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.

FIG. 1.

Limits of detection and linear dynamic range of TTS1 PCR using Burkholderia pseudomallei DNA extracts. (A) Probit nonlinear regression analysis of the real-time PCR assay's detection of the TTS1 gene target. (B) Dynamic range analysis of the real-time PCR assay's detection of the TTS1 gene target. The regression line represents data in the linear range. Error bars equal two standard deviations of the aggregate mean. The slope, correlation coefficient (R²), and PCR efficiency (E) are displayed.

FIG. 2.

Limits of detection and linear dynamic range of TTS1 PCR using Burkholderia pseudomallei-spiked human blood. (A) Probit nonlinear regression analysis of the real-time PCR assay's detection of the TTS1 gene target. (B) Dynamic range analysis of the real-time PCR assay's detection of the TTS1 gene target. The regression line represents data in the linear range. Error bars equal two standard deviations of the aggregate mean. The slope, correlation coefficient (R²), and PCR efficiency (E) are displayed.