The RPA assay was carried out with template DNA extracted from blood and DBS using a protocol described elsewhere (S2 Table ) [18 (link)]. In summary, a Twistamp exo kit (Product code#TAEXO02KIT, Twistamp exo kits, TwistDx, Cambridge, UK) was used to perform the assay. In a tube 420 nM of RPA primer, 120 nM of RPA Probe, and 1× rehydration buffer were mixed to prepare a 50 μL reaction volume which was added to the RPA lyophilized pellet. 14 mM of Mg acetate were added to the tube lids. Template DNA was added to the tubes, which were then closed and mixed thoroughly. The tubes were immediately placed into the tube scanner (Twista, TwistDx, Cambridge, UK) and incubated for 15 minutes at 42°C. At 20 s intervals, the emitted fluorescence signals were measured. Signal was interpreted utilizing a combined threshold and first derivative analysis. It took approximately 20 minutes to complete the RPA assay.
Twistamp exo kit
Manufactured by Twist Bioscience
Sourced in United Kingdom
The TwistAmp exo kit is a laboratory equipment designed for isothermal DNA amplification. It provides a simple and efficient method for generating large quantities of specific DNA sequences without the need for thermal cycling.
Automatically generated - may contain errors
Lab products found in correlation
Twista, by Twist Bioscience (4 mentions)
Twistamp basic kit, by Twist Bioscience (2 mentions)
Twistamp nfo kit, by Twist Bioscience (2 mentions)
Revertaid reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Lightcycler 480 instrument 2, by Roche (1 mentions)
Protoscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rpa exo probe, by Sangon (1 mentions)
Stratagene mx3005p thermocycler, by Thermo Fisher Scientific (1 mentions)
Nebuffer 3, by New England Biolabs (1 mentions)
Crrna, by Sangon (1 mentions)
Lbcas12a protein, by New England Biolabs (1 mentions)
Rna inhibitor, by New England Biolabs (1 mentions)
Rpa primers, by Sangon (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Esequant, by Qiagen (1 mentions)
Superscript 2 reverse transcriptase enzyme, by Thermo Fisher Scientific (1 mentions)
Endonuclease 4, by New England Biolabs (1 mentions)
C1000 touch thermal cycler, by Bio-Rad (1 mentions)
Cfx96 touch real time pcr system, by Bio-Rad (1 mentions)
Transcriptor rt, by Roche (1 mentions)
36 protocols using twistamp exo kit
1
RPA Assay for DNA Extraction
2
Real-time RPA for Staphylococcus aureus
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Real-time RPA was accomplished in the tube with 50 μl reaction volume, including 40.9 μl of Buffer A (rehydration buffer), 2.0 μl of each RPA primers (Sa -exo-F and Sa -exo-R, 10 μmol/L), 0.6 μl of exo probe (Sa -exo-P,10 μmol/L), and 2.5 μl of Buffer B (magnesium acetate, 280 mmol/L). Furthermore, 1 μl of genomic DNA was used for the specificity and sensitivity analysis, or 2 μl of sample DNA was used for the clinical sample diagnosis. In the process, the Genie III scanner device (OptiGene Limited, West Sussex, UK) and TwistAmpTM exo kit (TwistDX, Cambridge, UK) were applied.
3
Rapid and Sensitive Ebola Virus Detection
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A pair of primers and 1 probe were designed to specifically detect Ebola Zaire viruses including circulating strains from the 2014 EBOV outbreak (Table S1 ). The sequence spanning the amplification region was synthesized and cloned into the pSQ380-MS2 plasmid to generate pSQMS2-NP. Armored RNA was prepared from pSQMS2-NP. The RT-RPA assay was performed in a 50-μL volume using the TwistAmpTM Exo Kit (TwistDx, Cambridge, UK), 420 nM RPA primer F and R, 120 nM exo-probe, and 14 mM magnesium acetate. All reagents except for the template or sample RNA and magnesium acetate were prepared in a master mix, which was aliquoted into each tube of a 0.2-mL 8-tube strip containing a dried enzyme pellet13 (link). Magnesium acetate was pipetted into the tube lids. Subsequently, 2 μL sample was added to the tubes. The lids were closed, and the magnesium acetate was centrifuged into the tubes using a mini-spin centrifuge. The tubes were immediately used for amplification and detection. Ten-fold serial dilutions of armored RNA standard ranging from 108 to 101 molecules/μL were tested by the EBOV-RPA assay in 8 replicates. The Ct number was plotted against the number of molecules in each tube comprising the standard curve. The lowest concentration of the dilution that could be detected was determined as the detection limit.
