A method for highly sensitive and rapid detection of Pseudomonas aeruginosa, based on magnetic enrichment and magnetic separation, is described in this paper. The magnetic nanoparticles (MNPs) were applied to adsorb genome DNA after the sample was lysed. The DNA binding MNPs were directly subjected to polymerase chain reaction (PCR) to amplify gyrB specific sequence of Pseudomonas aeruginosa. The biotin labeled PCR products were detected by chemiluminescence when they were successively incubated with the probes-modified MNPs and alkaline phosphatase (ALP) labeled streptavidin (SA). Agarose gel electrophoresis analyses approved the method of in situ PCR to be highly reliable. The factors which could affect the chemiluminiscence were studied in detail. The results showed that the MNPs of 400 nm in diameter are beneficial to the detection. The sequence length and the binding site of the probe with a target sequence have obvious effects on the detection. The optimal concentration of the probes, hybridization temperature and hybridization time were 10 μM, 60 ºC and 60 mins, respectively. The method of in situ PCR based on MNPs can greatly improve the utilization rate of the DNA template ultimately enhancing the detection sensitivity. Experiment results proved that the primer and probe had high specificity, and Pseudomonas aeruginosa was successfully detected with detection limits as low as 10 cfu/mL by this method, while the detection of a single Pseudomonas aeruginosa can also be achieved.
Keywords: Chemiluminescence.; In Situ PCR; Magnetic Enrichment; Pseudomonas aeruginosa; gyrB.