Bst dna polymerase

The LAMP reaction occurred in a total volume of 25 μl that contained 2.5 μl of 10× Bst DNA polymerase reaction buffer, 1.6 mM of each of the inner primers (FIP and BIP), 0.2 mM of each outer primer (F3 and B3), 1.4 mM of dNTPs, 6 mM MgSO₄,1 μl of Bst DNA polymerase (New England Biolabs), 1 μl DNA template. The mixture was first incubated at 95°C for 5 min, then placing reaction tube on ice for 1 min, followed by adding 1 μl of Bst DNA polymerase, and the reaction was incubated at 65°C for 60 min and 80°C for 10 min to end the LAMP reaction. The reaction temperatures (58–67°C) and reaction times (10–60 min) were examined to obtain the best LAMP reaction conditions, and 67.5 ng/μl of the DNA concentrations was used in optimization and specificity tests of LAMP.
The conventional PCR method was developed based on the COI sequences of Guang-dilong. The PCR reaction was a 25-μl mixture containing 12.5 μl Taq PCR Mix, 1 μl DNA template, 1 μl of each primers (Table 1), reaction mix 7.5 μl, DNA polymerase 0.4 μl. The PCR cycle procedures were: 3 min at 95°C; then 35 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 45 s; and final extension at 72°C for 10 min. The PCR amplification products were detected by 1% agarose gel electrophoresis.

Both wild-type and vaccine viruses were detected by MEV-VP2-LAMP assay^{15 (link)}. LAMP-SNP reactions were performed in a total volume of 25 μL and contained 0.4 μL (10 pM) each of the F3 and B3 primers, 2.5 μL (10 pM) each of the FIP and BIP primers, 3.5 μL (10 mM) of dNTPs, 2.5 μL of 10 × Bst DNA polymerase buffer, 0.5 μL (8 U) of Bst DNA polymerase (New England BioLabs, Herts, UK), 2 μL of template DNA, 4 μL (100 mM) of MgSO₄, and 6 μL (20 μM) of betaine. The amplifications were performed at 65 °C in water bath for 60 min and were terminated by incubating the reactions at 80 °C in water bath for 10 min. The LAMP amplification products were analyzed by 2.0% (w/v) agarose gel electrophoresis. Besides agarose gel electrophoresis, the products of LAMP-SNP were judged by naked eyes with the addition of SYBR Green I (Solarbio, Beijing, China) dye into the tubes. Upon addition of the SYBR Green I dye to tubes after the LAMP-SNP reaction, the color changed to yellowish green in the positive reactions and remained reddish orange in a negative reaction.

The single RT-LAMP reactions for RdRp or N were performed in 25 μl reaction systems as previously described. Briefly, 0.4 μM of each outer primer (F3 and B3), 0.8 μM of each loop primer (LF* and LB), 1.6 μM of each inner primer (FIP* and BIP), 0.4 mM of biotin-14-dCTP, 1 μl (8 U) of Bst DNA polymerase (New England Biolabs, USA), 1 μl (10 U) of AMV Reverse Transcriptase (New England Biolabs, USA), 12.5 μl of 2 × reaction buffer [40 mM Tris-HCl (pH 8.8), 40 mM of KCl, 16 mM of MgSO₄, 20 mM of (NH₄)₂SO₄, 2 M of betaine, and 0.2% Tween-20] (HuiDeXin Bio-technique, Tianjin, China), and 1 × 10⁴ copies of the RNA template were added to a tube. The mixtures were incubated at 63°C for 1 h. Viral RNA from SARS-CoV (pseudo-virus), MERS-CoV (pseudo-virus), and double distilled water (DW) were used as negative controls (NCs). The mRT-LAMP reaction was performed in a one-step reaction in a 25 μl reaction system containing 12.5 μl of 2 × reaction buffer; 0.2 μM each outer primer, RdRp-F3, RdRp-B3, N-F3, and N-B3; 0.4 μM each loop primer, RdRp-LF*, RdRp-LB, N-LF* and N-LB; 0.8 μM each inner primer, RdRp-FIP*, RdRp-BIP, N-FIP* and N-BIP; 0.4 mM biotin-14-dCTP; 1 μl (8 U) of Bst DNA polymerase (New England Biolabs, USA); 1 μl (8 U) of AMV Reverse Transcriptase (New England Biolabs, USA); and 1 × 10⁴ copies of RNA template. The reaction conditions were carried out as described above.