Plasmid DNA cloned with HBV or HCV sequence was added to a LAMP reaction solution containing both HBV primers and the probe (FITC-labeled) and HCV primers and the probe (ROX-labeled) and amplified, after which low molecular weight PEI (Mw = 600; 0.2 µmol as a monomer) was added. As shown in Figure 2A , precipitate emitting green fluorescence characteristic of FITC was obtained in LAMP reaction solution containing the HBV template, precipitate emitting red fluorescence characteristic of ROX was obtained in LAMP reaction solution containing the HCV template, and precipitate emitting a color (orange) that was a combination of FITC green and ROX red fluorescence was obtained in LAMP reaction solution containing both templates. They could be observed using an ordinary UV illuminator or UV-LED (light emitted diode). When LAMP products (lambda DNA) not related to HBV and HCV were present, precipitate with no fluores-Sequence-specific visual detection method that utilizes precipitation titration of LAMP products by adding PEI 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. Figure 3 Precipitation titration of LAMP products by addition of PEI. (A) Effect of amount of PEI on sequence-specific incorporation of ROX-labeled lambda DNA recognition probes by DNA-PEI complex (Mw of PEI is 600). When 0.2 µmol to 1.0 µmol of PEI was added as a monomer, almost 100% of labeled probes hybridized to the LAMP products for lambda DNA was taken up by the DNA-PEI complex. On the other hand, when 0.4 µmol to 0.8 µmol of PEI was added as a monomer to a reaction solution in which an amplification reaction did not take place, a small amount (<20%) of labeled probes precipitated. Precipitation of this nonspecific probe did not take place when unrelated LAMP products (PSA) were present. (B) Effect of the Mw of PEI on sequence-specific incorporation of ROX-labeled lambda DNA recognition probes by DNA-PEI complex. When PEI with Mw 600 was used, the fluorescence intensity of supernatant decreased only when LAMP product for lambda DNA was present. As the Mw of the PEI used increased from 1,800 to 10,000, the fluorescence intensity of the supernatant decreased in the absence of a LAMP reaction since the formation of insoluble PEI-oligo DNA probe complex occurred. (C) Effect of amount of KCI on sequence-specific incorporation of ROX-labeled lambda DNA recognition probes by LAMP product-PEI (Mw = 600) complex. Normal LAMP reaction solution (control) contains 10 mM of KCI. Since formation of the PEI-LAMP product complex was inhibited by an increase in the amount of KCI added, the fluorescence intensity of supernatant increased regardless of the sequence of the LAMP product. (D) Effect of amount of LAMP product for lambda DNA on sequence-specific incorporation of ROX-labeled lambda DNA recognition probe by LAMP product-PEI complex (Mw of PEI = 600). Even when the amount of LAMP product was 1 µg per 25 µL, almost 100% of labeled probes (1 pmol) was taken up by PEI-DNA complex.