© 2020 All rights reserved
Bead-LAMP with commercial reagents is the most sensitive way to detect SARS-CoV-2 with reverse transcription loop mediated isothermal amplification. With fast sample inactivation and a simple bead enrichment step this method gives results in sensitivity compared to those achieved by gold standard qPCR. This page has the protocol, material list and a benchmarking assay you can use to confirm that your LAMP reactions are effective. See the limit of detection assay for our data on the sensitivity and specificity of this method.
LAMP requires six primers for optimal functioning. That is six short DNA oligonucleotides to synthesise or order, the names are F3, B3, LF, LB, FIP and BIP. For the ease of use, one can mix those in a pre-determined ratio to make a 10X primer mix to use with LAMP. You can make this mix with As1 primers against SARS-CoV-2 genome or against human ACTB transcript. Aliquot and label the finished primer mix with the name, date and name of the person who prepared it. We encourage to test every primer mix you receive for activity against the target template, especially when ordering from vendors you don’t have prior experience with.
|Oligo name||Add for 100 μl of mix|
|nuclease-free water||56 μl|
This protocol explains in detail how to make a diluted Ampure bead solution from commercial RNA magnetic beads and pre-defined bead dilution buffer. The bead dilution is used for bead-LAMP.
|Reagent||Amount to add (ml)||Final concentration|
|40% PEG||20||20 %|
|5M NaCl||19.4||2.5 M|
|1M Tris-HCl pH 8||0.4||10 mM|
|Tween™ 20 Surfact-Amps Detergent Solution||0.2||0.05|
|Ampure Dilution Buffer (see above)||4|
|Ampure RNAClean XP beads||1|
Commercial RT-LAMP assays are powered by enzymes that are produced by specialised companies. We found the LAMP kits from NEB to be very powerful in terms of performance and sensitivity, when used with our protocol. Therefore, we recommend purchasing the following reagents to anyone trying to get LAMP working in their settings as soon as possible.
Reagent Per 20 ul reaction (μl) Final concentration Colorimetric LAMP Kit with UTP/UDG (2x) 10 1X As1 Primer mix (10X) 2 1X E1 Primer mix (10X) 2 1X GuHCL (8M) 0.1 40 mM NEB LAMP dye 50X (optional) 0.4 1X Nuclease free water to a total of 20