Dna amplification kit

The reaction mixture (DNA Amplification Kit; Eiken Chemical Co., Ltd., Tochigi, Japan) for LAMP and MB-LAMP were 25 μL per reaction tube (Eiken Chemical Co., Ltd., Tochigi, Japan), containing the following reagents (final concentration): 20 mM Tris-HCl (pH = 8.8), 10 mM KCl, 10 mM (NH4)₂SO₄, 0.1% Tween 20, 0.8 M betaine, 8 mM MgSO₄, 1.4 mM each dNTP, and 8 U Bst DNA polymerase. Primer concentrations for LAMP were 40 pmol of FIP and BIP, 20 pmol of LF, and 5 pmol of F3 and B3; concentrations for MB-LAMP were 40 pmol of FIP and BIP, 20 pmol of LF, 2–10 pmol of LBP, and 5 pmol of F3 and B3. In addition, template genomic DNA (2 μL) was added. The LAMP was performed at the traditional temperature of 65 °C, and the optimum conditions for MB-LAMP were determined in the experiments described in this manuscript. We used the concentration of 3.2 × 10⁻⁴ mM and temperature of 63 °C once they were established as the optimal conditions.

The LAMP reactions were carried out in 25 μl reaction mixture (DNA Amplification Kit; EIKEN CHEMICAL CO., LTD, Tochigi, Japan) containing the following reagents (final concentration):20 mM Tris-HCl (pH = 8.8), 10 mM KCl , 10 mM (NH4)₂SO4, 0.1 % Tween20, 0.8 M betaine, 8 mM MgSO4, 1.4 mM dNTP each and 8U Bst DNA polymerase. The amount of primer needed for one reaction was 40 pmol for FIP and BIP, and 5 pmol for F3 and B3. Finally, an appropriate amount of template genomic DNA was added to the reaction tube. The reaction was carried out and monitored at 63 °C in a Loopamp real-time turbidimeter(LA320-C, Eiken Chemical Co., Ltd., Tokyo, Japan).

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)).