?url_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Adc&rft.relation=http%3A%2F%2Fd-scholarship-dev.library.pitt.edu%2F7076%2F&rft.title=Establishing+PCR+for+the+detection+of+Pseudomonas+aeruginosa+from+keratitis+patients&rft.creator=Hillenbrand%2C+Maria+Elizabeth&rft.description=Introduction%3A+Pseudomonas+aeruginosa+is+a+corneal+pathogen+and+may+cause+corneal+ulceration.+The+goal+of+this+study+was+to+determine+the+potential+of+PCR+for+detecting+P.+aeruginosa+in+corneal+specimens+from+patients+with+keratitis.+Study+Aims%3A+1)+To+establish+a+specific+real-time+PCR+assay+to+detect+P.+aeruginosa.+2)+To+determine+a+secondary+target+for+P.+aeruginosa+that+may+provide+a+universal+target+for+other+bacterial+pathogens.+3)+To+validate+both+assays+for+diagnostic+testing+with+true+positive+and+true+negative+clinical+samples.Methods%3A+1)+Analytical+studies+were+conducted+by+testing+P.+aeruginosa+and+other+bacteria+isolated+from+patients+with+keratitis+with+a+PCR+assay+designed+to+amplify+the+ecfX+gene+of+P.+aeruginosa.+The+outcome+parameters+were+limit+of+detection%2C+and+amplification+efficiency.+2)+Similarly%2C+P.+aeruginosa+isolates+were+tested+for+the+16S+rRNA+gene+using+the+same+parameters.+3)+Validation+of+both+assays+was+done+by+testing+20+cornea+samples+known+to+be+positive+for+P.+aeruginosa+and+20+clinical+samples+known+to+be+negative+for+P.+aeruginosa+DNA.+Descriptive+statistics+were+determined.+PAGE+analysis+was+performed+to+confirm+the+presence+of+amplified+product.Results%3A+1)+Amplification+efficiency+of+the+ecfX+assay+was+96.6%25%2C+with+a+limit+of+detection+of+33.6+copies+of+target+DNA%2F%C2%B5l.+All+21+P.+aeruginosa+isolates+were+detected%2C+with+no+detection+of+the+35+non-P.+aeruginosa+isolates.+2)+Amplification+efficiency+of+the+16S+rRNA+assay+was+103.4%25%2C+with+a+limit+of+detection+of+8.12+copies+%2F%C2%B5l.+All+21+P.+aeruginosa+isolates+were+detected.+3)+The+sensitivity%2C+specificity%2C+positive+predictive+value%2C+negative+predictive+value%2C+and+efficiency+for+the+ecfX+and+16S+rRNA+assays+were%2C+%5B75%25%2C+95%25%2C+94%25%2C+79%25%2C+and+85%25%5D%2C+and+%5B70%25%2C+100%25%2C+100%25%2C+77%25%2C+and+85%25%5D%2C+respectively.+PAGE+analysis+supported+specificity+of+the+DNA+amplified+products.+Conclusions%3A+Both+real-time+PCR+assays+used+in+this+study+detected+P.+aeruginosa+DNA+from+keratitis+patient+samples.+These+results+indicate+that+aside+from+culture%2C+PCR+may+be+a+useful+adjunct+method+in+the+diagnosis+of+keratitis+patients.+Public+Health+Relevance%3A+Real-time+PCR+can+be+used+to+detect+P.+aeruginosa+from+patients+with+keratitis+to+help+preserve+vision.&rft.date=2010-06-28&rft.type=University+of+Pittsburgh+ETD&rft.type=PeerReviewed&rft.format=application%2Fpdf&rft.language=en&rft.identifier=http%3A%2F%2Fd-scholarship-dev.library.pitt.edu%2F7076%2F1%2FMariaEHillenbrand042810.pdf&rft.identifier=++Hillenbrand%2C+Maria+Elizabeth++(2010)+Establishing+PCR+for+the+detection+of+Pseudomonas+aeruginosa+from+keratitis+patients.++Master's+Thesis%2C+University+of+Pittsburgh.++++(Unpublished)++