Rapid detection and quantification of Pseudomonas aeruginosa in bottled water by real time PCR

摘要

A SYBR Green I-based real time PCR method that provides rapid detection and quantification of Pseudomonas aeruginosa in bottled water has been developed. PCR primers were designed to target a 159 bp sequence of the gyrA gene in P. aeruginosa. The purified DNA amplified products were then inserted into pMD19-T Vector to construct recombined plasmids and transformed into competent Escherichia coli JM109 for replication. Target plasmids in white colonies selected by ampicillin screening were identified by colony PCR and DNA sequencing subsequently. The plasmid DNA was purified and diluted into serial gradient concentrations as the reference standard to predicting the load of P. aeruginosa contaminant in bottled water. Results of the bottled water sample analysis demonstrate that the method is rapid, accurate, and easy to manipulate and it can be employed in correlative detection industry.