protocol with commercial reagents

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RT-LAMP with commercially bought reagents is the easiest and fastest way to get started with SARS-CoV-2 detection with reverse transcription loop mediated isothermal amplification. This page includes the protocol, material list and a benchmarking assay that you can use to confirm your LAMP reactions are effective. See the limit of detection assay for our data on the sensitivity and specificity of this method. 


Some things to keep in mind before getting started:


LAMP primer mix 10X

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. 







  1. Prepare your ordered oligonucleotides. If they are not resuspended, resuspend them in nuclease-free water for 100 μM stock concentration.
  2. In a clean and template-free workspace, such as in a PCR hood, mix the oligonucleotides in the order outlined by the table below. Make sure to add the components from largest volume to the smallest volume.
    Oligo nameAdd for 100 μl of mix
    F32 μl
    B32 μl
    LF4 μl
    LB4 μl
    FIP16 μl
    BIP16 μl
    nuclease-free water56 μl
  3. Mix the finished primer mix by thoroughly pipetting up and down or by vortexing and briefly spinning down in a microfuge.
  4. Distribute the finished primer mix into Eppendorf tubes into conveniently sized aliquots, label the tubes and store at -20 ºC.

HNB solution preparation

In RT-LAMP assays, we use hydroxynaphtol blue as the dye that lends the reaction its easy and reliable colour readout. HNB is a dye commonly used in metallometric titrations for water purity assessment, as it changes colour from blue to pink in the presence of the magnesium cations. It is critical to use the trisodium salt of hydroxynaphtol blue. Using 3 mM stock solution of HNB is recommended for its stability and ease of pipetting even for a small number of reactions. 


  1. Weigh out 0.186 g of hydroxynaphtol blue trisodium salt powder on an analytical scale.
  2. Dissolve the powder in 100 millilitres of nuclease free water. Make sure that every bit of the powder is dissolved in the solution to get a precise 3 mM concentration of the dye.
  3. Filter the solution through a 0.22 µm sterile filter to make a sterile solution.
  4. Store the 100 ml of 3 mM solution in the fridge at 4 ºC.

LAMP reagents

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.


  • QuickExtract DNA solution 50ml (Lucigen QE09050), store at -20°C
  • WarmStart LAMP Kit (DNA & RNA) (NEB E1700L), store at -20°C
  • Antarctic Thermolabile UDG (NEB M0372L), store at -20°C
  • dUTP solution (NEB N0459S), store at -20°C
  • Magnesium sulfate (MgSO4) solution (NEB B1003S), store at -20°C
  • Nuclease free water (not DEPC-treated) 100ml (Thermofisher AM9938)



  1. Thaw an aliquot of QuickExtract DNA solution on ice and vortex before use. (50µl per sample).
  2. On ice, mix 50µl of original sample material (nasopharyngeal swab, gargle or saliva) with 50 µl of QuickExtract solution in PCR strips or in a 96-well plate; seal the plate (or close the PCR strips with the lids).
  3. Heat-inactivate QuickExtract:Sample mix at 95°C for 5 minutes, then cool on ice or store at -20°C
    [PAUSE] Sample can be stored at -20°C at this point for up to four weeks, potentially longer. Inactivated samples can now be handled in BSL-1 settings.


