Detection of Pseudomonas aeruginosa from clinical and environmental samples by amplification of the exotoxin A gene using PCR

Abstract

PCR was used to detect Pseudomonas aeruginosa from water samples by amplifying a 396-bp region of the exotoxin A (ETA) structural gene sequence. The identify of the amplified 396-bp fragment was confirmed by digesting it with PvuI restriction endonuclease, which produced the predicted 246- and 150-bp fragments. Specific primers amplified ETA-positive P. aeruginosa DNA, whereas other species of Pseudomonas and GC-rich bacteria did not yield any 396-bp fragment. The specificity and sensitivity of the assay were 100 and 96%, respectively, which confirms the assay's reliability for diagnostic and epidemiological studies. The assay can detect as few as 5 to 10 cells in a 10-ml water sample or 0.1 pg of P. aeruginosa DNA per reaction mixture (5 microliters) by ethidium bromide staining of an agarose gel. Ten-times-lower concentrations were detected by hybridization with a digoxigenin-labeled oligonucleotide probe internal to the PCR product. With this PCR method, ETA-positive P. aeruginosa was detected in animal cage water samples at a level of 40 cells per ml. This method is rapid and less cumbersome than other diagnostic methods for the identification of P. aeruginosa strains. The method described can be used to detect a low level of P. aeruginosa from environmental and clinical samples without the use of selective media or additional biochemical tests.