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

被引:161
作者
Novak, RT
Glass, MB
Gee, JE
Gal, D
Mayo, MJ
Currie, BJ
Wilkins, PP
机构
[1] CDC, Natl Ctr Infect Dis, Div Bacterial & Mycot Dis, Meningitis & Special Pathogens Branch, Atlanta, GA 30333 USA
[2] Flinders Univ S Australia, Royal Darwin Hosp, No Terr Clin Sch, Menzies Sch Hlth Res, Darwin, NT, Australia
关键词
D O I
10.1128/JCM.44.1.85-90.2006
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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 crossreactivity 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.
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收藏
页码:85 / 90
页数:6
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