FAQ

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Do you feel like you need more information on RT-LAMP before setting it into practice? You’re at the right place. We’ve compiled the questions we’re asked on a daily basis here on a concise page. If you’re not sure about something regarding LAMP, check this list first. If you still don’t have all the info you need, consider getting in touch with us on info@rtlamp.org or by filling out the form below. 

General questions

  • Why do you use DNA QuickExtract solution in the sample inactivation? Doesn't it only extract DNA?
  • We found that DNA QuickExtract is excellent at extracting viral RNA from virions and infected cells, and also serves well to maintain their integrity by inactivating native RNases. So yes, the DNA QuickExtract is a suitable and accessible reagent for this purpose.
  • Do I have to prepare my reactions on ice?
  • Assembling reactions on ice is necessary when using non-warmstart enzymes produced with the open-access protocols. For NEB WarmStart LAMP master mixes, assembling reactions on ice is not required but encouraged, since it helps to prevent issues such as non-specific amplification and protects the sample integrity.
  • I don't want to go through the hassle of buying everything on my own now, can I just buy all I need for these LAMP tests from somewhere?
  • We're pleased to offer now, in cooperation with the Vienna BioCenter Core Facilities, all-in-one reagent kits for RT-LAMP. They are available for research use and you can check them out at rtlamp.org/kits.
  • Do I have to pay you a licensing fee for using these protocols?
  • No, of course not. We want everyone in the world to have access to fast, sensitive, cheap, and frequent testing. For this, you can freely use our protocols. We do not give any guarantees however and you are encouraged to benchmark the methods in your settings to validate their effectiveness.
  • If I incubate the RT-LAMP reaction for longer than recommended, do I gain sensitivity?
  • You should in no case incubate your reactions for longer than 35 minutes. We have optimised our reaction for these conditions, and incubation times that are longer than the recommended amount are prone to generating false positives.
  • Why should I never open the LAMP reactions?
  • LAMP works much like PCR in that it multiplies DNA during the course of the reaction. Whereas PCR doubles the input DNA in each cycle, the amplification of LAMP is much more explosive. Thus, LAMP reactions generate much more DNA than PCR and opening the reaction tubes could cause droplets or aerosol of non-infectious but template-DNA containing solution to contaminate the workspace. In such a contaminated workspace, false positive results can start appearing. Our protocol minimises this risk thanks to using the dUTP/UDG contamination prevention system, but it's crucial not to open the reactions to prevent a possible issue. Contaminated items and rooms must be treated with 5 - 10 % bleach solution.
  • Why have you developed your protocols with specific commercial products, are you advertising them or do you have a deal with these companies?
  • The products used in our commercial protocols have been found to perform exceptionally well for their use in RT-LAMP assays. For an easy set-up of the reactions, and to guarantee quality-controlled reagents to people wanting to perform LAMP tests themselves we recommend the use of these reagents. We do not have any association with these companies and have no financial gain from using or writing about their products.
  • Is the RT-LAMP test a clinically accepted diagnostic method?
  • That depends on your local jurisdiction. LAMP is not a new method but is generally not as widely accepted as PCR when it comes to diagnostics. Check with your local health agency for the legal status. If you are running a diagnostics laboratory, you can start with self-validation of our RT-LAMP protocols to use in diagnostics. You can also write to your lawmakers and health officials to push for the official acceptance of RT-LAMP as a tool to aid in the management of the pandemic.

RT-LAMP

  • Which sample types is your RT-LAMP protocol compatible with?
  • Direct input RT-LAMP is compatible with a variety of popular sample media, for gargle and swabs. We have robustly tested HBSS and saline (0.9 % NaCl) and VTM as input media. Most other RT-LAMP protocols are compatible either only with one direct sample input, or just with extracted RNA in nuclease free water. For more information on sample compatibility, see our preprint Kellner et al., 2020 on bioRxiv.
  • The colour change is really hard for me to see in RT-LAMP. Can't I just use the colorimetric kit from bead-LAMP for direct sample input?
  • The problem with the colorimetric kit is in the colour change. The colour change, and thus the whole readout, is based on a change in pH that is caused during DNA amplification. But since various sample inputs have different starting pH, adding them to the colorimetric LAMP kit's mastermix might result in false positives or false negatives. Our direct input RT-LAMP method circumvents this by using a drop in free magnesium concentration in the sample as the thing that triggers colour change. If you have troubles seeing the colour change and suffer from some version of colour-blindness, you can use the web app colorimetry.net to better visualise the colour difference.
  • Do I need to use filter tips or can I use normal pipette tips without a filter?
  • We are aware of possible shortages in supply of filter tips. They are usually used for routine covid-19 testing at the moment for both manual and robotic approaches. Using filter tips is a contamination prevention precaution. Strictly adhering to all other contamination prevention rules and guidelines should enable one to use regular tips. Ultimately, the decision is yours.
  • Can I use more sample input in the RT-LAMP reaction?
  • Our reactions have been optimised for one fifth of reaction volume as an input. You can scale up the reaction volume itself to 20, 30 or even 50 microlitres, but the ratio of sample to reaction volume must stay the same to ensure an accurate result.
  • The colour of my RT-LAMP reaction changed upon the addition of the sample even before incubating it! What do I do now?
  • The sample collection medium likely contained some chemical ingredient that inhibited the colour readout with HNB. This is most often either a chaotropic agent such as guanidium or a metal chelator such as EDTA. This sample is then incompatible with RT-LAMP, and needs to be measured with bead-LAMP or use a different sample collection medium, such as saline (0.9 % NaCl).

bead-LAMP

  • I want to use bead-LAMP to pool my samples and cut down on the number of reactions. Can I pool these samples before inactivation to save on the amount of inactivation solution I use?
  • Yes, pooling samples before inactivation is just as valid as pooling them after inactivation. When pooling before inactivation, one has to adhere to proper protective precautions as with infectious sample handling. This approach helps you save money on the inactivation reagent used, but in case of a positive pool you will have to go back and inactivate the individual samples and re-test them, to find the positive sample from the pool.
  • How many samples can I pool at once?
  • That depends on many factors, such as the sensitivity you are aiming for and the percentage of positives that's in your sample sets. We recommend reading up on pooling strategies for SARS-CoV-2 testing. Pooling strategies for PCR are applicable for bead-LAMP. In general, we recommend pooling 10 samples.
  • My sample turned yellow before I even put it in the incubator / thermocycler / water bath, what is happening? And can I continue?
  • A positive result can never be produced before incubating the sample at 63°C, this is most likely a result of leftover ethanol being present in the wells before the addition of the bead-LAMP reaction mix. Make sure to remove all the ethanol you can from the wells by pipetting it out, and letting the beads dry for 3 - 5 minutes at room temperature. When you see a color change before incubation, you shouldn't continue – have to start over to get relevant results.
  • The colour of my bead-LAMP reaction is orange, somewhere in the middle between yellow and pink. How do I interpret this?
  • You should interpret every non-yellow result as a negative. Whenever you are not sure, compare the sample colour to the colour of a positive control. This is why we recommend everyone to run positive controls in every test.

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