ScholarWorks@중앙대학교

초록

To ensure high quality of food and water, the identification of traces of pathogens is mandatory. Rapid nucleic acid-based tests shorten traditional detection times while maintaining low detection limits. Challenging is the loss of nucleic acids during necessary purification processes, since elution off solid surfaces is not efficient. We therefore propose the development of a vanishing surface strategy in which cationic liposomes efficiently capture nucleic acids. A lipoplex is formed that can be easily centrifuged down and washed if needed. Adding the lipoplex to detergent solutions or nucleic acid amplification reactions dissolves the liposomes, releasing 100% of the nucleic acid into the reaction. After initial protocol optimization, it was applied to isolate DNA from Escherichia coli, Staphylococcus aureus, and adenovirus in buffer followed by qPCR detection. This enabled the detection of these pathogens down to concentrations of 1 CFU or 1 PFU/mL, respectively. Comparing it to a standard commercial DNA extraction kit, it was superior, as evidenced by lower Ct-values in the qPCR for all pathogen concentrations. Scaling up to larger volumes, samples containing bacteria were first concentrated through nitrocellulose filters (pore size = 0.45 μm). Tap water, lake water, and rinse water of fresh produce were investigated, leading to relevant limits of detection of 100 CFU in 100 mL of tap water, 1000 CFU in 100 mL of lake water, and 100 CFU in 10 g of iceberg lettuce, respectively. Since the liposome protocol is a homogeneous, simple incubation step, it is a valuable alternative to standard commercial nucleic acid extraction kits.

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