rt-lamp reaction

Calculate your reagent consumption according to this table

Reagent Per 10 ul reaction (μl) Final concentration
WarmStart LAMP Kit (2x) 5 1X
Primer mix (10x) 1 1X
HNB Dye solution (3 mM) 0.4 120 μM
dUTP (100 mM) 0.07 0.7 mM
Magnesium sulfate (100 mM) 0.07 0.7 mM
50x NEB LAMP Dye (optional) 0.2 1X
Thermolabile UDG (optional) 0.2 0.02 U/μl
WarmStart Bst2.0 DNA polymerase (8000 U/ml) 0.4 0.32 U/μl
Nuclease free water to a total of 8  
  1. Prepare the RT-LAMP reaction mix by mixing the following reagents in the given order (top to bottom) and quickly vortex and spin down in a microfuge.
  2. Dispense the reaction mix into PCR strips or 96-well plate on ice, 8 µl per well (sample).
  3. On ice, Add 2 µl of heat-inactivated QuickExtract lysate to RT-LAMP reaction and mix carefully by pipetting the reaction up and down. Seal PCR strips by capping them, and seal 96-well plates by applying a plastic transparent plate seal.
    [CRITICAL] This step should be done in a separate work area where no reagents are handled.
    [CRITICAL] If performing colorimetric detection, take an image of the PCR strips or 96-well plate with the mastermix and samples.
  4. Transfer the reaction to a suitable stable heat-source such as a heat block or a thermocycler and run reactions at 63°C for 35 minutes.
    [OPTIONAL] For real-time fluorescence data acquisition in qPCR thermocycler, we typically perform 35 cycles at 63°C with 1-minute cycle length and reading at the end of each cycle. NEB fluorescent dyes require data acquisition using a standard FAM or SYBR filter (494nm/518nm absorption/emission).
  5. After 35 minutes run-time, remove reactions and allow to cool briefly at room temperature. Then proceed to inspect reactions visually. Negative reactions are purple while positive reactions are sky-blue.
    If performing colorimetric detection, take a second image of the PCR strips or 96-well plate under the same conditions as you took the first one after the reaction.
  6. Discard the reactions immediately after noting down the results.

Readout methods

RT-LAMP color readout

Visual colorimetric

Hydroxynaphtol blue (HNB) is used in RT-LAMP reactions that do not include any purification step. The dye changes colour from purple to sky blue upon presence of target sequence and amplification. This can be visualised with the naked eye, and reactions can be scored directly after taking them out of the heating device.

RT-LAMP app enhanced color readout

App-assisted colorimetric

For people who find the colour change of HNB difficult to see with the naked eye, we recommend the use of a simple web app. Developed by Andrew Straw to increase the colour difference between positives and negatives and make the readout much simpler. Try it out on colorimetry.net

RT-LAMP fluorimetric detection


Amplification of DNA in RT-LAMP reactions can also be read out through the use of fluorescent intercalating dyes. These dyes, such as Syto9 or the NEB LAMP dye, enable the user to either monitor the progress of the reaction in real time through the use of a qPCR machine, or end-point detection with a plate reader or a similar fluorescence detection device.

Premixing information

Assembling each master mix for every testing session from scratch is not necessary, however freezing prepared master mixes increases the risk of running into sensitivity or specificity issues. We’ve tested the possible combinations and now we can tell you what you can and can’t premix for easier reaction assembly without compromising on the quality of the tests. 

LAMP reaction premix

ReagentPer 100 reactions (10 μl size)
WarmStart LAMP Kit (2x)500
dUTP (100 mM)7
Magnesium sulfate (100 mM)7
Thermolabile UDG (optional)20
WarmStart Bst2.0 DNA polymerase (120 000 U/ml)2.6

Add 5.35 µl of this mix per 1 reaction.

Note: A concentrated version of WarmStart Bst2.0 polymerase (NEB cat. nr. M0538M) is used to minimise the volume of the premix. 

Store at -20 °C.

LAMP primer premix

Reagent Per 100 reactions (10 μl size)
10x LAMP primer mix 100
nuclease-free water  100
 total  200

Add 2 µl of this mix per 1 reaction.

Note: You may use the As1, E1 or ACTB primers to make a LAMP primer premix. Make sure to label the mix adequately.

Store at -20 °C.

LAMP HNB premix

Reagent Per 100 reactions (10 μl size)
HNB Dye solution (3 mM) 40
nuclease-free water  25
 total  65

Add 0.65 µl of this mix per 1 reaction.

Store at 4 °C.

The premixes for commercial RT-LAMP enable you to have three tubes in your freezer and fridge and to assemble reactions easily using 5.35 µl of LAMP reaction buffer premix, 2 µl of LAMP primer premix and 0.65 µl of LAMP HNB premix per 10 µl reaction. We have observed these premixes to be stable for a month at the specified storage conditions, but they are most likely good even for longer.

See our other protocols