1.Development and validation of TaqMan real-time PCR for the detection of Burkholderia pseudomallei isolates from Malaysia
Mohd Ali, M.R ; Foo, P.C. ; Hassan, M. ; Maning, N. ; Hussin, A. ; Syed Ahmad Yunus, S.Z. ; Fauzi, M.H. ; Muhd Besar, A. ; Harun, A. ; Ismail, N. ; Chan, Y.Y.
Tropical Biomedicine 2019;36(2):379-389
Rapid detection of Burkholderia pseudomallei, the etiologic agent of melioidosis,
allows for timely initiation of appropriate treatment and better clinical outcomes. In the
current gold standard, the culture method is time consuming and suffers from low sensitivity.
Meanwhile, previously reported molecular assays are fast and sensitive, but their performance
on isolates from Malaysia, an endemic region of melioidosis is under reported. This study
designed oligonucleotides targeting orf2 of Type III secretion system (TTSS) genes cluster
for the detection of Malaysian B. pseudomallei isolates and evaluated the assay on 95 local
B. pseudomallei strains, 58 other microorganisms and 71 clinical specimens from patients.
The developed assay exclusively detected all tested B. pseudomallei isolates with a detection
limit of 20 fg per reaction (equivalent to ~2.5 copies). Subsequent testing on clinical samples
showed that the assay detected all confirmed specimens with the growth of B. pseudomallei
(n = 10/10). None of the negative specimens had a detectable signal of our TTSS-orf2 assay
(n = 0/61). In conclusion, the present study provides crucial preliminary data for a subsequent
study and should be considered as a potential alternative to current time-consuming culture
method for the detection of B. pseudomallei.
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