Rapid detection of Pseudomonas aeruginosa by polymerase spiral reaction (PSR)

Abstract:

[Background] Pseudomonas aeruginosa is an important water- and food-borne pathogenic bacterium causing acute enteritis, meningitis, sepsis and skin inflammation. It is important to detect rapidly P. aeruginosa for food safety. [Objective] A rapid and simple method for detection of P. aeruginosa using polymerase spiral reaction (PSR) technique was established. [Methods] The primers were designed according to the P. aeruginosa exotoxin A gene—ETA gene (toxA), the visual PSR method for rapid detection of P. aeruginosa comprised primer screening, accelerated primer introduction, reaction conditions optimization and color indicator screening. The specificity, sensitivity and reliability of the method were evaluated. [Results] The PSR assay to detect toxA of P. aeruginosa could fulfill within 40?min at 65 °C, and visualize the results by Calcein and HNB. This method specifically detected P. aeruginosa without showing cross-reaction with closely related Pseudomonas species or other bacteria. The sensitivity of the method was high, with detection limit 20 CFU/mL bacteria and 1.011 5 pg/μL genomic DNA. The detection of isolated strain of packaged drinking water showed that the PSR method was consistent with the results of traditional biochemical methods for the P. aeruginosa detection. [Conclusion] The established visual PSR method provides a potential method to be used for rapid detection in the field.

Key words:Pseudomonas aeruginosa, Polymerase spiral reaction (PSR), isothermal amplification, DNA rapid detection