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

Despite the advanced understanding of the disease, melioidosis, an infection caused by , continues to be of global interest. The bacterial virulence factor, type six secretion system-5 (T6SS-5), in particular, is an essential factor for 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 (C-5, D-5, A-5, -5, B-5, F-5, and 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 CFU/mL for all genes, excluding F-5, which was found at 10 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% ( = 33/35) clinical and 100% ( = 2/2) environmental isolates of . Whereas only four genes (C-5, D-5, F-5, and G-5) were amplified from , two genes (D-5 and B-5) were amplified from and zero target genes were amplified from . No amplification of any genes was obtained when tested against isolated DNA from non- species ( = 20), which include , , , and others. In conclusion, this multiplex PCR assay is sensitive, species-specific, rapid, and reliable to detect the virulent gene cluster T6SS-5 of .