Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprI and oprL

Abstract

A multiplex PCR test based on the simultaneous amplification of two lipoprotein genes, oprI and oprL, was designed and evaluated for its ability to directly detect fluorescent pseudomonads (amplification of oprI open reading frame, 249 bp) and Pseudomonas aeruginosa (amplification of oprL open reading frame, 504 bp) in clinical material. A collection of reference strains including 20 different species of fluorescent pseudomonads was tested. Positive PCR results for both genes were observed only for P. aeruginosa isolates (n ‫؍‬ 150), including strains of clinical and environmental origin, while only one gene, oprI, was amplified from the other fluorescent pseudomonads. All other bacteria tested (n ‫؍‬ 15) were negative by the amplification test. The lower detection level for P. aeruginosa was estimated to be 10 2 cells/ml. Preliminary evaluation on testing skin biopsy specimens from patients with burns (n ‫؍‬ 14) and sputum samples from cystic fibrosis patients (n ‫؍‬ 49) and other patients (n ‫؍‬ 19) showed 100% sensitivity and 74% specificity in comparison with culture. This multiplex PCR assay appears promising for the rapid and sensitive detection of P. aeruginosa in clinical specimens. Further evaluation of its specificity in longitudinal clinical studies is warranted.

Comparative genomic analysis of bovine, environmental, and human strains ofPseudomonas aeruginosa

“…Ten microliter of the Z-broth culture was streaked onto selective HiFluoro TM agar plates (Sigma). After incubation at 37°C for 2 days, fluorescent colonies were identified under UV light and were confirmed by oprI/oprL PCR as P. aeruginosa (De Vos et al, 1997). Biochemical identification of all strains of P. aeruginosa was performed, using the API 20NE test system (bioMerieux, France).…”

Relative expression ofPseudomonas aeruginosavirulence genes analyzed by a real time RT-PCR method during lung infection in rats

“…oprL gene codes for a structural membrane lipoprotein of P. aeruginosa. Its properties have been described in different settings to detect specifically P. aeruginosa in clinical expectoration or other samples by PCR [37] or real time PCR [38], but it was never tested as referent gene to quantify transcripts by RT-PCR. We demonstrate that oprL has the main characteristics required for this function [36] since amplification exhibits a parallel efficiency to those of target in tissue samples.…”

Applying a PCR-based open-reading frame typing method for easy genotyping and molecular epidemiological analysis of Pseudomonas aeruginosa

“…was used as a marker for Ps. aeruginosa (De Vos et al 1997). However, oprL is ubiquitous among Pseudomonas species, resulting in many false positives, even among specific Pseudomonas species.…”