The LAMP reaction system contained 1 × Isothermal Amplification Buffer (New England Biolabs, Shanghai, China), 1.2 mM dNTP (Sangon Biotech, Guangzhou, China), 6 mM MgSO4 (New England Biolabs, Shanghai, China), 320 U mL−1 Bst 2.0 DNA polymerase (New England Biolabs, Shanghai, China), 160 U mL−1 Bst 3.0 (New England Biolabs, Shanghai, China). Primers used for the LAMP are shown in Table S2 . A primer mix consisting of 1.6 μM of FIP and BIP, 0.2 μM of F3 and B3, and 0.4 μM of LF and LB was added to the reaction. RT-LAMP reactions were carried out in 0.2 mL PCR tubes. After adding the 1 μL sample, the final volume of the RT-LAMP reaction was 20 μL. In addition to the above reagents, the real-time LAMP reaction mixture contains 1 × LAMP Fluorescent Dye (New England Biolabs, Shanghai, China). The LAMP mixture was incubated at 65 °C for 50 min in a LongGene PCR System and the fluorescent signal was recorded every 1 min. The reaction mixture used in the system contains hydroxy naphthol blue (Aladdin, Shanghai, China).
Lamp fluorescent dye
Manufactured by New England Biolabs
Sourced in China, United States
The LAMP Fluorescent Dye is a reagent used in loop-mediated isothermal amplification (LAMP) assays. It is designed to emit fluorescence upon binding to double-stranded DNA generated during the LAMP reaction, allowing for real-time detection and monitoring of the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Bst 2.0 dna polymerase, by New England Biolabs (2 mentions)
Mgso4, by New England Biolabs (2 mentions)
Superase in rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Warmstart lamp master mix, by New England Biolabs (2 mentions)
Tween 20, by Merck Group (2 mentions)
Isothermal amplification buffer, by New England Biolabs (1 mentions)
Bst 3, by New England Biolabs (1 mentions)
Prepman ultra reagent, by Thermo Fisher Scientific (1 mentions)
Bst 2.0 warmstart dna polymerase, by New England Biolabs (1 mentions)
Takara ex taq kit, by Takara Bio (1 mentions)
Warmstart colorimetric lamp 2x master mix, by New England Biolabs (1 mentions)
Cfx96 touch thermal cycler, by Bio-Rad (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Perfection v700, by Epson (1 mentions)
Cfx connect, by Bio-Rad (1 mentions)
Polydimethylsiloxane pdms, by Dow (1 mentions)
Warmstart colorimetric lamp 2 master mix, by New England Biolabs (1 mentions)
Az 1512, by MicroChem (1 mentions)
Su 8 2075, by MicroChem (1 mentions)
Thermopol reaction buffer, by New England Biolabs (1 mentions)
7 protocols using lamp fluorescent dye
1
Rapid and Sensitive LAMP Detection
2
LAMP Assay for Rapid Detection
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All the primers were synthesized from Sangon Biotech (Shanghai, China). LAMP fluorescent dye and Bst 2.0 WarmStart® DNA Polymerase were purchased from New England BioLabs (Beijing, China). The TaKaRa Ex Taq kit was purchased from Takara Bio, Inc. (Beijing, China). The V8 juice was obtained from Campbell Soup Co. (Camden, NJ, USA). The PrepMan Ultra Reagent was obtained from Applied Biosystems (Foster City, CA, USA). All other compounds were obtained from Sangon Biotech (Shanghai, China).
3
Isothermal Detection of Filariasis Parasites
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LAMP reactions contained 1.6 μM each of primers FIP and BIP, 0.2 μM each of F3 and B3, 0.4 μM each of LF and LB, 12.5 μl of WarmStart Colorimetric LAMP 2X Master Mix (New England Biolabs Inc., USA) with 2 ul of template DNA, or H2O for non-template controls (NTCs) in a total volume of 25 μl. Reactions were incubated at the optimal temperature of 63 °C for 20–30 min (M. ozzardi) or 60 minutes (M. perstans) in a T100 Thermal Cycler (Bio-Rad Laboratories, USA). When a qPCR machine (CFX-96 Touch Thermal Cycler, Bio-Rad Laboratories, USA) was used to enable reaction dynamics to be monitored in real time, colorimetric reactions also contained 1X LAMP Fluorescent Dye (New England Biolabs Inc., USA) and were incubated at 63 °C for ~30 min (M. ozzardi: 36 cycles) or ~1 hr (M. perstans: 72 cycles) with a plate read step every 42 seconds. To record color changes, samples were scanned using an Epson Perfection v700 photo flatbed scanner (Epson America, Inc., USA).
