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Development of real-time PCR assays and evaluation of their potential use for rapid detection of Burkholderia pseudomallei in clinical blood specimens.
J Clin Microbiol. 2007 Sep; 45(9):2894-901.JC

Abstract

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.

Authors+Show Affiliations

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.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Evaluation Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

17634296

Citation

Supaprom, Chonthida, et al. "Development of Real-time PCR Assays and Evaluation of Their Potential Use for Rapid Detection of Burkholderia Pseudomallei in Clinical Blood Specimens." Journal of Clinical Microbiology, vol. 45, no. 9, 2007, pp. 2894-901.
Supaprom C, Wang D, Leelayuwat C, et al. Development of real-time PCR assays and evaluation of their potential use for rapid detection of Burkholderia pseudomallei in clinical blood specimens. J Clin Microbiol. 2007;45(9):2894-901.
Supaprom, C., Wang, D., Leelayuwat, C., Thaewpia, W., Susaengrat, W., Koh, V., Ooi, E. E., Lertmemongkolchai, G., & Liu, Y. (2007). Development of real-time PCR assays and evaluation of their potential use for rapid detection of Burkholderia pseudomallei in clinical blood specimens. Journal of Clinical Microbiology, 45(9), 2894-901.
Supaprom C, et al. Development of Real-time PCR Assays and Evaluation of Their Potential Use for Rapid Detection of Burkholderia Pseudomallei in Clinical Blood Specimens. J Clin Microbiol. 2007;45(9):2894-901. PubMed PMID: 17634296.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Development of real-time PCR assays and evaluation of their potential use for rapid detection of Burkholderia pseudomallei in clinical blood specimens. AU - Supaprom,Chonthida, AU - Wang,Dongling, AU - Leelayuwat,Chanvit, AU - Thaewpia,Wisansanee, AU - Susaengrat,Wattanachai, AU - Koh,Victor, AU - Ooi,Eng Eong, AU - Lertmemongkolchai,Ganjana, AU - Liu,Yichun, Y1 - 2007/07/18/ PY - 2007/7/20/pubmed PY - 2007/11/7/medline PY - 2007/7/20/entrez SP - 2894 EP - 901 JF - Journal of clinical microbiology JO - J Clin Microbiol VL - 45 IS - 9 N2 - 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. SN - 0095-1137 UR - https://www.unboundmedicine.com/medline/citation/17634296/Development_of_real_time_PCR_assays_and_evaluation_of_their_potential_use_for_rapid_detection_of_Burkholderia_pseudomallei_in_clinical_blood_specimens_ L2 - http://jcm.asm.org/cgi/pmidlookup?view=long&pmid=17634296 DB - PRIME DP - Unbound Medicine ER -