Lamp dna amplification kit

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).

Reagents (of analytical purity) Chelex-100 and betaine were purchased from Sigma, USA; manganese chloride, magnesium sulfate, potassium chloride, sodium hydroxide, EDTA, and ammonium sulfate from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); Tris-HCl from the Shanghai Shengke Biotechnology Co., Ltd. (Shanghai, China); Triton X-100 from the Beijing Meilaibo (MyLab) Medical Technology Co., Ltd. (Beijing, China); dNTPs from Pharmacia, USA; NP-40 from Fluka, USA; 2× Tap MIX Kit from the Tiangen Biochemical Technology Co., Ltd. (Beijing, China); Agarose from Amresco, USA; LAMP DNA Amplification Kit, LAMP reaction tube, and calcein (the fluorescent detection reagent) from the Eiken Chemical Co., Ltd. (Tochigi, Japan); the real-time turbidity meter (LA-320C) from the Rongyan Chemical Co., Ltd. (Tokyo, Japan); thermostatic metal bath (HB-2) from the Wealtech Corporation (Sparks, NV, USA); Spectrophotometer (NanoQ^TM) from the CapitalBio Technology Co., Ltd. (Beijing, China); gel imaging system (Quantun -ST5) from the Gel imager VILBER (Beijing, China); and PCR thermal cycler (ETC811) from the Gene Amplifier Suzhou Dongsheng Xingye Scientific Instrument Co., Ltd. (Suzhou, China).