Loopamp type a influenza detection kit

To compare the performance between liquid reagent (DNA amplification kit, Eiken Chemical Co.) and the dried reagent (contained in the microtubes provided in the Loopamp Type A Influenza detection kit), the DNA templates prepared from 14 V. parahaemolyticus reference strains were used. LAMP reaction with liquid reagent was performed according to the manufacture’s instruction. LAMP reaction with dried reagent is described above. The LAMP primer sets for the detection of tdh, trh1 and trh2 genes were used to conduct four LAMP assays targeting the tdh, trh1, trh2 and tdh plus trh2 genes as previously reported (Yamazaki et al., 2010 (link)). We judged the results using a turbidimetric system 1 h after the beginning of the reaction using the Loopamp EXIA LA-320A (Eiken Chemical Co.). Results were judged using the visual system after 1 h by a change in the color of the reaction solution. Finally, to assess the utility of the dried LAMP reagent in tropical countries, we tested its stability at high-temperatures. The reagents were exposed to temperatures of 30, 40, 50, and 60^∘C for 15 days; and after, a tdh LAMP assay using a standard tdh⁺V. parahaemolyticus strain was performed (Yamazaki et al., 2010 (link)).

The microtubes containing the dried reagent were taken from a commercially available kit for detection of Influenza virus (Loopamp Type A Influenza detection kit, Eiken Chemical Co., Ltd., Tokyo, Japan). The microtubes were transported, stored, and rehydrated at room temperature. Five μl of DNA template solution, 1.3 μl of tdh LAMP primer set (Yamazaki et al., 2010 (link)) and 23.7 μl of distilled water were added to make a final volume of 30 μl per reaction were added to the microtube. The cap was tightly secured and the microtube was inverted for 3 min in order to rehydrate the reagent which is located in the cap of the tube. The tube was heated at 65^∘C for 1 h (reaction) and at 80^∘C for 5 min (enzyme inactivation). Results of the reaction was judged using the visual system by the color change from brown to green in the reaction solution.