4
Real-time Recombinase Polymerase Amplification
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Real-time RPA was carried out as describled previously [26 (link), 27 (link)]. The Genie III scanner device (OptiGene Limited, West Sussex, UK) and TwistAmpTM exo kit (TwistDX, Cambridge, UK) was applied in the real-time RPA.
5
Rapid Isothermal DNA Amplification
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RPA was performed in a total volume of 50 μL, using a TwistAmp Exo kit (TwistDX Ltd., Cambridge, UK). Each reaction contained 29.5 μl TwistAmp rehydration buffer, 2.1 μl each RPA primer (210nM), 0.6 μl RPA probe (120nM), 12.2 μl nuclease-free water, 1 μl template and 2.5 μl magnesium acetate. All reagents except for the magnesium acetate and template DNA were pipette into a 0.2 ml reaction tube which contains a dried enzyme pellet. To start the reaction magnesium acetate and template DNA were added. Then the tube was placed in the TwistaTM incubator block (39 °C) and the fluorescence measurement was initiated to minitor the progression of RPA reactions. The preincubation was performed for 1 min and followed by incubation for 20 min, with brief mixing of the mixtures after a 4 min incubation step. In combination with the nuclease sensitive fluorophore/ quencher probes, a real-time DNA detection system was constituted. The labeled amplicon that generated in the reaction can be measured via 6-carboxyfluorescein (FAM) fluorescence using the Twista™ reader every 20 s. The RPA fluorescence data were assessed by taking a baseline relative fluorescence unit measurement.
6
Exo-RPA Reaction for Nucleic Acid Detection
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The exo-RPA reaction was performed in 50 μl using a TwistAmp™ exo kit (TwistDX, UK). A master mixture was prepared according to the manufacturer's instructions. The mixture contained 25 μl 2 × reaction buffer, 8.2 μl dNTPs (1.8 μM), 5 μl 10 × Probe E-mix, 2.1 μl forward Primer (10 μM), 2.1 μl reverse primer (10 μM), 0.6 μl TwistAmp™ exo probe (10 μM), 2.5 μl 20 × Core Reaction Mix, 1 μl 50 × EXO. Subsequently, 2.5 μl of 280 mM magnesium acetate and 1 μl nucleic acid were added to the mixture to initiate the amplification reaction on the LightCycler 480 Instrument II (Roche Diagnostics Corporation, IN, USA) for 40 cycles at 38°C for 20 min (30 s/cycle).
7
Real-time RPA for DNA Detection
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Real-time RPA was carried out with the TwistAmp Exo Kit (TwistDx, Cambridge, United Kingdom). The stock reaction mix was prepared as follows (working concentrations in parentheses): 29.5 μL rehydration buffer, 1 μL of extracted DNA template, 2.1 μL forward primer (10 μM), 2.1 μL reverse primer (10 μM), 0.6 μL probe (10 μM), 12.2 μL dH2O, and 2.5 μL magnesium acetate (280 mM). All reagents, except for the template or sample DNA and magnesium acetate, were prepared as a master mix, and added into a 0.2 mL tube containing a dried enzyme pellet. After adding 1 μL of plasmid template or sample DNA to the master mix tube, magnesium acetate was then pipetted into the tube lids, spun down briefly, and mixed well to start the reaction. The reaction mixture was first treated in a MiniAmp thermocycler at 37 °C for 4 min (brief mix, centrifugation, and vortex) and then incubated for 20 min at 37 °C, during which, the fluorescence (FAM) signal was collected every 30 s with QuantStudio1 (ABI, United States).
