title: PCR-based identification of Burkholderia pseudomallei.
abstract: DNA amplification techniques are being used increasingly in clinical laboratories to confirm the identity of medically important bacteria. A PCR-based identification method has been in use in our centre for 10 years for Burkholderia pseudomallei and was used to confirm the identity of bacteria isolated from cases of melioidosis in Ceará since 2003. This particular method has been used as a reference standard for less discriminatory methods. In this study we evaluated three PCR-based methods of B. pseudomallei identification and used DNA sequencing to resolve discrepancies between PCR-based results and phenotypic identification methods. The established semi-nested PCR protocol for B. pseudomallei 16-23s spacer region produced a consistent negative result for one of our 100 test isolates (BCC #99), but correctly identified all 71 other B. pseudomallei isolates tested. Anomalous sequence variation was detected at the inner, reverse primer binding site for this method. PCR methods were developed for detection of two other B. pseudomallei bacterial metabolic genes. The conventional lpxO PCR protocol had a sensitivity of 0.89 and a specificity of 1.00, while a real-time lpxO protocol performed even better with sensitivity and specificity of 1.00, and 1.00. This method identified all B. pseudomallei isolates including the PCR-negative discrepant isolate. The phaC PCR protocol detected the gene in all B. pseudomallei and all but three B. cepacia isolates, making this method unsuitable for PCR-based identification of B. pseudomallei. This experience with PCR-based B. pseudomallei identification methods indicates that single PCR targets should be used with caution for identification of these bacteria, and need to be interpreted alongside phenotypic and alternative molecular methods such as gene sequencing.
authors.affiliation: Division of Microbiology & Infectious Diseases, PathWest Laboratory Medicine WA, QEII Medical Centre, Nedlands, Australia.
authors.forename: Adam
authors.initials: A
authors.lastname: Merritt
authors.forename: Timothy J J
authors.initials: TJ
authors.lastname: Inglis
authors.forename: Glenys
authors.initials: G
authors.lastname: Chidlow
authors.forename: Gerry
authors.initials: G
authors.lastname: Harnett
mesh_terms: D015373:Bacterial Typing Techniques / Q000379:methods*; D016957:Burkholderia pseudomallei / Q000235:genetics* / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics; D005838:Genotype; D006801:Humans; D008554:Melioidosis / Q000175:diagnosis / Q000382:microbiology; D021141:Nucleic Acid Amplification Techniques; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial