Rapid and visually interpretable LAMP assay for detection of pathogenic Pseudomonas aeruginosa in artificial sputum.

BACKGROUND

Pseudomonas aeruginosa is one of the most common pathogens causing severe complications of lower respiratory tract infections. To rapidly and visually detect P. aeruginosa in sputum samples, we have developed a loop-mediated isothermal amplification (LAMP) assay targeting the oprM gene, a key component of the outer-membrane MexAB-OprM and MexXY-OprM efflux pumps.

METHODS AND

RESULTS

To evaluate the LAMP method, an artificial sputum sample spiked with P. aeruginosa was used; it was more efficient than PCR and showed no interference under the test conditions. The LAMP assay was optimised by varying the reaction temperature and amplification time for the oprM gene target. The assay demonstrated selective detection of P. aeruginosa compared with other bacterial strains isolated from sputum samples. The analytical limit of detection (LOD) was determined to be 2 CFU/mL. Validation with clinical isolates confirmed the assay's specificity and sensitivity, particularly in distinguishing P. aeruginosa strains from other bacterial species in artificial sputum and in indicating intrinsic antibiotic resistance, highlighting its potential for clinical diagnosis.

CONCLUSIONS

The developed oprM gene-targeted LAMP assay exhibits rapid, sensitive, and specific detection of P. aeruginosa in artificial sputum. The assay's low detection limit shows the reliable differentiation from other respiratory bacteria, making it a potentially effective diagnostic tool for early detection of P. aeruginosa infections.