Reaction tube

The LAMP reaction was conducted in a 25 µl reaction system (LAMP DNA Amplification Kit; Eiken Chemical Co., Ltd., Tochigi, Japan) including below reagents (eventual concentration): 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris–HCl (pH 8.8), 0.1% Tween 20, 0.8M betaine, eight mM MgSO₄, 1.4 mM each dNTP, 8 U Bst DNA polymerase and 10 pmol for F3 and B3, 20 pmol for LF and LB and 40 pmol for FIP and BIP. Eventually, one μl template genomic DNA was added to the reaction tube (Eiken Chemical Co., Ltd., Tochigi, Japan). Distilled water was used as the negative control. The reaction was performed at 60–67 °C for 50 min at 1 °C intervals, respectively. Followed by 80 °C for 5 min to terminate the amplification within a dry bath incubator (OSE-DB-02; Tiangen Biotech Beijing Co., Ltd., Beijing, China).

A 25 μl reaction volume was used for all LAMP reactions and contained the following components (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. The amount of primer per reaction was 40 pmol FIP and BIP, 20 pmol LB, 5 pmol F3 and B3. The appropriate amount of DNA template was included in the reaction volume. The LAMP assay proceeded in a reaction tube (Eiken Chemical Co., Ltd., Tochigi, Japan) for 60 min at 65°C.