Comparative Evaluation of Molecular Detection Performance of Pseudomonas aeruginosa based on Phylogenetic Markers 16S RNAr, recA, rpoB and ITS1

Abstract

P. aeruginosa may be involved in the poisoning of food. It is highly pathogenic to immunocompromised subjects or weakened, causing a high rate of morbidity and mortality. The aim of this study was to determine the phylogenetic marker suitable for molecular identification of Pseudomonas aeruginosa. The purity and concentration of the nucleic acids were determined by spectrophotometry. Sensitivity reactions using phylogenetic markers (16S RNAr, recA, rpoB, STS1) and the threshold detection of 42 strains were assessed by polymerase chain reaction (PCR). With an average absorption at 230 nm of 2.1, the DNA extracts has an average ratio (A260/A280) of 1.7. The threshold detection of Pseudomonas aeruginosa reference strain ATCC 27853 was 0.8 μg/ml for rpoB and 7.6 μg/ml for each of 16S markers RNAr and recA. The threshold detection of positive control strains CP2: 1125A and CP3: API was 1.2 μg/ml and 0.1 μg/ml for using rpoB gene, respectively. This threshold was respectively 12.3 μg/ml and 0.9 μg/ml for the recA gene. The sensitivity of the rpoB housekeeping gene was 97.4% followed by the recA and 16S RNAr with 87.2% and 82.1%, respectively. The phylogenetic resolution of the rpoB genes was higher than that of the 16S rRNA and recA genes. No sensitivity reaction was observed with ITS1 marker. The quality, purity of the nucleic acids and the choice of phylogenetic marker are among the most critical factors for PCR analysis.