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Journal of Clinical Microbiology, January 2006, p. 85-90, Vol. 44, No. 1
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.1.85-90.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Development and Evaluation of a Real-Time PCR Assay Targeting the Type III Secretion System of Burkholderia pseudomallei

Ryan T. Novak,1 Mindy B. Glass,1* Jay E. Gee,1 Daniel Gal,2 Mark J. Mayo,2 Bart J. Currie,2 and Patricia P. Wilkins1

Meningitis and Special Pathogens Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333,1 Menzies School of Health Research, Charles Darwin University, Northern Territory Clinical School, Flinders University, Royal Darwin Hospital, Darwin, Northern Territory, Australia2

Received 21 June 2005/ Returned for modification 19 July 2005/ Accepted 11 October 2005

Here we report on the development of a discriminatory real-time assay for the rapid identification of Burkholderia pseudomallei isolates and the evaluation of this assay for sensitivity against related species and detection in spiked human blood samples. The assay targets a 115-base-pair region within orf2 of the B. pseudomallei type III secretion system gene cluster and distinguishes B. pseudomallei from other microbial species. Assay performance was evaluated with 224 geographically, temporally, and clinically diverse B. pseudomallei isolates from the Centers for Disease Control and Prevention strain collection. This represents the first real-time PCR for rapid and sensitive identification of B. pseudomallei that has been tested for cross-reactivity with 23 Burkholderia mallei, 5 Burkholderia thailandensis, and 35 Burkholderia and 76 non-Burkholderia organisms which have historically presented diagnostic challenges. The assay performed with 100% specificity. The limit of detection was found to be 76 femtograms of DNA (equivalent to 5.2 x 103 genome equivalents per ml) in a single PCR. In spiked human blood, the assay could detect as few as 8.4 x 103 CFU per ml. This rapid assay is a valuable tool for identification of B. pseudomallei and may improve diagnosis in regions endemic for melioidosis.

* Corresponding author. Mailing address: Meningitis and Special Pathogens Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, CDC, MS-G34, 1600 Clifton Rd., N.E., Atlanta, GA 30333. Phone: (404) 639-4055. Fax: (404) 639-3023. E-mail: mglass{at}cdc.gov.

Journal of Clinical Microbiology, January 2006, p. 85-90, Vol. 44, No. 1
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.1.85-90.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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