Rapid detection of carbapenemase-producing Pseudomonas aeruginosa and Acinetobacter baumannii by loop-mediated isothermal amplification (LAMP)

Abstract

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) and carbapenem-resistant Acinetobacter baumannii (CRAB) are the leading causes of nosocomial infection at intensive care units. Rapid and sensitive detection of CRPA and CRAB infections is in high demand in order to provide timely and suitable antibiotic treatment. In this study, loop-mediated isothermal amplification (LAMP) was optimized as a distinct DNA-based diagnostic technique for rapid detection of the presence of blaVIM-2, blaIMP-1, and blaOXA-23 genes, critical components required for carbapenem resistance. Initially, experimental conditions for the LAMP reaction were optimized, including reaction temperature and the composition of reaction buffer. After blaVIM-2, blaIMP-1 and blaOXA-23 LAMP reactions were established, all experiments were processed under the same conditions. The detection limits of LAMP for blaVIM-2, blaIMP-1 and blaOXA-23 genes were then determined, and the minimum reaction times were evaluated for the detection of blaVIM-2, blaIMP-1 and blaOXA-23 by LAMP assay. With the detection limit of DNA concentration, the sensitivity of PCR and LAMP was compared, thereby suggesting a potential applicability of LAMP as a sensitive and urgent diagnostic method. Furthermore, positive LAMP reactions were directly detected with the naked-eye by monitoring the formation of a white precipitate. Finally, 120 clinical isolates of CRPA and 119 clinical isolates of CRAB were successfully tested for the presence of blaVIM-2, blaIMP-1, and blaOXA-23 genes by LAMP, which was determined to be more sensitive than PCR. Together, our results clearly demonstrate the usefulness of LAMP for the diagnosis of CRPA and CRAB.