AIM:To determine a species-specific real-time polymerase chain reaction ( PCR ) assay to detect Pseudomonas aeruginosa( PA) ,a secondary DNA target for PA that may provide a universal target for other bacterial pathogens,and validate both assays for diagnostic testing. METHODS:PCR detection was established against the ecfX PA gene and the 16S rRNA gene using known PA keratitis isolates. The outcome parameters for both assays were " limit of detection" ( LOD) ,amplification efficiency ( AE) ,and PAGE amplified product analysis. Both assays were validated against 20 true-positive clinical samples positive for PA DNA and 20 true-negative samples containing no PA DNA. Descriptive statistics and PAGE analysis were used as outcome parameters. RESULTS:AE of the ecfX assay was 96.6%,and LOD was 33. 6 copies of target DNA per microliter. AE of the 16S rRNA assay was 103.4%,and LOD was 8.12 copies per microliter. The sensitivity,specificity,positive predictive value,negative predictive value,and efficiency for the ecfX and 16S rRNA assays were ( 75%,95%,94%,79%,and 85%) ,and ( 70%,100%,100%,77%,and 85%) ,respectively. Both PCR assays were validated,followed by confirmation of DNA patterns from PAGE analysis. CONCLUSION:The PCR methodology described here may be a useful adjunct to standard methods in the diagnosis of PA keratitis.