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Development of Multiplex PCR Assay for Screening of T6SS-5 Gene Cluster: The Burkholderia pseudomallei Virulence Factor
- Source :
- Diagnostics, Vol 12, Iss 3, p 562 (2022)
- Publication Year :
- 2022
- Publisher :
- MDPI AG, 2022.
-
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.
- Subjects :
- Burkholderia pseudomallei
T6SS-5
optimization
multiplex PCR
Medicine (General)
R5-920
Subjects
Details
- Language :
- English
- ISSN :
- 12030562 and 20754418
- Volume :
- 12
- Issue :
- 3
- Database :
- Directory of Open Access Journals
- Journal :
- Diagnostics
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.32d023aefef40e4a428243304414047
- Document Type :
- article
- Full Text :
- https://doi.org/10.3390/diagnostics12030562