Development of a multiplex PCR assay for the detection and differentiation of Burkholderia pseudomallei, Burkholderia mallei, Burkholderia thailandensis, and Burkholderia cepacia complex

- A multiplex PCR assay was developed for differentiating Burkholderia pseudomallei complex based on a set of intrinsic beta-lactamase genes.
- The method demonstrated specificity for B. pseudomallei, B. mallei and B. thailandensis including mutants with altered drug resistance.
- The multiplex PCR proved to be highly sensitive, detecting 6023-9561 genomic equivalents (GE) per reaction.

Two species of Burkholderia pseudomallei complex (Bpc), B. pseudomallei and B. mallei, can cause severe life-threatening infections. Rapidly discerning individual species within the group and separating them from other opportunistic pathogens of the Burkholderia cepacia complex (Bcc) is essential to establish a correct diagnosis and for epidemiological surveillance. In this study, a multiplex PCR assay based on the detection of an individual set of chromosomal beta-lactamase genes for single-step identification and differentiation of B. pseudomallei, B. mallei, B. thailandensis, and Bcc was developed. Two pairs of primers specific to a distinct class of B metallo-beta-lactamase genes and a pair of primers specific to the oxacillin-hydrolyzing class D beta-lactamase gene were demonstrated to successfully discriminate species within Bpc and from Bcc. The assay sensitivity was 9561 genomic equivalents (GE) for B. pseudomallei, 7827 GE for B. mallei, 8749 GE for B. thailandensis and 6023 GE for B. cepacia.

The developed multiplex PCR assay based on the detection of class B metallo-beta-lactamase and oxacillin-hydrolyzing class D beta-lactamase genes allows for the one-step differentiation of Burkholderia pseudomallei, B. mallei, B. thailandensis and “cepacia” complex.145