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Development and evaluation of a real-Time PCR assay targeting the type III secretion system of Burkholderia pseudomallei

Novak, Ryan T., Glass, Mindy B., Gee, Jay E., Gal, Daniel, Mayo, Mark J., Currie, Bart J. and Wilkins, Patricia P. (2006). Development and evaluation of a real-Time PCR assay targeting the type III secretion system of Burkholderia pseudomallei. Journal of Clinical Microbiology,44(1):85-90.

Document type: Journal Article
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Title Development and evaluation of a real-Time PCR assay targeting the type III secretion system of Burkholderia pseudomallei
Author Novak, Ryan T.
Glass, Mindy B.
Gee, Jay E.
Gal, Daniel
Mayo, Mark J.
Currie, Bart J.
Wilkins, Patricia P.
Journal Name Journal of Clinical Microbiology
Publication Date 2006
Volume Number 44
Issue Number 1
ISSN 0095-1137   (check CDU catalogue open catalogue search in new window)
Start Page 85
End Page 90
Total Pages 6
Place of Publication Washington, United States
Publisher American Society for Microbiology
Field of Research 1108 - Medical Microbiology
0605 - Microbiology
0707 - Veterinary Sciences
HERDC Category C1 - Journal Article (DEST)
Abstract 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.
Keywords Burkholderia pseudomallei
Burkholderia mallei
Burkholderia thailandensis
northern Australia
Southeast Asia
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Additional Notes Copyright by the American Society for Microbiology

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