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Despite the advanced understanding of the disease, melioidosis, an infection caused by <i>Burkholderia pseudomallei</i>, continues to be of global interest. The bacterial virulence factor, type six secretion system-5 (T6SS-5), in particular, is an essential factor for <i>B. pseudomallei</i> 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 (<i>tss</i>C-5, <i>tag</i>D-5, <i>tss</i>A-5, <i>hcp</i>-5, <i>tss</i>B-5, <i>tss</i>F-5, and <i>vgr</i>G-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 <i>tss</i>F-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% (<i>n</i> = 33/35) clinical and 100% (<i>n</i> = 2/2) environmental isolates of <i>B. pseudomallei</i>. Whereas only four genes (<i>tss</i>C-5, <i>tag</i>D-5, <i>tss</i>F-5, and <i>vgr</i>G-5) were amplified from <i>Bukholderia thailandesis</i>, two genes (<i>tag</i>D-5 and <i>tss</i>B-5) were amplified from <i>Bukholderia stagnalis,</i> and zero target genes were amplified from <i>Bukholderia ubonensis</i>. No amplification of any genes was obtained when tested against isolated DNA from non-<i>Bukholderia</i> species (<i>n</i> = 20), which include <i>Staphylococcus aureus</i>, <i>Klebsiella pneumoniae</i>, <i>Enterococcus faecalis</i>, and others. In conclusion, this multiplex PCR assay is sensitive, species-specific, rapid, and reliable to detect the virulent gene cluster T6SS-5 of <i>B. pseudomallei</i>.