Figure 2

Sequence-specific visual detection method that utilizes precipitation titration of LAMP products by adding PEI. (A) Results of sequence-specific visual detection after adding PEI to LAMP reaction solution. After LAMP reaction in the presence of both FITC-labeled HBV probes and ROX-labeled HCV probes followed by addition of the prescribed amount (0.2 μmol as monomer) of PEI (Mw = 600), it was centrifuged for several seconds using a desk-top, low-speed centrifuge. The tube was then visually observed as is on a UV illuminator (365 nm). It was possible to differentiate the LAMP reaction by visualizing the presence of precipitate fluorescence and the color of the fluorescence. 1, LAMP reaction negative. 2, When LAMP reaction with PSA amplification (unrelated LAMP reaction) occurred. 3, When it contained HBV template nucleic acid. 4, When it contained HCV template nucleic acid. 5, When it contained both HBV and HCV template nucleic acids. (B) Diagram of principle of sequence-specific visual detection method that utilizes precipitation titration of LAMP products by adding PEI. First, a LAMP reaction is carried out using a LAMP primer set for two types of template nucleic acid and fluorescently labeled probes, which can hybridize to loop segments of each LAMP products. When a LAMP reaction corresponding to a certain fluorescently labeled probe progresses, the probe will sequentially hybridize to the loop segment generated during the reaction. On the other hand, an unrelated probe remains free in the solution. When an optimized amount of PEI is added after reaction for a set length of time, the positive charge of PEI neutralizes the negative charge of the DNA to form an insoluble LAMP product-PEI complex. At this stage, fluorescently labeled probes hybridized to LAMP products are taken up by the LAMP product-PEI complex together with the LAMP products. Since most of the PEI added is used for formation of the LAMP product-PEI complex, free oligo DNA probes cannot form a complex with PEI. When the generated insoluble complex is pelletized by centrifugation and the pellet is irradiated with excitation light, the labeled fluorescent dye hybridized to LAMP products produces fluorescence.