Open AccessDevelopment and Evaluation of a Real-Time PCR Assay Targeting the Type III Secretion System of Burkholderia pseudomallei
Author(s) -
Ryan T. Novak,
Mindy B. Glass,
Jay E. Gee,
Daniel Gal,
Mark Mayo,
Bart J. Currie,
Patricia P. Wilkins
Publication year - 2006
Publication title -
journal of clinical microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.349
H-Index - 255
eISSN - 1070-633X
pISSN - 0095-1137
DOI - 10.1128/jcm.44.1.85-90.2006
Subject(s) - burkholderia pseudomallei , microbiology and biotechnology , melioidosis , biology , secretion , real time polymerase chain reaction , burkholderia , virology , type three secretion system , bacteria , genetics , gene , virulence , biochemistry
Here we report on the development of a discriminatory real-time assay for the rapid identification ofBurkholderia 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 withinorf2 of theB. pseudomallei type III secretion system gene cluster and distinguishesB. pseudomallei from other microbial species. Assay performance was evaluated with 224 geographically, temporally, and clinically diverseB. pseudomallei isolates from the Centers for Disease Control and Prevention strain collection. This represents the first real-time PCR for rapid and sensitive identification ofB. pseudomallei that has been tested for cross-reactivity with 23Burkholderia mallei , 5Burkholderia thailandensis , and 35Burkholderia 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 × 103 genome equivalents per ml) in a single PCR. In spiked human blood, the assay could detect as few as 8.4 × 103 CFU per ml. This rapid assay is a valuable tool for identification ofB. pseudomallei and may improve diagnosis in regions endemic for melioidosis.