Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.3/81709

Title

Direct identification of Pseudomonas aeruginosa from blood culture bottles by PCR-enzyme linked immunosorbent assay using oprI gene specific primers

Author(s)

Kurupati, Prathiba;  Kumarasinghe, Gamini;  Poh, Chit Laa

Abstract

A PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed for direct identification of Pseudomonas aeruginosa from positive BACTEC blood culture bottles. PCR primers were designed to target a 249 bp sequence of the oprI gene in P. aeruginosa. Biotin-labeled probe (PA3) targeted to the species-specific motif were hybridized to the digoxigenin-labeled amplified products from P. aeruginosa and captured on streptavidin-coated microtiter plates. The specificity of the assay using the PA3 probe was investigated with a range of microorganisms, which are commonly isolated from blood culture bottles serving as negative controls. The PCR-ELISA assay was shown to be highly specific for the identification of P. aeruginosa and was 10-fold more sensitive than an agarose gel-based detection method using the same pair of primers, with a detection limit at 10 fg of template. The PCR-ELISA assay developed in this study is 100% sensitive and 100% specific as it correctly identified all 73 positive and 42 negative controls as well as 25 double blind clinical samples. It significantly reduces the time needed for the identification of P. aeruginosa from positive BACTEC blood cultures bottles from 2-3 days to 6-8 h.

Publication type

Journal article

Source

Molecular and Cellular Probes, Vol. 19, no. 6 (Dec 2005), pp. 417-421

Publication year

2005

FOR Code(s)

0304 Medicinal and Biomolecular Chemistry;  0601 Biochemistry and Cell Biology

Keyword(s)

Blood culture bottles;  OprI gene;  PCR-ELISA;  Pseudomonas aeruginosa

Publisher

Elsevier

ISSN

1096-1194

Publisher URL

http://dx.doi.org/10.1016/j.mcp.2005.07.005

Copyright

Copyright © 2005 Elsevier Ltd. All rights reserved.

Peer reviewed