Development and validation of TaqMan real-time PCR for the detection of Burkholderia pseudomallei isolates from Malaysia

MR Mohd Ali; PC Foo; M Hassan; N Maning; A Hussin; SZ Syed Ahmad Yunus; MH Fauzi; A Muhd Besar; A Harun; N Ismail; YY Chan

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Development and validation of TaqMan real-time PCR for the detection of Burkholderia pseudomallei isolates from Malaysia

Author: Mohd Ali, M.R ^{1
,

2} ; Foo, P.C. ³ ; Hassan, M. ⁴ ; Maning, N. ⁴ ; Hussin, A. ⁴ ; Syed Ahmad Yunus, S.Z. ⁵ ; Fauzi, M.H. ⁶ ; Muhd Besar, A. ⁷ ; Harun, A. ⁷ ; Ismail, N. ⁷ ; Chan, Y.Y. ⁸
Author Information

1. Department of Medical Microbiology &
2. Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia
3. Acarology Unit, Infectious Disease Research Center (IDRC), Institute for Medical Research (IMR), Complex National Institutes of Health (NIH), Section U13 Setia Alam, 40170 Shah Alam, Selangor, Malaysia
4. Department of Pathology, Hospital Raja Perempuan Zainab II, 15586 Kota Bharu, Kelantan, Malaysia
5. Bacteriology Unit, Infectious Disease Research Center (IDRC), Institute for Medical Research (IMR), Complex National Institutes of Health (NIH), Section U13 Setia Alam, 40170 Shah Alam, Selangor, Malaysia
6. Department of Emergency Medicine, School of Medical Science, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia
7. Hospital Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia
8. Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia
Publication Type:Journal Article
From:Tropical Biomedicine 2019;36(2):379-389
CountryMalaysia
Language:English
Abstract: 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.
Full text:8.2019my1022.pdf