Bst 2.0 warmstart dna polymerase

Three sets of Gap-LAMP primers were prepared in separate reaction tubes. Gap-LAMP reaction was performed in a total volume of 25 μL. The reaction was containing 0.2 μM for each outer primer (B3 and F3), 0.8 μM for each inner primer (BIP and FIP), 1 M betaine (Sigma-Aldrich, St. Louis, MO), 1× isothermal amplification buffer (20 mM Tris-HCl, 50 mM KCl, 10 mM (NH₄)₂SO₄, 2 mM MgSO₄ and 0.1% Tween 20), 2 mM MgSO₄ (New England Biolabs, Ipswich, MA) and 0.9 mM dNTP for SEA primer set or 1.1 mM dNTP for normal and THAI primer sets. The reaction mixture was pre-heated at 95°C for 15 min and cooled on ice. Then, 8 U Bst 2.0 WarmStart DNA Polymerase (New England Biolabs) and 0.16 μg/μL malachite green (Sigma-Aldrich) were added and followed by incubating at 64°C for 60 min. After the incubation step, reaction tubes were placed under visible light for 15 min. Positive reaction with amplified DNA products showed light blue color solution. In contrast, the negative reaction without amplification showed a colorless solution.

All LAMP primers (outer, inner, and loop) were designed based on previously described sequences of the Mtb-IS6110 gene (GenBank accession number: X17348) (Figure 2, Table S1). The LAMP-specific primer design software PrimerExplorer V5 (Eiken Chemical Company Ltd., Tokyo, Japan) was used to design the primers listed in Table S1. The LAMP assay reaction mixture (25 µL) contained IS-Forward Internal Primer (FIP) and IS-Backward Internal Primer (BIP) (1.6 μM), loop primers (IS-Forward Loop Primer (FLP) and IS-Backward Loop Primer (BLP)) (0.8 μM), IS-Forward Outer Primer (FOP) (F3) and IS-Backward Outer Primer (BOP) (B3) (0.2 μM), deoxynucleoside triphosphates (dNTPs; 2 mM), betaine (0.8 M; Sigma-Aldrich, St. Louis, MO, USA), Tris-HCl (20 mM; pH 8.8), potassium chloride (KCl; 10 mM), ammonium sulfate ((NH4)2SO4; 10 mM), magnesium sulfate (MgSO4; 9 mM), Triton X-100 (0.1% v/v), Bst 2.0 WarmStart DNA Polymerase (8 U; New England Biolabs, Ipswich, MA, USA), and the DNA template (5 μL) [24 (link)]. Agarose gel (1% w/v) electrophoresis was used to determine the optimal reaction temperature and time. The positive control was the genomic DNA of Mtb H37Ra reference strain (ATCC, Manassas, VA, USA), while the negative control was diethyl pyrocarbonate (DEPC)-treated water.

LAMP assay based on an 839 bp Loa loa-specific repetitive DNA sequence (GenBank accession no.M34259.1) was performed at CIETUS, Salamanca, Spain, using the set of primers and reaction conditions previously described elsewhere by our group [27 (link)]. Briefly, LAMP reactions were carried out with a total of 15 μL reaction mixture containing 40 pmol of each FIP and BIP primers, 5 pmol of each F3 and B3 primers and 0.57 μL of Bst 2.0 Warm Start DNA polymerase (New England Biolabs Ltd., Hitchin, UK) with 1 μL of extracted DNA as a template. Reactions were incubated at 65 °C for 50 min in a heating block followed by heated at 80 °C for 5–10 min to stop the reaction. LAMP results were visually detected by color change (green: positive; orange: negative) by adding 1 μL of 1:10 diluted 10,000× concentration SYBR Green I (Invitrogen, Waltham, MA, USA) to the reaction tubes. When required, LAMP products were monitored using 1.5 % agarose gel electrophoresis and visualized under UV light in a transilluminator (UVP BioDoc-It² Imager, Analytik Jena).

