Development and Validation of a Specific and Sensitive Quantitative PCR Assay for the Detection of the oprD Cassette in Pseudomonas aeruginosa Clinical Isolates

Carbapenem resistance in Pseudomonas aeruginosa is frequently mediated by the mutational inactivation or complete loss of the oprD porin gene. However, extreme genetic polymorphism within oprD limits traditional PCR amplification, a bottleneck for molecular surveillance. This study aimed to design and validate a novel quantitative PCR (qPCR) primer set flanking the 5′ and 3′ untranslated regions to reliably amplify the complete oprD cassettes across divergent clinical isolates. The assay demonstrated optimal amplification at 54 °C and an analytical limit of detection (LoD) between 10−4 and 10−5 ng of genomic DNA mass. This assay successfully matched 94.09% (1608/1709) of globally distributed P. aeruginosa strains via in silico BLASTn analysis. In a blinded validation cohort of 100 clinical isolates (50 P. aeruginosa and 50 non-P. aeruginosa controls), the qPCR assay achieved 100.0% analytical specificity without cross-reactivity. Among the 50 P. aeruginosa isolates including 40 carbapenem-resistant (CRPA) and 10 carbapenem-susceptible (CSPA), the qPCR assay exhibited 96.0% sensitivity, successfully amplifying oprD cassettes in 95.0% (38/40) of CRPA and 100.0% (10/10) of CSPA strains without phenotypic bias (p > 0.9999). Notably, when combined with sequencing, the two qPCR-negative CRPA isolates were confirmed to harbor true biological oprD deletions. This optimized assay provides a robust molecular surveillance tool for detecting oprD cassettes to facilitate the epidemiological genetic characterization in both CRPA and CSPA isolates.