Abstract
Despite the advanced understanding of the disease, melioidosis, an infection caused by Burkholderia pseudomallei, continues to be of global interest. The bacterial virulence factor, type six secretion system-5 (T6SS-5), in particular, is an essential factor for B. pseudomallei that is associated with internalization and intracellular survival of the pathogen. To detect the virulence gene cluster, this study has successfully developed a novel seven-gene (tssC-5, tagD-5, tssA-5, hcp-5, tssB-5, tssF-5, and vgrG-5) multiplex PCR assay. The optimum annealing temperature for this assay ranged between 59 and 62 °C. The limit of detection for this assay was 103 CFU/mL for all genes, excluding tssF-5, which was found at 105 CFU/mL of the bacterial concentration. In sensitivity and specificity tests, this multiplex assay was able to amplify all of the seven target genes from 93.8% (n = 33/35) clinical and 100% (n = 2/2) environmental isolates of B. pseudomallei. Whereas only four genes (tssC-5, tagD-5, tssF-5, and vgrG-5) were amplified from Bukholderia thailandesis, two genes (tagD-5 and tssB-5) were amplified from Bukholderia stagnalis, and zero target genes were amplified from Bukholderia ubonensis. No amplification of any genes was obtained when tested against isolated DNA from non-Bukholderia species (n = 20), which include Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecalis, and others. In conclusion, this multiplex PCR assay is sensitive, species-specific, rapid, and reliable to detect the virulent gene cluster T6SS-5 of B. pseudomallei.
Highlights
Burkholderia pseudomallei is a gram-negative environmental saprophyte with high genetic diversity [1]
In Malaysia, it is estimated that more than 2000 patients die due to melioidosis per year, which is much higher than the death caused by tuberculosis or dengue fever based on the reported incidence and mortality rate [6]
The purpose of the present study was to develop a sensitive, speciesspecific, rapid, and reliable multiplex PCR assay for the detection of seven genes of the T6SS-5 virulence gene cluster
Summary
Burkholderia pseudomallei is a gram-negative environmental saprophyte with high genetic diversity [1] It is the causative agent of melioidosis, a neglected tropical and potentially fatal disease affecting both humans and animals. In Malaysia, it is estimated that more than 2000 patients die due to melioidosis per year, which is much higher than the death caused by tuberculosis or dengue fever based on the reported incidence and mortality rate [6]. This indicates that the pathogen is highly virulent
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