The consensus sequence of E. canis gltA from selected samples was used to generate LAMP primers using the online LAMP primer designing software, Primer Explorer version 5 (http://primerexplorer.jp/e/) (Table 1).
LAMP reaction mixtures were prepared by mixing 2.5 µL (1×) 10× isothermal amplification buffer, 6 mM MgSO₄, 1.4 mM dNTP mix, a primer mix containing 0.2 µM F3/B3 primers, 1.6 µM FIP/BIP primers, and 0.4 µM LF/LB primers, 8 U Bst 2.0 WarmStart^® DNA Polymerase (New England Biolabs, Inc., Ipswich, MA, USA), 1 µL colori-fluorometric indicator (CFI), 2 µL DNA, and nuclease-free water to achieve a final volume of 25 µL. CFI contains 3 mM hydroxylnaphthol blue (HNB; MP Biomedicals, Aurora, OH, USA) and 0.35% v/v GelGreen (10 000 × Sol, Biotium, Hayward, CA, USA) dissolved in distilled water [17 (link)]. For optimizing LAMP condition, six PCR-positive and two PCR-negative samples were subjected to a LAMP assay for 60 min with varying temperatures between 60 °C to 65 °C. A negative control (nuclease-free water) was included in each run. The reaction was then terminated by heating at 80 °C for two minutes. After determining the optimum temperature, reaction time was varied to 30, 45, and 60 min.

The reaction buffer is composed of 50 mM NaCl, 10 mM (NH₄)₂SO₄, 10 mM KCl, 8.4 mM MgSO₄, 0.8 mM of each dNTP [New England Biolabs (NEB)], 0.1% Synperonic F108 (Sigma-Aldrich), 500 μg/mL BSA (NEB), 2 μM Netropsin (Sigma-Aldrich).
DNA oligonucleotides were acquired with HPLC purification (IDT). Their sequences were the following: input, 5′-dithiol CATTCAGGATCG-3′, template, 5′-C*G*A*TCCTGAATG-CGATCCTGAA-3′; pseudo-template, 5′-T*T*T*TTCGATCCTGAATG-3′; reporter, 5′-Cy5 *A*T*TCAGAATGCGATCCTGAAT BHQ2-3′, where * indicates phosphorothioate groups. Just before the experiment’s start, enzymatic mixture was added, 10× solution of which contained: Bst 2.0 WarmStart DNA Polymerase (NEB): 8% of (/20) solution in diluent A (NEB); Nb.BsmI nicking enzyme (NEB): 40%; ttRecJ exonuclease (provided by collaborators): 15% of (/140) solution in diluent A; BSA, 20 mg/ml: 25%; diluent A: 12%.
The following DNA concentrations were used: template – 50 nM, pseudo-template – 10 nM, reporter – 50 nM. All the solutions were prepared in TE buffer.
The reaction mixture was injected in both microfluidic channels. The device was placed on a heating plate (Tokai hit) at 38 °C and the fluorescence signal was measured using an inverted fluorescence microscope (Olympus IX71, 1.25× objective), equipped with CoolLED pE-2 (CoolLED) excitation system. Fluorescence images were processed using the ImageJ software^{23 (link)}.

DNA staining dye 20 × EvaGreen was purchased from Biotium (Fremont, CA). Deoxynucleotide (dNTP) solution mix (10 mM of each), Bst 2.0 WarmStart DNA polymerase (8 U/μL), Mg₂SO₄ (100 mM), 10 × Isothermal Amplification Buffer (200 mM Tris–HCl, 500 mM KCl, 100 mM (NH₄)₂SO₄, and 20 mM MgSO₄, 1.0% Tween 20 and pH 8.8 at 25 °C) were purchased from New England BioLabs (Ipswich, MA). The ribonuclease RNase H2 (50 U at 2 U/μL) (Dilution Buffer included), primers, CHB and molecular beacon probes, and the pUCIDT (Amp) plasmid containing 300-bp B. burgdorferi recA gene sequence, or 300-bp Enterovirus 71 (EV71) VP1 gene sequence were purchased from or synthesized by Integrated DNA Technologies (Coralville, IA). DNA was extracted from each tick individually, using a MACHEREY–NAGEL nucleospin tissue kit (MACHEREY–NAGEL GmbH & Co. KG, PA, USA). Maestrogen UltraSlim LED blue light illuminator was purchased from Fisher Scientific (Pittsburgh, PA).