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 pathogen...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.展开更多
基金Supported by The Pennsylvania Lions Club and The Charles T. Campbell Foundation. A core grant for Vision Research NIH EY008098 provided expertise within the molecular moduleResearch to Prevent Blindness has provided continued support of the ophthalmology department
文摘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.
基金Supported by The Pennsylvania Lions Club and The Charles T. Campbell Foundation. A core grant for Vision Research NIH EY008098 provided expertise within the molecular module,and Research to Prevent Blindness has provided continued support of the ophthalmology department~~
