Rapid identification of imipenem-resistant pseudomonas aeruginosa by loop-mediated isothermal amplification based detection of OprL and OprD2 genes

Objective: The incidence of imipenem-resistant Pseudomonas aeruginosa (IRPA) continues increasing rapidly. Sensitive approaches for detecting IRPA infection are warranted to improve the ability to select appropriate antibiotic treatment. Methods: In this study, we optimized a rapid DNA-based diagnostic technique, the visual loopmediated isothermal amplification (LAMP) technique for detecting of the outer membrane lipoprotein (OprL) gene and the loop 2 of outer membrane protein (OprD2) gene. Results: The target DNA was amplified and detected by LAMP method within 60 min at an isothermal temperature of 65°C. The sensitivity of LAMP, which had a detection limit of 17.41 μg/L DNA, was 10-fold greater than that of conventional PCR. Furthermore, positive LAMP results were directly read by the naked-eye. Thus, the visual LAMP method can be suggested as a simple and rapid diagnostic assay. Finally, 62 clinical isolates including 39 P. aeruginosa and 23 negative isolates were detected for OprL and OprD2 genes by visual LAMP, which was showed to be more specific than conventional PCR. Conclusion: The LAMP method is a rapid, simple, sensitive and specific assay for the detection of OprL and OprD2 genes.