Thermopol buffer

Tetrachloroauric (III) acid trihydrate (HAuCl₄ 3H₂O, Sigma-Aldrich, St. Louis, MO, USA), trisodium citrate (Na₃C₆H₅O₇, Sigma-Aldrich, St. Louis, MO, USA), and GO (Graphene Supermarket, Calverton, NY, USA) were used as purchased. Bst DNA polymerase, 10X of ThermoPol buffer, 100 mM magnesium sulfate (MgSO₄), and 10 mM dNTP were obtained from New England BioLabs (Ipswich, MA, USA). SYBR green I fluorescent nucleic acid was purchased from LONZA (Rockland, ME, USA). Ultrapure water was used throughout this study.

The reaction mixture (25 μL) contained 4 μM primer and template. The dNTP concentrations were 200 μM (each) in corresponding buffer. ThermoPol® buffer (New England Biolabs) was used for the Vent (exo-) and Deep Vent (exo-) polymerases. A buffer supplied by the manufacturer was used in the reactions with Taq polymerase. Five units of enzyme were used for all polymerases. The reaction mixtures were heated to 95 °C for 1 min and subsequently cooled to 55 °C for 1 min. PEX reactions were performed at 72 °C (if not specified) for varying amounts of time. The reaction was stopped by precipitation with 2% LiClO₄ in acetone.

The LAMP reaction was initially performed using the Loopamp DNA Amplification Kit (Eiken Chemical, Japan) to optimize the reaction temperature. The self-assembled 2 × LAMP reaction mixture consists of 2x ThermoPol^® buffer (New England Biolabs, Ipswich, MA, USA), 12 mM of magnesium sulfate, MgSO₄ (New England Biolabs, Ipswich, MA, USA), 2.8 mM of dNTPs (First Base Laboratories, Seri Kembangan, Malaysia) and 0.8 M of betaine (Sigma, St. Louis, MO, USA) was prepared. Briefly, a 25 µl LAMP reaction contained 12.5 µl of 2 × reaction mixture, 1 µl of Bst DNA polymerase (8 U), 1 µl of fluorescent dye, 5 pmol each of F3 and B3 primers, 40 pmol each of FIP and BIP primers, 20 pmol each of the LF and LB primers, and varying volumes of samples and nuclease-free water. A no-template control was included in each test. To determine the optimum reaction temperature, the reaction tubes were incubated at 61 °C, 63 °C, and 65 °C for 30 to 60 min either in a Loopamp Realtime Turbidimeter (Eiken Chemical, Taito-ku, Japan) or in a water bath. This was followed by an enzyme inactivation step at 80 °C for 5 min. Following the successful LAMP reaction, the use of a self-assembled LAMP reaction buffer was investigated.

EXPAR templates, DNA targets and DNA probe functionalized on AuNPs were purchased from Integrated DNA Technologies. The sequences of DNA used are shown in Table 2. The Vent (exo-) polymerase, Nt.BtsNBI nicking enzyme, the ThermoPol buffer, the NEBuffer 3.1, BSA, SSB proteins, dNTPs and the Streptavidin magnetic beads, were purchased from New England BioLabs. The Biotin-11-dUTP was obtained from Biotium. Ethylene glycol, propylene glycol, betaine, DMSO, trehalose, TMAC, and HAuCl₄, sodium citrate dehydrate, 4-mercaptobenzoic acid (MBA) were purchased from Sigma-Aldrich.

gDNA was extracted from single A. meraukensis mosquito bodies by two methods, the Invitrogen PureLink gDNA kit (used in accordance with the manufacturer’s protocol) and a previously published method (52 (link)). Single A. farauti mosquitoes were similarly processed as controls. The gDNA extracts were used in PCRs with the ITS2 primers as quality control and subsequently tested for integrated virus sequence with a range of primers based on KRBV NS4 and NS5 sequences. Six primer sets were used in 12 combinations to prevent false-negative results due to potential introns in the primer binding regions (Table 5). PCRs were performed with Taq DNA polymerase and Thermopol buffer (New England Biolabs) in accordance with the manufacturer’s protocol.

Total RNA was extracted from cells using Qiazol (Qiagen) and following the manufacturer’s protocol. Reverse transcription was carried out using 1 μg of RNA and 4 μl of iScript Reverse Transcription Supermix (BIO-RAD) in a final volume of 20 μl using the following PCR program: 5 min at 25 °C; 20 min at 46 °C; 1 min at 95 °C. AS specific primer (IDT) were designed to amplify only one ASE and were resuspended together at 1 μM. Primers used and predicted amplicon are available in Additional file 1: Table S2. Each reaction was composed of ThermoPol buffer (NEB), dNTPs, primers, cDNA and TAQ (NEB) and were incubated using the following program: 2 min at 94 °C; 34 cycles: 30 s at 94 °C; 30 s at 55 °C; 1 min at 72 °C; 2 min at 72 °C (final elongation). PCR products were resolved using a Caliper LC-90 capillary electrophoresis (Caliper LifeSciences).