Multiplex amplification assisted real-time nanopore accurate sequencing for rapid and on-site identification of Pseudomonas aeruginosa in packaged drinking water

The occurrence of Pseudomonas aeruginosa (P. aeruginosa) contamination in packaged drinking water has become a significant public health concern. Accurate, specific and rapid detection of P. aeruginosa in drinking water is crucial to safeguard public health. In this study, we developed a multiplex PCR assisted real-time nanopore sequencing method for rapid and accurate identification of target P. aeruginosa. Three primer sets of long amplicons with non-overlapping sequences were designed. By combining real-time sequencing, the time-varying current signals captured by nanopore proteins were used for real-time identification of the sequence information of the measured strand using the portable nanopore gene sequencing device. Rapid and on-site identification of P. aeruginosa in packaged drinking water were achieved through real-time bioinformatics analysis. The total time of sequence-based identification could be completed in less than 220 min from sampling to the final results output. Interestingly, with our designed multiplex PCR assisted strategy, the core sequencing operations were completed in just 5 min, and the detection limit could be achieved as 10² CFU/250 mL without pre-culture treatment. Of greater significance, this multiplex PCR assisted real-time sequencing strategy can be applied for the identification of other common bacteria by designing corresponding long amplicon primer sets, making it a potential universal method for rapid and accurate bacteria identification in food safety.