4
Quantitative Cell-based LAMP Assays
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We performed bulk RT-LAMP and DNA logic assays on purified RNAs, cells and mixtures of cells in triplicate using a Bio Rad CFX Connect quantitative PCR (qPCR) machine. We incubated the reactions at 65°C and monitored FAM, HEX and/or SYBR fluorescence channels. Each reaction comprised a total volume of 10 μl, consisting of 1.6 μM each FIP/BIP primer, 0.2 μM each F3/B3 Primer, 0.4 μM each LoopF/B Primer, 1× WarmStart LAMP Master Mix (New England Biolabs) and 0.5 U/μl SUPERase•In™ RNase Inhibitor (Invitrogen). We added DNA complexes at varying concentrations given in Supplementary Table S2 . LAMP primer and logic gate sequences are shown in Supplementary Table S1 . In reactions without any DNA complexes, we included LAMP Fluorescent Dye (New England Biolabs) as a general LAMP indicator. For experiments performed on purified RNAs, we added in vitro transcribed KRT19, VIM and/or PTPRC RNAs at the concentrations shown in Supplementary Table S2 . For experiments performed on cells, we also included 2.5% Tween-20 (Sigma Aldrich) in the reaction buffer to act as a lysis reagent. We then added intact, unstained cells immediately before starting an experiment at a final concentration of 50 cells/μl per cell type.
5
Graphene-based LAMP Assay for SARS-CoV-2
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Monolayer graphene obtained through chemical vapor deposition (CVD) was sourced from Graphenea (San Sebastian, Spain). Polydimethylsiloxane (PDMS) was purchased from Dow Corning (Midland, MI, USA). Several other chemicals, including AZ-1512, SU-8 2075, diethyl pyrocarbonate (DEPC), hexamethyldisilazane, and (1-methoxy-2-propyl) acetate (SU-8 developer), were acquired from MicroChem (Westborough, MA, USA). Additionally, anhydrous iron (III) chloride (FeCl3) powder and phosphate-buffered saline (PBS; pH 7.4) were obtained from Sigma-Aldrich (St. Louis, MO, USA). WarmStart Colorimetric LAMP 2× Master Mix and LAMP Fluorescent Dye were purchased from New England Biolabs Inc. (Ipswich, MA, USA), and the primers synthesized and purified by Bionics Inc. (Seoul, South Korea). The template for the LAMP assay, consisting of a plasmid containing sequences of the SARS-CoV-2 envelope (E) and RNA-dependent RNA polymerase (RdRP) genes, was sourced from GenScript (Piscataway, NJ, USA).
6
Optimizing LAMP Reaction Conditions
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LAMP reaction was performed according to the method developed by Notomi et al. (2000 (link)). The reaction mixture had a final volume of 25 μL, comprising ThermoPol Reaction Buffer (NEB, #B9004S), MgSO4 (NEB, #B1003S), dNTPs (Sangon Biotech, #B500055‐0500), external primers (F3 and B3), internal primers (biotin‐FIP and BIP), Bst 2.0 DNA polymerase (NEB, #M0537S), LAMP fluorescent dye (NEB, #B1700S), LF, template and diethyl pyrocarbonate (DEPC)‐treated water (Sangon Biotech, #B501005‐0500). To prepare the optimal reaction system for LAMP, the final concentration of dNTPs was set to 1.0, 1.2, 1.4, 1.6, 1.8 and 2.0 mM; the final concentration of Mg2+ was set to 0–6 mM; and the internal‐to‐external primer ratios were set to 2:1, 4:1, 6:1, 8:1, 10:1, 12:1, 14:1 and 16:1. The system was optimized at 58, 60, 62, 64 and 66°C, and loop primers were added to accelerate the reaction. The reaction mixture was amplified on the CFX96 Touch Real‐Time PCR Detection System (Bio‐Rad) at 58–66°C for 1 h and terminated at 85°C for 10 min. The amplified product was analysed via fluorescence signal acquisition.
7
Multiplexed RT-LAMP and DNA Logic Assays
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We performed bulk RT-LAMP and DNA logic assays on purified RNAs, cells, and mixtures of cells in triplicate using a Bio Rad CFX Connect qPCR machine. We incubated the reactions at 65 °C and monitored FAM, HEX, and/or SYBR fluorescence channels. Each reaction comprised a total volume of 10 µL, consisting of 1.6 µM each FIP/BIP primer, 0.2 µM each F3/B3 Primer, 0.4 µM each LoopF/B Primer, 1X WarmStart LAMP Master Mix (New England Biolabs), and 0.5 U/µL SUPERase•In™ RNase Inhibitor (Invitrogen). We added DNA complexes at varying concentrations given in Table S2. LAMP primer and logic gate sequences are shown in Table S1. In reactions without any DNA complexes, we included LAMP Fluorescent Dye (New England Biolabs) as a general LAMP indicator. For experiments performed on purified RNAs, we added in vitro transcribed KRT19, VIM, and/or PTPRC RNAs at the concentrations shown in table S2. For experiments performed on cells, we also included 2.5% Tween-20 (Sigma Aldrich) in the reaction buffer to act as a lysis reagent. We then added intact, unstained cells immediately before starting an experiment at a final concentration of 50 cells/µL per cell type.
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