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

Authors :
Noreafifah Semail
Azian Harun
Ismail Aziah
Nik Mohd Noor Nik Zuraina
Zakuan Zainy Deris
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.

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