Development of Multiplex PCR Assay for Screening of T6SS-5 Gene Cluster: The Burkholderia pseudomallei Virulence Factor.

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 ( tss C-5, tag D-5, tss A-5, hcp -5, tss B-5, tss F-5, and vgr 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 10 3 CFU/mL for all genes, excluding tss F-5, which was found at 10 5 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 ( tss C-5, tag D-5, tss F-5, and vgr G-5) were amplified from Bukholderia thailandesis , two genes ( tag D-5 and tss B-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 .