Open AccessDevelopment of Real-Time PCR Assays and Evaluation of Their Potential Use for Rapid Detection of Burkholderia pseudomallei in Clinical Blood Specimens
Author(s) -
Chonthida Supaprom,
Dongling Wang,
Chanvit Leelayuwat,
Wisansanee Thaewpia,
Wattanachai Susaengrat,
Victor Wee Hong Koh,
Eng Eong Ooi,
Ganjana Lertmemongkolchai,
Yichun Liu
Publication year - 2007
Publication title -
journal of clinical microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.349
H-Index - 255
eISSN - 1070-633X
pISSN - 0095-1137
DOI - 10.1128/jcm.00291-07
Subject(s) - burkholderia pseudomallei , melioidosis , taqman , biology , microbiology and biotechnology , polymerase chain reaction , burkholderia , blood culture , real time polymerase chain reaction , bacteria , virology , antibiotics , gene , genetics
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 toBurkolderia pseudomallei . The analytical sensitivity and specificity of the assays were assessed with 91 differentB. pseudomallei isolates, along with 96 isolates and strains representing 28 other bacterial species, including the closely relatedBurkholderia /Ralstonia . The two assays performed equally well with both purified DNA and crude cell lysates, with 100% analytical specificity for the detection ofB. 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 ofB. pseudomallei , and this may be of particular use in the early diagnosis and treatment of septicemic melioidosis.