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011, USA

Correspondence
Ricky L. Ulrich
Ricky.Ulrich{at}AMEDD.ARMY.MIL

Received 30 September 2005
Accepted 13 January 2006

Burkholderia mallei is the causative agent of human and animal glanders and is a category B biothreat agent. Rapid diagnosis of B. mallei and immediate prophylactic treatment are essential for patient survival. The majority of current bacteriological and immunological techniques for identifying B. mallei from clinical samples are time-consuming, and cross-reactivity with closely related organisms (i.e. Burkholderia pseudomallei) is a problem. In this investigation, two B. mallei-specific real-time PCR assays targeting the B. mallei bimAma gene (Burkholderia intracellular motility A; BMAA0749), which encodes a protein involved in actin polymerization, were developed. The PCR primer and probe sets were tested for specificity against a collection of B. mallei and B. pseudomallei isolates obtained from numerous clinical and environmental (B. pseudomallei only) sources. The assays were also tested for cross-reactivity using template DNA from 14 closely related Burkholderia species. The relative limit of detection for the assays was found to be 1 pg or 424 genome equivalents. The authors also analysed the applicability of assays to detect B. mallei within infected BALB/c mouse tissues. Beginning 1 h post aerosol exposure, B. mallei was successfully identified within the lungs, and starting at 24 h post exposure, in the spleen and liver. Surprisingly, B. mallei was not detected in the blood of acutely infected animals. This investigation provides two real-time PCR assays for the rapid and specific identification of B. mallei.

Abbreviations: CT value, crossing threshold value; EPF, end-point fluorescence; MGB probe, minor groove binding probe; p.e., post exposure.






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