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Journal of Clinical Microbiology, September 2007, p. 2894-2901, Vol. 45, No. 9
0095-1137/07/$08.00+0     doi:10.1128/JCM.00291-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Development of Real-Time PCR Assays and Evaluation of Their Potential Use for Rapid Detection of Burkholderia pseudomallei in Clinical Blood Specimens{triangledown}

Chonthida Supaprom,1,{dagger} Dongling Wang,3,{dagger} Chanvit Leelayuwat,1 Wisansanee Thaewpia,2 Wattanachai Susaengrat,2 Victor Koh,3 Eng Eong Ooi,3 Ganjana Lertmemongkolchai,1* and Yichun Liu3*

Department of Clinical Immunology, Centre for Research and Development in Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand,1 Khon Kaen Regional Hospital, Ministry of Public Health, Khon Kaen 40000, Thailand,2 Defense Medical and Environmental Research Institute, DSO National Laboratories, 27 Medical Drive, 13-01 Singapore, Republic of Singapore 1175103

Received 6 February 2007/ Returned for modification 5 June 2007/ Accepted 6 July 2007

The early initiation of appropriate antimicrobial therapy is critical for improving the prognosis of patients with septicemic melioidosis. Thus, the use of a rapid molecular diagnosis may affect the outcome of this disease, which has a high mortality rate. We report the development of two TaqMan real-time PCR assays (designated 8653 and 9438) that detect the presence of two novel genes unique to Burkolderia pseudomallei. The analytical sensitivity and specificity of the assays were assessed with 91 different B. pseudomallei isolates, along with 96 isolates and strains representing 28 other bacterial species, including the closely related Burkholderia/Ralstonia. The two assays performed equally well with both purified DNA and crude cell lysates, with 100% analytical specificity for the detection of B. pseudomallei. The limit of detection was 50 fg of DNA (equivalent to six bacterial genomes) per PCR for both assay 8563 and 9438. We also evaluated these assays with DNA extracted from blood specimens taken from 45 patients with culture-confirmed septicemic melioidosis or other septicemias. Of the 28 melioidosis blood specimens, assays 8653 and 9438 gave sensitivities of 71% (20/28) and 54% (15/28), respectively. Effectively, all fatal cases of septicemic melioidosis were detected by 8653. For the 17 non-melioidosis blood specimens, specificities of 82% (14/17) and 88% (15/17) were obtained for assays 8653 and 9438, respectively. The real-time PCR assays developed in this study provide alternative, rapid molecular tools for the specific detection of B. pseudomallei, and this may be of particular use in the early diagnosis and treatment of septicemic melioidosis.

* Corresponding author. Mailing address for Yichun Liu: Defense Medical and Environmental Research Institute, DSO National Laboratories, 27 Medical Drive, 13-01 Singapore, Republic of Singapore 117510. Phone: 65 64857254. Fax: 65 64857262. E-mail: lyichun{at}dso.org.sg. Mailing address for Ganjana Lertmemongkolchai: Department of Clinical Immunology, Centre for Research and Development in Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand. Phone: 66 (0) 4320 3825. Fax: 66 (0) 4320 3826. E-mail: ganja_le{at}kku.ac.th

{triangledown} Published ahead of print on 18 July 2007.

{dagger} These authors contributed equally to this study.

Journal of Clinical Microbiology, September 2007, p. 2894-2901, Vol. 45, No. 9
0095-1137/07/$08.00+0     doi:10.1128/JCM.00291-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Hodgson, K., Engler, C., Govan, B., Ketheesan, N., Norton, R. (2009). Comparison of Routine Bench and Molecular Diagnostic Methods in Identification of Burkholderia pseudomallei. J. Clin. Microbiol. 47: 1578-1580 [Abstract] [Full Text]  
  • Chantratita, N., Meumann, E., Thanwisai, A., Limmathurotsakul, D., Wuthiekanun, V., Wannapasni, S., Tumapa, S., Day, N. P. J., Peacock, S. J. (2008). Loop-Mediated Isothermal Amplification Method Targeting the TTS1 Gene Cluster for Detection of Burkholderia pseudomallei and Diagnosis of Melioidosis. J. Clin. Microbiol. 46: 568-573 [Abstract] [Full Text]  


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