Warmstart rtx reverse transcriptase

The RT-LAMP primer sets used in this study were designed by Lamb et al. (2020) [20 (link)] against the nonstructural protein 3 (NSP3) coding region of ORF1ab. The RT-LAMP reaction for swab and saliva was conducted in a total volume of 25 μL of 1X isothermal amplification buffer, 1.4 mM deoxynucleoside triphosphates (dNTPs), 6 mM MgSO4, 1.6 µM FIP/BIP, 0.2 µM F3/B3, 0.4 µM Loop F/B primers, 0.32 U/µL Bst 2 WarmStart DNA polymerase (New England Biolabs, Ipswich, Massachusetts, USA), 15 U/µL WarmStart RTx Reverse Transcriptase (New England Biolabs), 8 µL of nuclease-free water, and 2 µL of viral RNA. A no-template control (NTC) with the same water used for the reaction contained substituted viral RNA. The reaction mixtures were incubated using a PCR thermocycler at 63 °C for 45 min for the swab samples and at 65 °C for 60 min for the saliva samples followed by enzyme inactivation at 80 °C for 10 min. Amplification products were fractionated by 3% agarose gel electrophoresis, stained with fluorescent dye ethidium bromide, and visualized under ultraviolet (UV) light.

All reactions were completed in
triplicate. Each 10 μL experiment contained: 1 μL 10×
isothermal buffer [New England Biolabs (NEB)], 0.6 μL of MgSO₄ (100 mM stock), 1.4 μL of dNTPs (10 mM stock of each
nucleotide), 0.6 μL of BSA (20 mg/mL stock), 0.8 μL of
betaine (5 M stock), 0.25 μL of SYTO 9 green (20 μL stock),
0.25 μL of NaOH (0.2 M stock), 0.042 μL of Bst 2.0 DNA
polymerase (120,000 U/mL stock, NEB), 0.1 μL of RiboLock RNAse
Inhibitor (40 U/μL stock, Thermo Fisher), 0.3 μL of WarmStart
RTx reverse transcriptase (15,000 U/mL, NEB), 1 μL of 10×
LAMP primer mix (20 μM FIP and BIP, 10 μM LB and LF, 2.5
μM F3 and B3), 1 μL of RNA sample, and the remaining solution
was topped up to 10 μL with nuclease-free water. Reactions were
conducted at 63 °C for 35 min. One melting curve from 63 to 97
°C was conducted to confirm the specific amplification of the
reaction at a ramp of 0.2 °C/s. Reactions were conducted with
a LightCycler 96 instrument (Roche Diagnostics) in 96-well plates.

A LAMP primer set (Table 1) targeting gene N (Nucleocapsid phosphoprotein) of SARS-CoV-2 located at the region between nucleotides 28,501–28,709 of the genome (LC547533.1) was used for this study (Zhang et al., 2020 (link)). The specificity of the primer set is shown in Figure S1. The real-time reverse transcriptase LAMP (rRT-LAMP) assay was carried out in 20 μl of master mixture containing 0.2 μM of F3, 0.2 μM of B3, 1.6 μM of FIP, 1.6 μM of BIP, 0.8 μM of LF, 0.8 μM of LB (Integrated DNA Technologies, Leuven, Belgium), 0.35 mM of dATP, 0.35 mM dGTP, 0.35 mM of dCTP, various concentrations of dTTP and dUTP ranging from 0 to 0.35 mM (Thermo Fisher Scientific, Roskilde, Denmark), 0.25 M betaine (Sigma-Aldrich, Denmark), 6 U of Warmstart^® RTx Reverse Transcriptase, 8 U of Bst Warmstart^® 2.0 DNA polymerase, 1× isothermal amplification buffer (New England Biolabs), 0.01 U of Cod-UNG, 2 µM of SYTO 9, sterilized water, and RNA template.
The Cod-UNG-rRT-LAMP reactions were incubated at 25°C for 5 min, then performed on an Mx3005P system (Stratagene, AH Diagnostics, Denmark) at 55°C for 5 min followed by 65°C for 60 min, and terminated by heating to 90°C for 5 min. The fluorescence signal was recorded every minute of amplification.