title: Development and evaluation of a real-time PCR assay targeting the type III secretion system of Burkholderia pseudomallei.
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 10(3) genome equivalents per ml) in a single PCR. In spiked human blood, the assay could detect as few as 8.4 x 10(3) CFU per ml. This rapid assay is a valuable tool for identification of B. pseudomallei and may improve diagnosis in regions endemic for melioidosis.
authors.affiliation: Meningitis and Special Pathogens Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, CDC, Atlanta, GA 30333, USA.
authors.forename: Ryan T
authors.initials: RT
authors.lastname: Novak
authors.forename: Mindy B
authors.initials: MB
authors.lastname: Glass
authors.forename: Jay E
authors.initials: JE
authors.lastname: Gee
authors.forename: Daniel
authors.initials: D
authors.lastname: Gal
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Mayo
authors.forename: Bart J
authors.initials: BJ
authors.lastname: Currie
authors.forename: Patricia P
authors.initials: PP
authors.lastname: Wilkins
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015373:Bacterial Typing Techniques; D016957:Burkholderia pseudomallei / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D005069:Evaluation Studies as Topic; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial