期刊
DIAGNOSTICS
卷 12, 期 3, 页码 -出版社
MDPI
DOI: 10.3390/diagnostics12030562
关键词
Burkholderia pseudomallei; T6SS-5; optimization; multiplex PCR
资金
- Ministry of Higher Education Malaysia [FRGS/1/2019/SKK11/USM/02/5]
Despite the advanced understanding of melioidosis, caused by Burkholderia pseudomallei, it continues to be a global concern. This study has developed a sensitive and rapid multiplex PCR assay to detect the virulence gene cluster of B. pseudomallei, which shows high sensitivity and specificity.
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 degrees C. The limit of detection for this assay was 10(3) CFU/mL for all genes, excluding tssF-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 (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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据