8
Real-time RPA Assay for Virus Detection
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A real-time RPA assay was performed in a 50 µL volume using the TwistAmp Exo kit (TwistDx, Cambridge, UK). The reaction mix comprised 29.5 µL rehydration Buffer, 8.2 µL nuclease-free water, 2.5 µL magnesium acetate (280 mM), 2.1 µL of each primer (10 µM), 0.6 µL probe (10 µM), and 5 µL template (or 1 µL for samples treated with the rapid extraction protocol), which was added into the lid of the reaction tube containing the freeze-dried pellet. Water was used instead of the DNA template for the negative control. For RNA viruses tested for cross reactivity, 8.2 µL (500 U) of RevertAid reverse transcriptase (Thermo Scientific, Regensburg, Germany) was used instead of nuclease-free water. The tube was closed, centrifuged, mixed, centrifuged, and placed immediately into the T8-ISO Instrument (Axxin, Fairfield, Australia). The incubation temperature was 42 °C for 15 min. A mixing and centrifuging step was conducted at 320 s after the test start. The FAM fluorescence signal was recorded in real time. The TT was determined using the T8 Desktop Application (version 2.8.0.0, Axxin) based on the first derivative values.
9
SARS-CoV-2 RT-RPA Assay Protocol
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A lyophilized pellet containing RPA reagents from the TwistAmp© Exo Kit (TwistDx™ Ltd. San Diego, CA, USA) was rehydrated in a total volume of 42.5 μL comprised of 29.5 μL TwistAmp Primer Free Rehydration buffer, 0.13 μM Exo-IQ probe (Eurofins Genomics LLC, Louisville, KY, USA), 10 U/μL ProtoScript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA), 0.25 U/μL thermostable RNase H (New England Biolabs), 0.44 μM each SARS_RPA_F1 and SARS_RPA_R1 primers [38 (link)] (Eurofins Genomics LLC, Louisville, KY, USA), and 6.2 μL PCR-grade water. Each 25 μL reaction was comprised of 21.25 μL of the above mixture, 2.5 μL extracted sample and 14 nM magnesium acetate (MgOAc) (TwistDx™). The RNA extract and MqOAc were added, spatially separated, to the tube lid. Following brief centrifugation to start the RT-RPA reaction, the tubes were placed in a TS16-ISO instrument (Axxin, Fairfield, Victoria, Australia), preheated to 42 °C. Fluorescence was monitored within the FAM channel at 20% LED level. After 300 s, the samples were removed from the instrument, briefly vortexed and centrifuged, then returned for further incubation (1620 s). A threshold for amplification (618.619 RFU) was established three standard deviations above the mean fluorescence output of the NTCs throughout the assay duration.
10
Exo-RPA Assay for Rapid DNA Amplification
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Exo-RPA trials were conducted by a TwistAmp Exo kit (TwistDx, United Kingdom). The reactions were conducted in 50 μl using the RPA lyophilized pellet, which offered all components required for DNA amplification. For the Exo-RPA assay, 29.5 μl of rehydration buffer was blended with 10.8 μl of ddH2O, 2.1 μl of forward/reverse primer (10 μM), 1 μl of exo probe (10 μM), and 2 μl of templates as the master mix. The total volume of 47.5 μl of the reaction solution was distributed into the tube of freeze-dried pellet. At the start of amplification, the catalytic agent was infused into the tube lids, and the tubes were closed carefully. Then, the isolated tandem reagent tubes were mixed to homogeneity, centrifuged, and placed in a tube scanner to ensure that the amplification reaction began simultaneously. All tandem tubes were kept at 39°C for 20 min, followed by fluorescence recording (Ex: 492 nm/Em: 518 nm) every 20 sec (ABI QuantStudio1, USA).
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