Development of Real-Time Multiplex PCR Assay for the Detection and Differentiation of Burkholderia mallei and Burkholderia pseudomallei

Abstract

Objective of the study was to develop a real-time multiplex PCR assay for the detection and differentiation of B. mallei and B. pseudomallei, characterized by high sensitivity and specificity. Materials and Methods. The primers and probes were designed to detect the species-specific sequence of the fliР gene of B. mallei and gp68 gene of B. pseudomallei, respectively. Species specificity was tested with a panel of 56 B. pseudomallei strains, 14 B. mallei strains and 34 strains of closely or distantly related species. To define the analytical sensitivity of the assay, the serially diluted bacterial suspension at concentrations of 109 –102 cells /ml was used. Conclusions. The multiplex PCR assay with two primer pairs and fluorescently-labeled probes, allowing for simultaneous detection and differentiation between B. mallei and B. pseudomallei was designed. Species-specific for glanders agent, B. mallei, fragment of fliP gene, which encodes protein of flagellin biosynthesis, and species-specific gene region of B. pseudomallei, encoding gp68 protein, were identified as DNA targets. Testing of Burkholderia and non-Burkholderia bacterial species revealed 100 % specificity of the amplification assay. The minimum detection limit of the designed multiplex PCR test-system was 1·103 cells/ml for B. mallei, and 1·104 cells/ml for B. pseudomallei.

Keywords

• glanders
• melioidosis
• pcr assay
• real-time pcr
• burkholderia mallei
• burkholderia pseudomallei